key: cord- -qcjbtflt authors: carrero, javier antonio title: confounding roles for type i interferons during bacterial and viral pathogenesis date: - - journal: international immunology doi: . /intimm/dxt sha: doc_id: cord_uid: qcjbtflt although type i interferons (ifn-i) were initially defined as potent antiviral agents, they can also cause decreased host resistance to some bacterial and viral infections. the many antiviral functions of the ifn-i include direct suppression of viral replication and activation of the immune response against viruses. in addition to their antiviral effects, ifn-i are also protective against several extracellular bacterial infections, in part, by promoting the induction of tnf-α and nitric oxide. in contrast, there is a negative effect of ifn-i on host resistance during chronic infection with lymphocytic choriomeningitis virus (lcmv) and acute infections with intracellular bacteria. in the case of lcmv, chronic ifn-i signaling induces adaptive immune system suppression. blockade of ifn-i signaling removes the suppression and allows cd t-cell- and ifn-γ-mediated resolution of the infection. during acute intracellular bacterial infection, ifn-i suppress innate immunity by at least two defined mechanisms. during francisella infection, ifn-i prevent il- upregulation on γδ t cells and neutrophil recruitment. following listeria infection, ifn-i promote the cell death of macrophages and lymphocytes, which leads to innate immune suppression. these divergent findings for the role of ifn-i on pathogen control emphasize the complexity of the interferons system and force more mechanistic evaluation of its role in pathogenesis. this review evaluates ifn-i during infection with an emphasis on work carried out ifn-i-receptor-deficient mice. the type i interferons (ifn-i) are an extensive family of pleiotropic cytokines that all signal through the ubiquitously expressed ifn-i receptor, termed the 'ifn-α receptor' (ifnar) ( ) . the activity of these cytokines was discovered in the s because of their ability to 'interfere' with viral infection ( ) . molecular cloning techniques and genome sequencing have led to the identification of an extensive number of members of the ifn-i family. in mice, the two best analyzed members of this family are the cluster of ifn-α subtypes and ifn-β molecule. genetic ablation of ifnar (ifnar -/-) in the mouse is sufficient to prevent signaling by all members of the ifn-i family and is the biologically defining activity that groups the ifn-i molecules into one family ( ) . ifnar is composed of two chains (ifnar and ifnar ) that coordinately activate the kinases jak and tyk (tyrosine kinase ) upon ifn-i binding. jak (janus kinase ) and tyk phosphorylate stat and stat that, together with interferon regulatory factor (irf ), form the interferon-stimulated gene factor (isgf ) complex ( ) . isgf binds to interferonstimulated response elements to cause upregulation of over genes. the type ii interferon receptor, ifngr -ifngr , which binds ifn-γ, the only type ii interferon, activates jak and jak , leading to stat phosphorylation and homodimerization ( ) . because of the similarities in signaling, many of the genes that are upregulated by ifn-i are also upregulated by ifn-γ, providing some degree of redundancy between the ifn-i and ifn-γ signaling pathways ( ) . however, there are a large number of genes unique to ifn-i. although many of the ifn-i-specific genes have defined antiviral functions, the role of many remains unresolved. the antiviral activity of ifn-i was initially defined with conditioned supernatant inhibition of viral growth and then with purified cytokines. however, it was the generation of mice deficient in ifnar signaling that permitted examination of infections under more physiological conditions ( ) . this review will focus on the role of ifn-i during murine infection with viral and bacterial pathogens. there is also extensive work on the role ifn-i in the pathogenesis of autoimmunity and cancer, and more recently, in fungal and protozoan infections ( ) ( ) ( ) . although this work has contributed to our understanding of the biological activities of ifn-i, it is beyond the scope of this review. because of the extent of the ifn-i literature, this review will mostly limit itself to experiments that have examined viral and bacterial pathogenesis in ifnar -/mice with a particular emphasis on work that has examined lethality and pathogen burden. four major themes will be covered: first, there is a broad summary of the (generally) protective role of ifn-i in mice infected with viruses. second is the recent finding that ifn-i promote the suppression of adaptive immunity seen during chronic infection with lymphocytic choriomeningitis virus (lcmv). third, there is an examination of the divergent results that have been obtained using different bacterial pathogens. finally, there is a detailed examination of the role of ifn-i during listeria monocytogenes infection. the consensus in the field is that ifnar signaling is protective against most types of viral infection. table summarizes some of the results obtained after infecting wild-type and ifnar -/mice with multiple species and strains of viruses. in most cases, the absence of systemic ifnar signaling by the mouse led to an increase in viral titer, lethality, or both compared with controls. ifn-i is important in handling all major genetic classes of viruses including single-stranded rna (ssrna; +/-stranded), double-stranded rna and double-stranded dna viruses, and acute retroviruses (ssrna-rt). the two exceptions to the strict requirement for ifnar are influenza and dengue virus infections. in the case of influenza and potentially other respiratory viruses, the type iii interferon system (which comprises ifn-λ subtypes and signals using il- r -ifnlr ) plays a dominant role in restricting acute epithelial cell infection, thereby limiting the requirement of ifn-i signaling ( , ) . in dengue, ifnγ-mediated protection is dominant over ifn-i, although the combined ifnar -/-× ifngr -/mice are more susceptible than the ifngr -/mice ( ) . the effects of ifn-i that limit viral infection are extensive, but several aspects are important to consider. ifn-i signaling enhances the susceptibility of virally infected cells to undergo programmed cell death, thereby limiting viral replication ( , ) . dendritic cells (dcs) exposed to ifn-i become activated and secrete proinflammatory cytokines that lead to activation of the adaptive immune response ( ) . nk cells become potent killers of virally infected cells after exposure to ifn-i ( , ) . ifn-i has direct effects on adaptive αβ t cells and sensitizes them to activation via the tcr ( , ) . ifn-i production by plasmacytoid dcs promotes b-cell activation and production of antiviral antibody ( , ) . in general, these effects of ifn-i signaling are beneficial to the host, as they lead to control of viral replication and spread. the importance of host ifn-i signaling is further reinforced by viral evolution. viruses have evolved extensive immune-evasion strategies many of which center around inhibition of the host ifn-i response ( , ) . however, the biological responses to ifn-i do not always lead to beneficial outcomes to the host. in the case of viral infections, the best studied example of the negative role of ifn-i are chronic viral infections, in particular infection with lcmv. a long-standing finding in the lcmv field is that small genetic changes can convert an acutely infective strain of lcmv (armstrong b) into a chronically infective strain (armstrong b clone ; cl ) ( ) . several hallmarks of the negative effects of chronic viral infection have been discovered using this system. mice become chronically infected with lcmv because of t-cell 'exhaustion' that prevents normal clearance ( ) . several factors have been implicated in the suppression of t-cellmediated clearance of chronic lcmv; most salient among them are il- and pd- (programmed cell death ). il- is known to antagonize inflammatory activation on multiple immune cell types and its neutralization prevents chronic infection with lcmv ( ) . pd- , a member of the cd /ctla family of t-cell regulators, is upregulated on exhausted t cells found in chronically infected mice. its ligands, pd- l and pd- l, are broadly expressed and inducible by interferons ( ) . the interaction of pd- with pd-l acts to limit t-cell activity during chronic infection. blockade of the pd- -pd-l interaction using mabs derepresses cd t-cell activity and leads to enhanced adaptive immune responses to lcmv infection ( ) . in two recent publications, the effects of il- and pd- in limiting the response to lcmv infection have been causally linked to ifn-i signaling ( , ) . during the initial stages of infection with cl , the absence of ifn-i signaling allows for an increased viral titer and delayed clearance during the acute phase of the response ( , ) . wild-type mice control the primary infection well but become chronic carriers. ifnar -/mice also become chronic carriers albeit at higher viral loads. the cl strain induces higher levels of ifn-α and ifn-β than the acutely infective armstrong strain ( ) . the major early producer of ifn-i are plasmacytoid dcs that are infected with the virus ( ) . the presence of ifn-i is associated with a prolonged signature of interferon-inducible genes in spleen cells ( ) . despite having higher early titers of lcmv, ifnar -/mice show reduced il- in serum and reduced pd-l expression on myeloid cells. in wild-type mice, blockade of ifnar signaling with neutralizing mabs replicates this effect, leading to reduction of il- and pd-l . furthermore, neutralization of ifnar after the establishment of chronic infection leads to reduced viral burden ( ) . therefore, ifnar plays a major role in the establishment of chronic infection with lcmv and neutralization of ifnar has therapeutic potential for patients harboring chronic viral infections. the response of ifnar -/mice to bacterial infections varies depending on the species and route of infection. table summarizes some of the findings of ifnar -/mice infected with several important pathogenic bacteria. in streptococcus, escherichia coli, and helicobacter infections, ifnar -/mice have higher titers and/or lethality than wild-type controls. during brucella, francisella, salmonella, chlamydia, mycobacterium, or yersinia infections, ifnar -/mice control infection better than wild-type. the ifnar -/mice are more resistant to l. monocytogenes given systemically. a recent report has shown that during oral infection with listeria, ifnar signaling may be protective. based on these initial studies, the simplest conclusion is that ifnar signaling is beneficial during extracellular bacterial infection and detrimental during intracellular bacterial infection. type i ifn signaling provides increased protection during streptoccocus infection by promoting upregulation of tnf-α, ifn-γ and nitric oxide ( ) . this is associated with restriction of bacterial growth. ifnar -/mice have bacteremia, increased systemic titers, and decreased survival. in vitro, streptococcus spp. induce ifn-β production through cell-type-specific signaling pathways ( ) . strepcococcus can trigger ifn-i via the complex of irf , stimulator of interferon genes (sting) and tank-binding kinase (tbk ). in streptococcus-infected macrophages, ifn-β is induced through the irf -sting-tbk complex and this is partially dependent on myd (ifn-i can also be produced in a myd -independent pathway). in contrast, streptococcusinfected dcs induce ifn-β through irf and myd . more mechanistic studies are still required to resolve the molecular triggers and in vivo cellular sources of ifn-i during streptoccocus infection. helicobacter pylori-infected ifnar -/mice have higher titers, but the mechanism of ifn-i action in this infection remains unresolved ( ) . further work needs to be done on the extracellular bacterial infections to determine how ifn-i is protective and what distinguishes ifn-i from ifn-ii in these types of infections. following brucella infection, there is ifnar-dependent upregulation of tnf-related apoptosis-inducing ligand (trail) and splenic apoptosis that is associated with increased susceptibility to infection ( ) . ifnar -/mice also express more ifn-γ and nitric oxide. in the case of salmonella enterica and chlamydia muridarum, ifn-i signaling sensitizes the infected macrophage to undergo cell death ( , ) . prevention of macrophage cell death during s. enterica infection led to decreased bacterial titer. ifnar −/− mice infected with francisella have decreased titers and lethality compared with controls. this is attributed to the inhibitory effect of ifn-i signaling on il- a/f expression ( ) . ifnar -/mice express more il- a/f, have an expansion of il- + γδ t cells and increased neutrophils at the site of infection. finally, treatment with ifn-i agonists such as poly(i:c) also promotes negative outcomes during bacterial infection. in the case of mycobacterium tuberculosis, intranasal delivery of poly(i:c) throughout the course of infection led to increased inflammatory infiltrates and necrosis of lung tissue that was dependent of ifnar signaling ( ) . similar detrimental effects of poly(i:c) treatment are also seen following streptoccocus pneumoniae, staphylococcus aureus and l. monocytogenes infections ( ) (see below). the first example of the detrimental effect of ifn-i during bacterial infection was discovered following l. monocytogenes infection. to date, it remains the best examined system, yielding information on the mechanisms of ifn-i induction, cellular sources and targets of ifn-i, and the nature of biological outcomes. macrophages infected with l. monocytogenes induce expression of ifn-i that is dependent on bacterial expression of the pore-forming toxin listeriolysin o (llo) ( , ) . llo is important for the bacterial egress from the nascent phagosome to the cytosol ( ) . llo alone does not induce strong levels of ifn-i production by the infected macrophage, suggesting that the presence of cytosolic bacteria is the driver of ifn-i production ( ) . this is reinforced by experiments demonstrating that bacillus subtilis expressing llo gain access to the cytosol and also strongly induce ifn-responsive genes. the major molecular driver of ifn-i induction by l. monocytogenes is the cyclic dinucleotide c-di-amp ( ) . the cyclic dinucleotides were initially discovered in bacteria as a second messenger system that also doubles as a pathogen-associated molecular pattern ( ) . interestingly, c-di-amp is actively exported from the bacteria and the induced expression of a c-di-amp synthesizing enzyme (di-adenylate cyclase) increases ifnb gene expression by infected macrophages ( ) . a sensor for cyclic dinucleotides has been identified as the helicase ddx , which recruits sting, tbk and irf (see above) to drive upregulation of ifn-i genes ( ) . splenic macrophages (cd b + cd c -pdca -b -) and tnf/inos-producing dcs (tip-dcs; cd b + cd c + ly c + ) produce ifn-i following l. monocytogenes infection in vivo ( , ) . mice lacking ccr expression, which do not recruit tip-dcs to the spleen, have reduced expression of ifn-α following l. monocytogenes infection ( ) . to date, there is no clear demonstration that a l. monocytogenes-infected myeloid cell population is producing ifn-i in vivo. on the basis of experiments using immunofluorescent colocalization, tip-dcs appear not to be infected ( ) . the work that identified ifn-i production by splenic macrophages did not evaluate the infected status of the cells ( ) . therefore, at this time, the connection between the molecular mechanisms of induction and in vivo cellular sources of ifn-i cannot be definitely established. listeria monocytogenes causes apoptotic cell death of macrophages that is enhanced by ifnar signaling. within h of infection, bone marrow-derived macrophages upregulate ifn-β and phosphorylate stat ( ) . deletion of ifnar on macrophages raises their resistance to l. monocytogenesmediated killing significantly. the death induced by l. monocytogenes is dependent on bacterial expression of llo ( ) . since llo is essential for virulence, it is unclear if it has a direct role in killing the infected macrophage or is only important for allowing egress of the bacteria to the cytosol. ifnar-dependent macrophage death is also found following infection of mice with l. monocytogenes ( ) . a population of tnf-α-producing cd b + macrophages is depleted following infection of wild-type mice. this population is maintained in ifnar -/mice, demonstrating a role for ifn-i in sensitization of macrophage death. it is not known at this time if the macrophages that die in vivo are infected by the bacteria. the most profound ifnar-dependent effect seen in mice infected with l. monocytogenes is the extensive depletion of white-pulp lymphocytes via apoptotic cell death ( ) . in wildtype mice, apoptosis begins in the periarteriolar lymphoid sheath (t-cell area) and extends to the entire white pulp in a dose-dependent manner ( ) . removal of ifnar significantly limits the number of apoptotic profiles and the extent of apoptotic death in any given white pulp ( ) . treatment of t cells with ifn-α sensitizes them to llo-induced apoptosis suggesting that secreted llo may be a killer molecule in vivo ( ) . additionally, ifn-i upregulate trail on nk cells and trail receptor (dr ) on the t cells and macrophages, providing a second potential mechanism for interferon-mediated lymphocyte and macrophage killing ( ) . trail -/mice harbor lower bacterial burdens than wild-type counterparts, have decreased splenic lymphocyte apoptosis, and increased accumulation of myeloid cells in the spleen following l. monocytogenes infection. the reduction in lymphocyte death seen following l. monocytogenes infection is the major reason that ifnar -/mice are more resistant to infection ( ) . several lines of evidence support this conclusion. first, mice deficient in lymphocytes (scid/ rag mice) are highly resistant to l. monocytogenes infection ( , ) . second, mixed bone marrow chimeras that create mice that are ifnar + in all cells except lymphocytes are also resistant to l. monocytogenes infection ( ) . this demonstrates the dominance of ifn-i signaling effects on lymphocytes. third, induction of ifn-i using poly(i:c) increases the susceptibility to infection of wild-type but not scid mice ( , ) . finally, l. monocytogenes infection induces myeloid cell expression of il- that is dependent on ifnar expression by lymphocytes ( ) . the upregulation of il- is a negative regulator of pathogen handling and il- -/mice are more resistant to infection despite having normal lymphocyte apoptosis ( , ) . as an aside, the work on ifnar effects on l. monocytogenes infection was conducted on three different genetic backgrounds with different susceptibilities to infection. in all three strains-c bl/ ( ), s ( ) and balb/c ( )-ifnar signaling was detrimental to the outcome of infection. this demonstrates that ifnar effects are dominant over the genetic susceptibilities of the mouse strains to l. monocytogenes infection. further work needs to be done to determine if this applies to other bacterial infection models. the initial paradigm of the ifn-i system is that it provides antiviral protection that sometimes goes awry in certain autoimmunities. this simplified view has been replaced with a more complex and interesting role for the interferons in regulating immune responses. chronic viral infections are teaching us that while early ifn-i is important in controlling viremia, pathogens that can overcome this initial control benefit from the immune regulation that takes place following long-term interferon induction. the clinical relevance of this can be seen during infection of patients with hiv, where chronic ifn-i leads to trail-mediated t-cell death and poor disease outcome ( ) . future work needs to be done to determine the applicability of ifn-i modulation as a therapeutic to important chronic human viral infections. another important area of research is the interface between viral and bacterial coinfections. the clinical importance of severe bacterial infections occurring after a primary viral infection is well established ( ) . respiratory bacterial infections are more dangerous to patients when they occur following infection with viruses such as influenza and respiratory syncytial virus. this observation has been replicated in mouse models of infection ( , ) . however, the interaction between viral and bacterial infection is not always deleterious. infection with herpesvirus induces prolonged ifn-γ production that leads to protection against infection with l. monocytogenes and yersinia pestis ( ) . the main distinguishing feature between the two potential outcomes (acute versus chronic virus) and (detrimental versus beneficial) appears to center on the balance between ifn-i and ifn-γ effects. this reinforces the need to understand the molecular effects of these cytokines during bacterial infection. in the bacterial world, ifn-i were once believed to be relatively unimportant. this idea was reversed by research on l. monocytogenes. careful examination of additional bacterial infections has demonstrated that both route and tropism of bacterial infections matter in the requirement of ifn-i. future studies will be needed to determine cellular sources, molecular triggers and biological outcomes of ifn-i for many classes of bacterial pathogens. it will be interesting to see if bacteria have evolved mechanisms to manipulate the ifn-i system like some viruses do. recently, it has been shown that the balance of ifn-i and ifn-ii may be important in the outcome of human mycobacterial infections ( ) . reminiscent of chronic lcmv infection, il- is also a key player in the ifn-i-mediated suppression of mycobacterial immunity. future work will be needed to determine if chronic ifn-i production is a common determinant of negative outcomes in infectious diseases. finally, we need a better understanding of how the genes specific for ifn-i lead to different outcomes from their close cousin, ifn-ii. national institute of allergy and infectious diseases (ai ). type i interferons (alpha/beta) in immunity and autoimmunity the jak-stat pathway at twenty mechanisms of type-i-and type-ii-interferon-mediated signalling how cells respond to interferons systematic identification of type i and type ii interferon-induced antiviral factors functional role of type i and type ii interferons in antiviral defense type i interferon: friend or foe? type i interferons and the innate immune response-more than just antiviral cytokines interferons, immunity and cancer immunoediting lambda interferon renders epithelial cells of the respiratory and gastrointestinal tracts resistant to viral infections induction and function of type i and iii interferon in response to viral infection interferon-dependent immunity is essential for resistance to primary dengue virus infection in mice, whereas t-and b-cell-dependent immunity are less critical interferon function is not required for recovery from a secondary poxvirus infection effects of type i interferons on friend retrovirus infection alpha/beta interferons regulate murine gammaherpesvirus type i interferons and infection latent gene expression and reactivation from latency the role of alpha/beta and gamma interferons in development of immunity to influenza a virus in mice the role of interferon in influenza virus tissue tropism critical role for alpha/beta and gamma interferons in persistence of lymphocytic choriomeningitis virus by clonal exhaustion of cytotoxic t cells type i interferons are essential in controlling neurotropic coronavirus infection irrespective of functional cd t cells type i interferons produced by hematopoietic cells protect mice against lethal infection by mammalian reovirus theiler's virus infection of sv mice that lack the interferon alpha/beta or interferon gamma receptors alpha/beta interferon protects against lethal west nile virus infection by restricting cellular tropism and enhancing neuronal survival type i interferons in host defense type interferons and the virus-host relationship: a lesson in detente coordinated and distinct roles for ifn-alpha beta, il- , and il- regulation of nk cell responses to viral infection regulation of effector and memory t-cell functions by type i interferon type i interferon-mediated stimulation of t cells by cpg dna plasmacytoid dendritic cells promote rotavirusinduced human and murine b cell responses plasmacytoid dendritic cells induce plasma cell differentiation through type i interferon and interleukin mechanisms of evasion of the type i interferon antiviral response by flaviviruses recent advances in understanding viral evasion of type i interferon molecular basis of viral persistence: a single amino acid change in the glycoprotein of lymphocytic choriomeningitis virus is associated with suppression of the antiviral cytotoxic t-lymphocyte response and establishment of persistence virus persistence in acutely infected immunocompetent mice by exhaustion of antiviral cytotoxic effector t cells interleukin- and the interleukin- receptor pd- and its ligands in tolerance and immunity restoring function in exhausted cd t cells during chronic viral infection persistent lcmv infection is controlled by blockade of type i interferon signaling blockade of chronic type i interferon signaling to control persistent lcmv infection myd and sting signaling pathways are required for irf -mediated ifn-β induction in response to brucella abortus infection type i ifns enhance susceptibility to chlamydia muridarum lung infection by enhancing apoptosis of local macrophages type i ifn signaling constrains il- a/f secretion by gammadelta t cells during bacterial infections type i interferon sensitizes lymphocytes to apoptosis and reduces resistance to listeria infection mice lacking the type i interferon receptor are resistant to listeria monocytogenes type i interferon production enhances susceptibility to listeria monocytogenes infection dynamic roles of type i and type ii ifns in early infection with mycobacterium tuberculosis the type i ifn response to infection with mycobacterium tuberculosis requires esx- -mediated secretion and contributes to pathogenesis type i interferon induces necroptosis in macrophages during infection with salmonella enterica serovar typhimurium opposing roles for interferon regulatory factor- (irf- ) and type i interferon signaling during plague type i ifn signaling is crucial for host resistance against different species of pathogenic bacteria nod contributes to mouse host defense against helicobacter pylori via induction of type i ifn and activation of the isgf signaling pathway route of infection determines the impact of type i interferons on innate immunity to listeria monocytogenes type i interferon production induced by streptococcus pyogenes-derived nucleic acids is required for host protection intranasal poly-ic treatment exacerbates tuberculosis in mice through the pulmonary recruitment of a pathogen-permissive monocyte/macrophage population poly i:c enhances susceptibility to secondary pulmonary infections by gram-positive bacteria production of type i ifn sensitizes macrophages to cell death induced by listeria monocytogenes a specific gene expression program triggered by gram-positive bacteria in the cytosol listeriolysin o: a phagosome-specific lysin c-di-amp secreted by intracellular listeria monocytogenes activates a host type i interferon response innate sensing of bacterial cyclic dinucleotides: more than just sting the helicase ddx recognizes the bacterial secondary messengers cyclic di-gmp and cyclic di-amp to activate a type i interferon immune response characterization of the interferon-producing cell in mice infected with listeria monocytogenes a fluorescence reporter model defines "tip-dcs" as the cellular source of interferon β in murine listeriosis tnf/inos-producing dendritic cells mediate innate immune defense against bacterial infection ifn-beta increases listeriolysin o-induced membrane permeabilization and death of macrophages lymphocyte apoptosis during early phase of listeria infection in mice reduced apoptosis and ameliorated listeriosis in trail-null mice mechanisms and immunological effects of apoptosis caused by listeria monocytogenes regulation of macrophage ia expression in mice with severe combined immunodeficiency: induction of ia expression by a t cell-independent mechanism lymphocytes are detrimental during the early innate immune response against listeria monocytogenes both innate and acquired immunity to listeria monocytogenes infection are increased in il- -deficient mice hiv- immunopathogenesis: how good interferon turns bad how do viral infections predispose patients to bacterial infections? type i interferon induction during influenza virus infection increases susceptibility to secondary streptococcus pneumoniae infection by negative regulation of γδ t cells viral infection augments nod / signaling to potentiate lethality associated with secondary bacterial infections herpesvirus latency confers symbiotic protection from bacterial infection type i interferon suppresses type ii interferon-triggered human anti-mycobacterial responses i wish to thank dr emil r. unanue for his support and insightful discussions. the content is solely the responsibility of the author and does not necessarily represent the official views of the national institutes of health. key: cord- -knbf m x authors: rodrigues, merlyn r.; lennette, david a.; arentsen, juan j.; thompson, charla title: methods for rapid detection of human ocular viral infections date: - - journal: ophthalmology doi: . /s - ( ) - sha: doc_id: cord_uid: knbf m x recent methods for detection of viruses in clinical specimens include immunofluorescence, immunoperoxidase, immune adherence hemagglutination, radioimmunoassay, enzyme-linked immunosorbent assay (elisa), and immunoelectron microscopy. some are useful for the detection of traces of viral antigens but are more complicated and timeconsuming than others. simple techniques of immunofluorescence and negative stain electron microscopy are used for the rapid detection of viruses in human adenoviral, herpetic, rubella, molluscum contagiosum, and vaccinial infections. in , conjunctival specimens obtained from patients with epidemic keratoconjunctivitis (ekc) were examined by direct immunofluorescence. the specimens were conjunctival swabs or scrapings from patients with acute follicular keratoconjunctivitis. adenovirus groupantigen specific, fluorescein conjugated rabbit antiserum was used. titration of this conjugate, dilated in % mouse brain suspension in pbs ph . , gave an optimum dilution for use of : . at this dilution, antigen-positive cells stained brilliantly ( +), while antigen-negative cells were only faintly stained but were readily discernible ( + or less). conjunctival scrapings were prepared on clean microscope slides and air dried as rapidly as was feasible-preferably on the returnair intake grill of a laminar-flow biologic safety cabinet. following thorough air drying, the. slides were volume r march ocular viral infections acetone fixed at room temperature for ten minutes. the slides with the conjugate diluted in brain suspension were incubated for minutes at c in a moist chamber, followed by a ten-minute wash in pbs ph . with intermittent agitation. the slides were examined at a magnification of x , using standard exciter/barrier filter combinations for fluorescein with w hgarc illumination. scrapings were scored as positive if any cells of normal morphology were observed with typical granular cytoplasmic fluorescence. scrapings were read as negative if no such cells were found and as inconclusive if only atypical fluorescence and fewer than cells were noted. controls.-fl uorescein -ia be led preserum from the animals used to produce the antibodies became commercially available only after the study was completed. before the study was conducted on clinical specimens, the working titer of the conjugates was established at varying dilutions by staining uninfected cells, homologous-infected cells, and heterologous-infected cells. at a working dilution that produced brightto-brilliant fluorescence with the homologous agents, there was negligible background fluorescence with either uninfected or heterologousinfected (herpes simplex virus [hsv- ], herpes simplex virus , and herpes zoster with adenovirus) cells. the conjugates were stored at -soc after reconstitution, and only small amounts of working dilutions were prepared to avoid repeated freezing and thawing. the clinical specimens were examined with absorbed-conjugate con-trois that were retested approximately monthly with uninfected, homologous-infected, and heterolologous-infected cell cultures. during the study, conjugated normal rabbit serum was used as a control conjugate at the same dilution as the virus-specific conjugates. the intention was to determine whether accurate results could be obtained by immunofluorescence without multiple controls. cell culture isolation.-specimens of conjunctival scrapings for viral isolation were collected in -ml quantities of hank's balanced salt solution, with . % gelatin added previously, dispensed and sterilized in -gm vials. the specimens were refrigerated until delivered to the virology laboratory, usually within hours. they were treated by adding antibiotics (amphotericin b and psnb, a standard mixture of penicillin, streptomycin, neomycin, and bacitracin), then inoculated in . ml volumes into two tubes each of human embryonic lung or kidney fibroblast cells and primary rhesus monkey kidney cells. primary human embryonic kidney cells were used when available, as were hela cells. cell cultures were held for a minimum of one month; any cultures that showed nonspecific degeneration earlier were passed to fresh cells of the same type. no further ''blind" passages were made. if typical adenovirus cytopathic effects were observed, the cells were held until the effects were estimated to involve well over half of the cell sheet, then harvested for titration and neutralization by the intersecting serum pool scheme. final serotyping was confirmed using monotypic antisera at a dilution providing approximately antibody units against to tcidso. adequate smears and 'scrapings from patients with dendritic corneal lesions were fixed in acetone for ten minutes at room temperature, air dried, inoculated with a : dilution of conjugate in brain suspension for minutes at c, and washed for ten minutes in pbs ph . . with fluorescence micros--copy of the slides it was usually possible to differentiate type and type herpes simplex viruses if type-specific conjugates were used. lens aspirate from a -year-old patient with clinical ocular rubella was examined by immunofluorescence and negative stain electron microscopy. indirect immunofluorescence was performed with two human sera, pretested by a standard rubella hemagglutination-inhibition antibody (hal) test. the positive serum had a titer of : , and the negative serum, a titer of less than : , by hal these sera were then used in an indirect immunofluorescence test on rubella-infected and uninfected bhk-cells, a susceptible line used for laboratory propagation of rubella virus. titration of the positive serum revealed a working titer of : ; working titer is defined as a dilution that gives brilliant ( +) fluorescence of infected bhk- cells and minimal (±) fluorescence of noninfected cells. the conjugate used was a : antihuman igg rabbit serum. the lens smears were fixed in acetone for ten minutes at room temperature, ringed with fast-drying enamel and overlaid with positive and negative sera, incubated for minutes at c, washed in pbs ph . (also used to dilute the serum), stained with the antihuman igg for minutes at c, washed, mounted in polyvinyl alcohol ph . , and examined with ultraviolet illumination at x . the conjunctival and corneal scrapings were fixed in . % buffered glutaraldehyde. the suspension was placed in drops in formvar-coated grids that were then stained for approximately seconds in % phosphotungstic acid at ph . . the specimens were examined by electron microscopy. the patients had conjunctival hyperemia and folliculosis (fig ) . early subepithelial corneal infiltrates were present in % of the patients, preauricular nodes were present in %, and conjunctival pseudomembranes were present in approximately %. of specimens from patients with ekc, positive immunofluorescence for adenovirus was present in cases. cytoplasmic fluorescence was marked (fig ) , but stippled nuclear fluorescence was also observed (fig ) . cultures and typing showed that of these were a hybrid type - , one was type , and one was type . in three persons with herpetic dendritic keratitis (fig and ) , positive immunofluorescence was present (fig ) ; all were herpes simplex hominis type . herpes simplex virions obtained from scrapings of dendritis lesions measured nm in diameter and displayed mostly intact capsids (fig ) . a -year-old boy with ocular rubella had unilateral cataract (fig ) and microphthalmos. smears of the cataractous lens revealed positive immunofluorescence with the positive serum in the form of diffuse and granular cytoplasmic fluorescence, and diffuse streaks of amorphous extracellular material. molluscum contagiosum lid lesions displayed circumscribed nodules with a central umbilication (fig ) . scrapings stained with hematoxylin-eosin revealed large basophilic cytoplasmic inclusions in the granular layer. n~gative stain electron microscopy disclosed brick-shaped virions (fig ) measuring x nm with the usual interlacing pattern of surface threads. in one patient with a vaccinia lid lesion (fig ) , both mulberry (m) and capsule (c) forms were observed (fig and ). recent advances in electron microsaopy as well as immunologic techniques have facilitated the rapid detection of viruses in clinical specimens. a hapten-sandwich procedure was reported for immunospecific labeling of cell surface antigens with markers visible by scanning electron microscopy (sem). antihapten antibody was used to link hapten-modified tobacco mosaic virus and bushy stunt virus. viral identification by sem of preparations stained with fluorescein-labeled antibody was described by springer et al, using hemagglutination of chicken erythrocytes by influenza virus as a model. transmission electron microscopy has been widely used for the study of viral structure and morphogenesis, although the low concentration of viral particles in some clinical used for the preparation of highly concentrated suspensions of mosaic viruses and human adenovirus type . the advantage of this technique over conventional negative staining is the formation of two-dimen-sional and three-dimensional crystalline arrays of viruses. the packing arrangement of the viruses could be varied according to the type of negative stain and ph used during the preparation. doane et al described a two-hour embedding procedure for intracellular detection of viruses in tissue culture as well as tissue biopsy specimens. the total processing time by this method required two hours compared with an average of to hours by the standard method. however, there was slight decrease of specimen detail with less than optimal staining compared with the standard techniques. doane reported that identification of viris by immunoelectron microscopy was useful for the study of viral antigens and antibodies. he was able to identify elusive viruses including rubella, hepatitis-associated antigens, rhinovirus, and coronavuus. gardner and mcquillin have described the value of immunofluorescence in the diagnosis of rubella, adenoviral infections, and measles. immunofluorescence of human viral infections, particularly herpes simplex, has been reported. s the most common causes of viral keratoconjunctivitis are herpes simplex, adenovirus, and chlamydia. the most frequent types of adenoviruses encountered in ocular infections are types and ; types (pharyngoconjunctival fever), , and are sporadically seen. viral isolations occur during ekc outbreaks, usually in the spring and fall in the united states. clinically, adenovirus ekc is characterized by the acute "onset of a follicular conjunc-tivitis, hyperemia, photophobia, and lacrimation. diffuse epithelial keratitis followed by subepithelial infiltrates are a frequent finding. all of these features were present in the cases · studied. however, the clinical manifestations of the patients with hybrid type - infection resembled ekc caused by other types of adenoviruses. there was no evidence of systemic involvement. outbreaks of ekc frequently occur in schools, swimming pools, outpatient departments, and hospitals. the epidemics occur from contamination of eye solutions, instruments, and from infected fingers of doctors and nurses. • three of the patients in this study were ophthalmologists who had acquired the infection from examining patients with adenoviral infections. simultaneous nosocomial and community outbreaks of ekc with types and adenovirus were recently described. in a recent epidemic of ekc at a vietnamese refugee camp in florida, adenovirus type was recovered in % of cases cultured within two weeks of onset of infection.u dawson et ap described adenovirus-like particles in the conjunctiva of one patient and in the corneal epithelium of another by transmission electron microscopy of tissue culture preparations. they found that among patients with ekc caused by adenovirus type , had persistent corneal erosions during the acute stage of the disease, had conjunctival scarring, and had inflammatory membranes. an outbreak of adenovirus type in the united states was described by hierholzer and associates and by burns and potter. an epidemic of ekc caused by adenovirus type occurred in london. in this study, volume rfi adenovirus type was isolated from patients using human embryonic kidney cells. another re-port described difficulties encountered in typing adenovirus related to types to in an outbreak of keratoconjunctivitis in bristol. the same problem was encountered in this study, since the hybrid type [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] showed characteristics of type determined by neutralization and of type by hemagglutination inhibition. in , ellison and associates compared methods available at that time for the laboratory diagnosis of ocular adenovirus type infections. they used fluorescent antibody staining, examination of conjunctival cytology by the giemsa and papanicolaou stains, and standard virus isolations and antibody titers. they believed that virus culture was the most reliable way to identify adenoviral infections, since attempts at more rapid techniques using fluorescence antibody methods were unsuccessful. more recently, however, vastine and asso-ciates used a direct immunofluorescence technique for the diagnosis of acute adenoviral keratoconjunctivitis with positive results in patients with epidemic keratoconjunctivitis. in an earlier report, schwartz et ap described positive immunofluorescence studies in all patients with adenoviral isolation. the fluorescence staining was predominantly present in the cytoplasm, but speckled nuclear fluorescence was also noted. in the present study, positive results with immunofluorescence and cultures were obtained in patients examined within the first week of infection, negative results were found in persons with advanced or chronic disease. thus, fluorescent antibody staining provides a more rapid and sensitive method for the detection of adenoviruses in infected conjunctival cells than viral culture, which usually requires one to eight weeks for virus identification. the direct immunofluorescent method is more rapid (requiring less than one hour) than the indirect technique, which takes a few hours to perform. adequate controls are essential for the reliability of immunofluorescence and should be easier with the new commercial preserum conjugates. frenkel and piekarski ° emphasized the problems in the diagnosis of toxoplasma organisms and others by immunofluorescence with appropriate positive and negative controls. the present study showed a good relation of viral isolates to positive immunofluorescence; the latter was confirmed by culture. adenovirus virions were also noted in conjunctival scrapings by negative stain electron microscopy. kajima and associates were unable to demonstrate adenovirus by this method, although the organisms were demonstrated by conventional thin section electron microscopy. this could be due to more superficial smears or scrapings in their cases. in certain areas, adenoviral infection may be confused with keratoconjunctivitis (ahc) produced by enterovirus type . in ahc, the presence of conjunctival hemorrhage, slight folliculosis, and fre-quent pain in the early stage distinguishes this condition from ekc. in keratoconjunctivitis caused by echovirus , associated gastrointestinal symptoms, fever, headache, and lymphadenopathy are present at an early stage, but conjunctival folliculosis was not reported. the two main antigenic types of herpes virus hominis are type , commonly associated with infections of the eye, mouth, skin, and upper body, and type , associated with genital infections. neumann-haefelin et al reported patients with hsv type and three with type ocular infections. in the present study, the typical virions of herpes simplex keratitis were readily identified both by immunofluorescence and by negative stain transmission electron microscopy. the patients clinically manifested typical dendritic corneal lesions, which stained with fluorescein. dawson and togni observed virus particles in corneal epithelium and stroma of human corneal buttons. the virus may replicate in the corneal stroma without any overt inflammation. collin and abelson recently described a case of apparent herpes simplex keratouveitis where the virus was demonstrated by transmission electron microscopy in the cornea removed at the time of a third corneal transplant, indicating that the virus persisted for a considerable period of time. in a case of herpetic iritis, virus was found in the anterior chamber by immunofluorescence and electron microscopy. in specimens of molluscum, the virions displayed typical brickshaped structures and were the largest viruses observed. the mol-luscum lid lesion was a centrally umbilicated lid nodule. light microscopy showed typical cytoplasmic inclusions in the stratum corneum and granulosum. rapid techniques for the detection of viruses in specimens from patients with epidemic keratoconjunctivitis, herpes simplex keratitis, rubella, vaccinia, and molluscum were used. these included direct and indirect immunofluorescence and negative stain electron microscopy. in , conjunctival specimens from patients with ekc showed positive immunofluorescence in cases. the latter were from subjects with onset of infection of up to one week. cultures and typing showed that of these were hybrid type - , one was type , and one was type . herpes simplex hominis type was recovered in culture and demonstrated by direct immunofluorescence and negative stain electron microscopy in three patients with herpetic dendritic keratitis. in one patient with ocular rubella, lens aspirate showed positive indirect immunofluorescence. in patients with vaccinial and molluscum lid lesions, virions were demonstrated by negative stain electron microscopy. hapten-sandwich labeling: ii. immunospecific attachment of cell surface markers suitable for scanning electron microscopy viral identification by scanning electron microscopy of preparations stained with fluorescein-labeled antibody application of electron microscopy to the diagnosis of virus infection doane fw: lndentification of viruses by immunoelectron microscopy a negative staining-carbon film technique for studying viruses in the electron microscope: i preparative procedures for examining icosahedral and filamentous viruses two-hour embedding procedure for intracellular detection of viruses by electron microscopy rapid virus diagnosis application of immunofluorescence adenoviruses rapid diagnosis of herpes virus hominis infections by immunofluorescent antibody techniques community and hospital outbreaks of epidemic keratoconjunctivitis simultaneous nosocomial and community outbreak of epidemic keratoconjunctivitis with types and adenovirus epidemic keratoconjunctivitis at a vietnamese refugee camp in florida adenovirus type infections in the united states potter mh: epidemic keratoconjunctivitis due to adenovirus type epidemic keratoconjunctivitis and chronic papillary conjunctivitis in london due to adenovirus type epidemic adenovirus keratoconjunctivis cytologic diagnosis of adenoviral epidemic keratoconjunctivitis by direct immunofluorescence the demonstration of toxoplasma and other organisms by immunofluorescence: a pitfall electron microscopy as a diagnostic procedure for viral infections of the eye. twentysecond concilum herpes simplex eye infections: clinical manifestations, pathogenesis and management herpes simplex virus in human cornea, retrocorneal fibrous membrane, and vitreous herpetic iritis: demonstration of virus in the anterior chamber by fluorescent antibody techniques and electron microscopy acknowledgment peter laibson, md, provided a specimen of a herpetic dendrite, and brian altman, md, provided the specimen of lens aspirate from a patient with ocular rubella. serum and antisera were obtained from microbiolo· gical associates, the research resources branch of the national institute of allergy and infectious diseases, and miles laboratories. key: cord- -plz ja e authors: dussurget, olivier; bierne, hélène; cossart, pascale title: the bacterial pathogen listeria monocytogenes and the interferon family: type i, type ii and type iii interferons date: - - journal: front cell infect microbiol doi: . /fcimb. . sha: doc_id: cord_uid: plz ja e interferons (ifns) are secreted proteins of the cytokine family that regulate innate and adaptive immune responses to infection. although the importance of ifns in the antiviral response has long been appreciated, their role in bacterial infections is more complex and is currently a major focus of investigation. this review summarizes our current knowledge of the role of these cytokines in host defense against the bacterial pathogen listeria monocytogenes and highlights recent discoveries on the molecular mechanisms evolved by this intracellular bacterium to subvert ifn responses. listeria monocytogenes is a pathogenic gram-positive bacillus responsible for a foodborne disease in humans and animals called listeriosis (vázquez-boland et al., ) . this highly versatile bacterium can be isolated from multiple sources such as human and animal feces, soil, water, plants and food. as a common contaminant of fruits, vegetables, seafood, meat and cheese, it represents a major economic problem for the food industry. infection usually originates from ingestion of contaminated food (schlech et al., ) and may cause febrile gastroenteritis in otherwise healthy persons (ooi and lorber, ) . in contrast, in immunocompromised individuals it leads to a severe invasive disease, which manifests itself as septicemia, meningitis and encephalitis. in the specific case of pregnant women, infection may cause fetal loss or neonatal bacteremia and meningitis. in the united states, incidence of listeriosis ranged from . to . cases per million population between and (cartwright et al., ) . in france, incidence was . per million between and , and increased risk of listeriosis was noticed in people with underlying diseases, such as chronic lymphocytic leukemia (goulet et al., ) . while relatively rare, listeriosis is among the most deadly foodborne diseases with mortality rates reaching up to % depending on the clinical manifestations (lorber, ) . in addition to the immunological status of the patient, the clinical outcome of the disease depends on the pathogenic potential of the infecting bacteria. l. monocytogenes strains of serovars / a, / b, and b account for % of human cases and serovar b alone is associated to most outbreaks (swaminathan and gernersmidt, ) . the capacity of l. monocytogenes to survive and multiply within the gastrointestinal tract is critical for the initial infection, persistence and transmission. l. monocytogenes is well adapted to this environment and produces multiple factors to compete with microbiota and counteract antimicrobial peptides, acidity, hyperosmolarity, hypoxia, bile and iron deprivation (gahan and hill, ) . crossing of the intestinal epithelium is thought to occur by invasion of enterocytes, in particular goblet cells and m cells of the peyer's patches. invasion of enterocytes requires the specific interaction between the listeria surface protein inla and its cellular receptor e-cadherin (lecuit et al., ; disson et al., ) , which can take place at sites of cell extrusion at the tip and other locations of intestinal villi (pentecost et al., ; nikitas et al., ) . indeed, as recently shown, upon interaction with e-cadherin, listeria preferentially crosses the intestinal barrier by transcytosis through goblet cells (nikitas et al., ) . entry through ileal peyer's patches via m cells does not rely on inla. it has been reported to require listeria invasion protein inlb (chiba et al., ) . after translocation, bacteria reach lymph nodes, the liver and spleen and finally secondary target sites of infection, including the central nervous system and the placenta. a remarkable feature of l. monocytogenes is its capacity to invade non-professional phagocytic cells such as enterocytes, hepatocytes and trophoblast cells. the exceptional repertoire of virulence factors necessary for entry, survival and multiplication has been extensively studied cossart, ) . expression of many virulence genes relies on the transcriptional activator prfa, whose role is pivotal for l. monocytogenes transition from saprophytic to intracellular lifestyle (freitag et al., ; toledo-arana et al., ) . elimination of l. monocytogenes is mostly based on the capacity of the host to mount an efficient cellular immune response to infection (mackaness, ; shi and pamer, ) . in particular, the fate of infection depends on the level of macrophage activation and on listeria ability to counteract bactericidal mechanisms of host cells (shaughnessy and swanson, ; corr and o'neill, ; stavru et al., ) . bacterial escape from the phagosome and avoidance of autophagy for intracytosolic replication and cell-cell spread have been well characterized. they have been shown to depend on five major virulence factors: the secreted pore-forming toxin listeriolysin o (llo) and two phospholipases c (plca and plcb) for vacuolar escape, the surface protein acta for actin-based motility and both acta and a surface protein of the internalin family, inlk, for autophagy evasion (cossart, ; dortet et al., ) . other strategies of immune escape that lead to the modulation of cytokine expression occur through a variety of mechanisms. modifications of l. monocytogenes peptidoglycan by the n-deacetylase pgda and the o-acetyl transferase oata prevent lysozyme-dependent release of microbe-associated molecular patterns (mamps), activation of pathogen-recognition receptors (prrs) and subsequent production of pro-inflammatory cytokines (boneca et al., ; aubry et al., ; rae et al., ) . the toxin llo induces dephosphorylation of histone h and deacetylation of histone h , which correlate with decreased expression of pro-inflammatory genes, such as the chemokine gene cxcl (hamon et al., ) . the secreted internalin inlc inhibits inflammation by interacting with ikk-α, a component of the iκb-kinase complex, which is essential for nf-κb activation and expression of proinflammatory genes (gouin et al., ) . other evasion mechanisms remain to be characterized, such as the control of the expression of il- by the surface internalin inlh (personnic et al., ) . l. monocytogenes also has the capacity to modulate interferon (ifn) production during infection. type i ifn production by infected cells can be controlled by listeria multidrug efflux pumps mdrm and mdrt, via the secretion of the second messenger cyclic-di-amp (crimmins et al., ; woodward et al., ; schwartz et al., ) . synthesis of type iii ifn has also recently been shown to be tuned by listeria nucleomodulin lnta (lebreton et al., ) . our knowledge concerning the role of the ifn cytokine family during listeriosis has rapidly expanded in the last few years and will be the focus of this review. ifns form a family of proteins secreted by many cell types in response to infection. they were originally named for their capacity to interfere with viral proliferation (isaacs and lindemann, ) . this diverse family is composed of three groups of cytokines, namely type i-, type ii-, and type iii-ifns, which are important components of innate immune responses ( table ) . type i-ifns consist of ifn-α, ifn-β, ifn-δ, ifn-ε, ifn-ζ, ifnκ, ifn-ν, ifn-τ, and ifn-ω (levy et al., ) . type ii-ifn is composed of a single cytokine, ifn-γ (pestka et al., ) . type iii-ifns are ifn-λ , ifn-λ , and ifn-λ (formerly il- , il- a, and il- b) and ifn-λ (kotenko, ; prokunina-olsson et al., ) . type i-and type iii-ifns have similar signal transduction systems (see below) and are phylogenetically closer from each other than type ii-ifn (pestka et al., ) . sequence conservation and chromosome location suggest that type i-ifn genes evolved from a single ancestor through duplication. however, the extent of type i-ifn gene diversification varies greatly depending on the species (pestka et al., ) . generally, a single gene encodes the type i-ifn in fish. in contrast, multiple gene duplications and diversification led to the emergence of sub-types of type i-ifns in mammals ( table ) . gene duplication varies also within each sub-type. a single ifn-β gene is found in the human and mouse genomes (decker et al., ; honda et al., ; durbin et al., ) . in contrast ifn-α genes and one pseudogene and ifn-α genes and three pseudogenes are found in the human and mouse genomes, respectively (van pesch et al., ; durbin et al., ) . a single gene encodes type ii-ifn and four genes encode type iii-ifns in human (decker et al., ; levy et al., ) . of note, ifn-λ and ifn-λ are pseudogenes in mice, which prevents the study of these cytokines in this animal model ( table ) (fox et al., ). ifns are important components of the immune system, which generally trigger cellular protective defenses in response to infection or tumor formation. type-ii ifn (ifn-γ) is a paradigm for this, being an important mediator of innate and adaptive immune responses with a key role in clearance of viral and bacterial pathogens and in tumor control. ifn-γ was first described as an antiviral protein (wheelock and sibley, ) , but is now known to exhibit broader biological activities, non-redundant with that of other types of ifns. the crucial role of ifn-γ in immunity to infection is reflected by the phenotype of mice lacking the ifn-γ receptor or the ifn-γ gene, which are highly susceptible to mycobacterium bovis bcg infection (dalton et al., ; kamijo et al., ) . genetic deficiencies resulting in the loss of ifn-γ production or signaling in mice lead to increased susceptibility to infections by other intracellular pathogens, such as l. monocytogenes (see below), salmonella typhimurium and some viruses harty and bevan, ; jouanguy et al., ) . these defects also lead to the loss of tumor control (kaplan et al., ) . patients with deficiencies in the ifn-γ pathway, for instance by mutation in the gene for the ifn-γ receptor , are characterized by severe infections with viruses and intracellular bacteria including l. monocytogenes, salmonella sp. and mycobacteria (jouanguy et al., ; newport et al., ; roesler et al., ; van de vosse et al., ) . ifn-γ mediates macrophage activation, i.e., increased phagocytosis and production of pro-inflammatory cytokines, of microbicidal reactive oxygen and nitrogen species, leading to clearance of intracellular pathogens (schoenborn and wilson, ) . in addition, ifn-γ controls differentiation of t cells in th effector cells, antigen processing and presentation by antigen-presenting cells, which participate to cellular immunity against intracellular pathogens (schroder, ; hu and ivashkiv, ). the immunostimulatory and immunomodulatory properties of ifn-γ have therapeutic implications. indeed, ifn-γ is used in patients with chronic granulomatous disease to reduce infection and mortality, **protein length in amino-acids and protein modifications (g: glycosylation, p: phosphorylation). *** genes and a pseudogene in the human genome, genes and three pseudogenes in the mouse genome. although the clinical benefit has not been demonstrated in all studies (holland, ) . type i-ifns are produced in responses to viruses, many bacteria and parasites. however, in contrast to type ii ifns, these cytokines are not always protective against bacterial infections. indeed, the role of type i-ifns in response to bacterial infection is complex and depends on the microorganism (decker et al., ; monroe et al., ; carrero, ) . they contribute to resistance of the host against infection by extracellular bacteria, such as escherichia coli, helicobacter pylori, streptococcus agalactiae and s. pneumoniae (mancuso et al., ; watanabe et al., ) . in contrast, they are associated with suppression of innate immune responses and increased susceptibility of the host to infection by l. monocytogenes (see below), brucella abortus, chlamydia muridarum, francisella novicida, salmonella enterica, staphylococcus aureus, and yersinia pestis (auerbuch et al., ; carrero et al., ; o'connell et al., ; qiu et al., ; martin et al., ; henry et al., ; de almeida et al., ; patel et al., ; robinson et al., ; archer et al., ) . these different effects on infection are likely linked to the wide range of cellular responses induced by their downstream effectors, the products of ifn-stimulated genes (isgs) (schoggins et al., ) . although type i-ifns have long been known to induce antiviral response in the infected host (isaacs and lindemann, ) , they can also induce apoptosis, autophagy, differentiation and migration, inhibit proliferation as well as angiogenesis and mediate cellular damage, inflammation or autoimmunity (trinchieri, ) . as a result, type i-ifns have a therapeutic potential that can be used to treat tumors and viral infections (pestka, ; heim, ; wilson and brooks, ) , while being detrimental for the host in response to a subset of pathogens. type iii-ifns have been discovered in (kotenko et al., ; sheppard et al., ) and their activities have been less extensively characterized than those of type i-and type ii-ifns. however, several studies suggest that type i and type iii-ifns share common biological activities (levy et al., ; zheng et al., ) . although type iii-ifns respond to different stimuli, use different receptors and are not always expressed by the same cells as type i ifns (see below), engagement of type i-and type iii-ifn receptors leads to similar transcriptional responses. like type i-ifns, type iii-ifns have been involved in antiproliferative and antiviral responses (iversen and paludan, ; mordstein et al., ; durbin et al., ; hamming et al., ) . recently, type iii-ifns have been shown to be induced in response to bacterial pathogens, but their downstream effects are not yet characterized (pietilä et al., ; lebreton et al., ; bierne et al., ) . transcription of ifn genes is induced rapidly in response to microbial infection. type i-ifns can be produced by all cells, while type iii-ifns are secreted by specific cell types, including dendritic and epithelial cells. type i-and type iii-ifns activation is initiated by detection of mamps by prrs such as endosomal transmembrane toll-like receptors and cytosolic receptors (stetson and medzhitov, ; monroe et al., ) . upon recognition of mamps, prrs trigger diverse signaling pathways that involve adaptor proteins and cytosolic or organelle-bound protein scaffolds activating kinases converging to phosphorylation of transcription factors and their subsequent translocation into the nucleus (figure ) . irf , irf , irf , irf , irf , and irf are important for transcription of the ifn-α genes, with irf considered as the master regulator of ifn-α response (honda et al., ; tailor et al., ; levy et al., ) . regulation of the ifnβ gene is more complex. activated irf , irf , ap- , and nf-κb bind to the enhancer/promoter regions of the ifn-β gene and participate to the formation of the enhanceosome, which alters chromatin structure and allows transcription (panne et al., ; panne, ) . in contrast, irfs and nf-κb independently activate transcription of type iii-ifn genes (iversen and paludan, ) . regulation of the ifn-γ gene expression is different from that of type i and type iii-ifn genes. nk cells and nkt cells are effectors of the innate immune response and primary sources of ifn-γ. mature nk and nkt cells quickly react to infection by inducing ifn-γ secretion. upon recognition of ligands expressed on infected cells, nk cell activating-receptors trigger signaling cascades involving adaptor proteins and protein tyrosine kinases leading to activation of ras/sos, plc-γ and mapk pathways and induction of ifn-γ production (schoenborn and wilson, ) . in addition to receptors, il- , il- , il- , il- , and type i-ifns also contribute to induction of ifn-γ production by nk cells (newman and riley, ; schoenborn and wilson, ; marçais et al., ) . similarly, il- and il- induce ifn-γ production by nkt cells (godfrey and berzins, ) . in nk and nkt cells, the ifn-γ gene locus is transcriptionally permissive within accessible chromatin and allows rapid ifn-γ expression upon activation of transcription factors, such as ap- , nf-κb, stat , and t-bet (glimcher et al., ; schoenborn and wilson, ; lazarevic et al., ) . in addition, naive cd and cd t cells can differentiate into th cd effector t cells and cd cytotoxic t lymphocytes capable of ifn-γ secretion (wilson et al., ) . ifn-γ production by cd and cd t cells depends on il- , il- and ifn-γ itself and share many signaling pathways with nk cells. multiple transcription factors act at the ifn-γ promoter, e.g., ap- , atf- /c-jun, c/ebp, eomes, ets- , nfat, nf-κb, runx , stats and t-bet (schoenborn and wilson, ; samten et al., ; wilson et al., ; lazarevic et al., ) . moreover, distal regulatory elements modify the chromatin and remodel the ifn-γ gene locus to facilitate ifn-γ production (wilson et al., ). ifns are rapidly secreted upon infection and then bind to their receptors on the surface of target cells (table ) . type i-ifns bind the ubiquitous ifnar receptor, which consists of two chains, ifnar and ifnar (piehler et al., ) . type iii-ifns bind and signal through a different receptor complex, made of two chains: ifnlr (also known as il- rα) and il r . this receptor is expressed primarily by epithelial cells and hepatocytes (iversen and paludan, ) . thus, the physiological roles of type i-and type iii-ifns are distinct because of the different distribution of their receptors in tissues, type iii-ifns acting predominantly at mucosal surfaces (mordstein et al., ; durbin et al., ) . type i-and type iii-ifns use different receptors but trigger the same jak-stat signal transduction cascade involving tyk , jak , stat , and stat albeit with different kinetics (figure ) (marcello et al., ) . ultimately, stat , stat , and irf form a transcription factor complex, referred to as isgf , which translocates to the nucleus and binds to ifn-stimulated responsive elements (isre) in the promoter of isgs (schindler et al., ) . type ii-ifn, uses a heterodimeric receptor consisting of ifnγr and ifnγr chains, expressed by many cell types (bach et al., ) . ifn-γ activates jak , jak and stat , leading to transcription of genes bearing a γ-activation sequence (gas) in their promoter (figure ) (schindler et al., ) . mamps activate prrs of host cells such as epithelial cells and macrophages (figure ) . infection induces a robust type i-ifn response. in mice, macrophages have been identified as the major source of ifn-β (stockinger et al., ) . in vitro, ifn-β production by bone-marrow-derived murine macrophages has been shown to require bacterial escape from the phagosome and activation of cytosolic surveillance pathways (o'riordan et al., ) . induction of ifn-β depends on the adaptor protein sting and the cytosolic prr ddx , which are activated by bacterial secondary messengers c-di-amp and c-di-gmp and by bacterial dna (woodward et al., ; burdette et al., ; sauer et al., ; parvatiyar et al., ; archer et al., ) . sting is a direct receptor for cyclic-dinucleotides, including the cellular second messenger cyclic gmp-amp (cgamp) which is produced by the cytosolic sensor cgamp synthase (cgas) upon interaction with microbial dna (ablasser et al., ; gao et al., ; wu et al., ; sun et al., ; schoggins et al., ) . interestingly, type i-ifn production requires activation of the rig-i helicase by listeria rna in non-immune cells lacking a functional sting signaling pathway (abdullah et al., ; hagmann et al., ) . another cytosolic prr, the leucine-rich repeat-containing protein lrrfip , has also been implicated in ifn-β production by mouse primary peritoneal macrophages in response to listeria infection, possibly by sensing double stranded dna and rna (yang et al., ) . while production of ifn-β in response to listeria infection is independent from tlrs in bone-marrowderived macrophages (mccaffrey et al., ; stockinger et al., ; o'connell et al., ) , tlr- contributes significantly to ifn-β secretion by peritoneal macrophages, suggesting that specific macrophage populations have evolved different recognition strategies in response to listeria infection (aubry et al., ) . listeria infection has recently been shown to induce type iii-ifn gene expression in cells of epithelial origin, such as intestinal and trophoblast cells and hepatocytes (lebreton et al., ; bierne et al., ) . similar to type i-ifn, type iii-ifn induction is triggered by intracellular listeria . listeria infection also triggers a rapid and robust ifn-γ response. after intravenous infection of mice with l. monocytogenes, nk and t cells are the main sources of ifn-γ (thale and kiderlen, ; bou ghanem et al., ) . ifn-γ producing v δ + -γδ t cells are other murine immune cells induced at an early stage of listeria infection in mice inoculated intraperitoneally (hamada et al., ) . using oral infection of mice, the natural route of infection in permissive hosts, l. monocytogenes has been shown to induce ifn-γ production by intraepithelial lymphocytes of the small intestine (okamoto, ) . more recently, human e-cadherin (hecad) expressing mice, a mouse line permissive for listeria oral infection (lecuit et al., ) , were used to study cells involved in intestinal mucosal immunity. infection induced ifn-γ production in nk cells of the small intestine (reynders et al., ) . the production of ifn-γ by immune cells promotes bacterial clearance and is thus critical in controlling primary l. monocytogenes infections (zenewicz and shen, ) . injection of neutralizing monoclonal anti-ifn-γ antibodies in mice infected intraperitoneally with l. monocytogenes inhibits macrophage activation and increases the mortality rate (buchmeier and schreiber, ) . in addition, resistance of ifn-γ gene or ifn-γ receptor knock-out mice infected intravenously with l. monocytogenes is frontiers in cellular and infection microbiology www.frontiersin.org april | volume | article | severely impaired harty and bevan, ) . recent work using cell-type specific inactivation of stat in mice elegantly demonstrated the key role of ifn-γ and stat in macrophage activation and clearance of listeria (kernbauer et al., ) . interestingly, the role of stat was extremely different after infection of immunized mice. stat signaling in t cells and dendritic cells was critical for adaptive immunity to listeria, while ifn-γ-activated macrophages were not essential anymore once memory cells were produced. upon oral infection of hecad mice with listeria, ifn-γ contributes to the control of bacterial burden in the intestine and of bacterial dissemination to other organs. for instance, blocking ifn-γ with neutralizing antibodies increases listeria load in the small intestine, the mesenteric lymph nodes and in the spleen of mice infected orally (reynders et al., ) . in contrast to ifn-γ, type i-ifn is beneficial to l. monocytogenes. mice lacking type i-ifn receptor or irf are more resistant to listeria intraperitoneal or intravenous infection (auerbuch et al., ; carrero et al., ; o'connell et al., ; garifulin et al., ; jia et al., ) . the role of type i-ifns in increasing host susceptibility could be explained by modulation of components of the immune response involved in controlling bacterial growth such as induction of t cell apoptosis, resulting in greater il- secretion by phagocytic cells, in turn dampening the innate immune response (carrero and unanue, ) , the downregulation of ifn-γr (rayamajhi et al., ; kearney et al., ) , or neutrophil recruitment (brzoza-lewis et al., ) . as shown recently, sting-dependent activation of type i-ifn reduces the adaptive immune response to l. monocytogenes (archer et al., ) . in contrast, recent studies showed that type i-ifns can also play a beneficial role for the host during listeria infection, pointing to the infection route and the timing of type i-ifn production as determinative factors (pontiroli et al., ; kernbauer et al., ) . interestingly, different strains of l. monocytogenes have been shown to vary greatly in their capacity to induce ifn-β (reutterer et al., ; schwartz et al., ) . the lo strain hyperinduces ifn-β (reutterer et al., ) . this strain bears a nonfunctional brta (also named tetr), the transcriptional repressor of the multidrug efflux pump mdrt (schwartz et al., ; yamamoto et al., ) . in listeria, mdrt allows secretion of cdi-amp, which triggers ifn-β. thus, derepression of mdrt in the lo strain promotes ifn-β production. of note, high expression of mdrt in lo correlates with both induction of ifn-β and lower virulence. another listeria multidrug resistance transporter, mdrm, has been involved in the stimulation of ifn-β production, possibly by secreting c-di-amp (crimmins et al., ; woodward et al., ; witte et al., ) . the role of type iii-ifns during listeriosis remains to be determined. since listeria colonizes several tissues of epithelial origins, such as the liver, intestine and placenta, it is tempting to speculate that ifn-λs play a role in the interaction of listeria with epithelia. however, a prerequisite to address this question is the establishment of a new animal model, i.e., a mouse line expressing a human e-cadherin, thus permissive for listeria infection of epithelia (lecuit et al., ) and impaired in type iii-ifn responses, such as il rα knockout mice (mordstein et al., ) . one should keep in mind that the mouse model is not optimal to address the role of type iii-ifn in human listeriosis. indeed, ifn-λ is a pseudogene in mice, while human cells produce this cytokine upon infection with l. monocytogenes. in addition, the type iii-ifn receptor is expressed at very low levels in the mouse liver and the ifn-λ response of the mouse liver is very weak (mordstein et al., ) . in line with this, it has been recently shown that mouse hepatocytes, in contrast to human hepatocytes, are not responsive to ifn-λ (hermant et al., ) . the beneficial or detrimental effects of ifns on listeria infection rely on the functional properties of their downstream effectors. indeed, ifns elicit expression of hundreds of interferonstimulated genes (isgs), which encode proteins involved in a broad range of cellular functions (reviewed in macmicking, ) . however, while about , isgs have been identified so far (rusinova et al., ) , their functions in immunomodulation remain to be characterized. to date, the contribution of interferon-induced proteins on listeria infection has mostly been studied in the context of the ifn-γ pathway. the antilisterial activity of ifn-γ in phagocytic cells involves induction of oxidative and nitrosative defences, via increased expression of enzymes that control production of reactive oxygen and nitrogen species, such as nox /cybb, duox , and inos/nos (macmicking, ). these enzymes play an important role in protecting infected cells against listeria cytoinvasion (myers et al., ; lipinski et al., ) . the assembly of these enzymes requires ifn-γ-inducible guanosine triphosphatases (gtpases) of the gbp (guanylate binding protein) family (boehm et al., ) , which not only participate to oxidative pathways but also regulate autophagy (kim et al., ) . several gbps have been shown to protect cells from listeria infection by coordinating a potent oxidative and vesicular trafficking program (kim et al., ) . ifn-γ also induces the expression of many nuclear genes encoding mitochondrial respiratory chain machinery, via activation of the nuclear receptor errα (estrogen-related receptor α). errα contributes to mitochondrial ros production and efficient clearance of l. monocytogenes (sonoda et al., ) . a family of ifn-γ-induced chemokines (cxcl , cxcl , cxcl ) displays direct antimicrobial activity against l. monocytogenes (cole et al., ) . in dendritic cells, one of the ifn-γ-associated isgs is the immunoregulatory enzyme indoleamine , -dioxygenase (ido), a key enzyme of the tryptophan metabolism. ido is proposed to play a role in the containment of listeria within granulomatous structures, thus avoiding massive t cell activation (popov et al., ) . the function of type i ifn-associated isgs in listeria infection is less documented. zwaferink et al. have observed that upregulation of inos/nos by ifn-β promotes necrotic death of macrophages (zwaferink et al., ) . additionally, several interferon-inducible proteins belong to inflammasomes; thus, type i ifn may potentiate inflammasome activation and cell death by pyroptosis (malireddi and kanneganti, ). yet, the link between these effectors and the observed harmful effects of type i ifns on the host is still unclear. likewise, the role of of interest, a subset of isgs is amongst the most induced genes in the intestinal tissue of gnotobiotic humanized mice infected orally with l. monocytogenes (archambaud et al., ) . however, which type of ifns triggers this response and for which function on the intestinal mucosa remain to be explored. in addition, ifn-independent pathways may contribute to expression of these isgs. listeria has evolved several mechanisms to avoid immune detection and evade ifn responses. it has been demonstrated that deacetylation of listeria peptidoglycan by the deacetylase pgda confers resistance to host lysozyme, thus preventing release of mamps, such as dna, rna and lipopeptides, that trigger ifnβ production (boneca et al., ) . listeria pgda mutants are rapidly killed in murine macrophages, which produce lysozyme, and induce a strong secretion of ifn-β compared to wildtype listeria. the role of pgda is not limited to the control of type i-ifn production as a pgda mutant hyperinduces proinflammatory cytokines as well. modification of peptidoglycan by pgda is an extremely efficient mechanism of immune escape used by listeria, which correlates with its critical role in virulence. remarkably, listeria has evolved a sophisticated strategy to modulate, either negatively or positively, the expression of isgs in epithelial cells, by targeting a chromatin-repressive complex, bahd (bierne et al., ; lebreton et al., lebreton et al., , . indeed, listeria infection promotes, albeit via an unknown mechanism, the targeting of bahd at the promoter of a set of isgs, thereby downregulating type i-and type iii-ifn responses. on the other hand, listeria can produce a nucleomodulin, lnta, which when secreted by intracellular bacteria, enters the nucleus of infected cells, binds bahd and inhibits its function (lebreton et al., , . thus, lnta stimulates ifn responses. consistent with the presence of hdac / in the bahd -associated complex, the level of acetylation of lysine on histone h , which is a mark of active chromatin, increases at the promoters of isgs in the presence of lnta. when, in which host conditions, and how lnta targets bahd specifically at isgs remains an open question. the lnta-mediated stimulation of type iii-ifn responses might support localized pro-bacterial conditions, as was proposed for ifn-i responses. we have an extensive knowledge of the molecular and cellular mechanisms involved in listeria-host interactions. yet, our understanding of the immune response to listeria, and more specifically the role ifns and of their downstream effectors, is far from complete and often relies on studies performed in cultured cells or in mice. however, murine and human listeriosis differ in many aspects (lecuit, ; hoelzer et al., ) . for instance, e-cadherin, the major receptor for listeria in epithelial cells, is not functional for listeria uptake in the mouse. thus, the route of entry of listeria is not strictly the same in mice and humans. moreover, isgs induced in response to infection are not identical in mice and humans. additionally, murine hepatocytes do not respond to type iii-ifns (hermant et al., ) , precluding the study of these ifns during infection by human hepatotropic pathogens, such as l. monocytogenes. altogether, species-specific differences provide limits to the use of mouse models in characterizing ifn pathways engaged during listeria infection in humans, especially in key epithelial organs such as the gut, liver and placenta. it will be important to perform future studies using adapted animal models, such as humanized mice permissive to oral infection or transgenic mice with human xenografts (walters et al., ) , since the effect of type i-ifn on listeria infection depends on the route and time of infection (pontiroli et al., ; kernbauer et al., ) and type iii-ifn requires bacterial interaction with epithelia . finally, numerous isgs are induced upon listeria infection in vitro, but the relevant isgs and their cellular functions remain to be identified. validation of isgs identified in cultured cells in adequate in vivo models or deduced from analyses of patient samples, will be required to address the complex role of ifns and bacterial subversion strategies and provide new insights into listeria pathogenesis. rig-i detects infection with live listeria by sensing secreted bacterial nucleic acids cgas produces a - -linked cyclic dinucleotide second messenger that activates sting impact of lactobacilli on orally acquired listeriosis sting-dependent type i ifn production inhibits cell-mediated immunity to listeria monocytogenes both tlr and trif contribute to interferon-β production during listeria infection oata, a peptidoglycan o-acetyltransferase involved in listeria monocytogenes immune escape, is critical for virulence mice lacking the type i interferon receptor are resistant to listeria monocytogenes the ifn gamma receptor: a paradigm for cytokine receptor signaling human bahd promotes heterochromatic gene silencing activation of type iii interferon genes by pathogenic bacteria in infected epithelial cells and mouse placenta two 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signaling for immunity to listeria monocytogenes infection a family of ifn-gamma-inducible -kd gtpases protects against bacterial infection ifn-λs ifn-λs mediate antiviral protection through a distinct class ii cytokine receptor complex t-bet: a bridge between innate and adaptive immunity bacteria tune interferon responses by playing with chromatin structural basis for the inhibition of the chromatin repressor bahd by the bacterial nucleomodulin lnta a bacterial protein targets the bahd chromatin complex to stimulate type iii interferon response human listeriosis and animal models a transgenic model for listeriosis: role of internalin in crossing the intestinal barrier induction and function of type i and iii interferon in response to viral infection duox -derived reactive oxygen species are effectors of nod -mediated antibacterial responses listeriosis cellular resistance to infection ifn-inducible gtpases and immunity to intracellular pathogens interferon-inducible effector mechanisms in cellautonomous immunity role of type i interferons in inflammasome activation, cell death, and disease during microbial infection type i ifn signaling is crucial for host resistance against different species of pathogenic bacteria regulation of mouse nk cell development and function by cytokines interferons α and λ inhibit hepatitis c virus replication with distinct signal transduction and gene regulation kinetics staphylococcus aureus activates type i ifn signaling in mice and humans through the xr repeated sequences of protein a a specific gene expression program triggered by gram-positive bacteria in the cytosol induction of type i interferons by bacteria what have we learned from the il receptor knockout mouse? localized reactive oxygen and nitrogen intermediates inhibit escape of listeria monocytogenes from vacuoles in activated macrophages whatever turns you on: accessory-celldependent activation of nk cells by pathogens a mutation in the interferon-gamma-receptor gene and susceptibility to mycobacterial infection transcytosis of listeria monocytogenes across the intestinal barrier upon specific targeting of goblet cell accessible e-cadherin type i interferon production enhances susceptibility to listeria monocytogenes infection immune activation of type i ifns by listeria monocytogenes occurs independently of tlr , tlr , and receptor interacting protein but involves tnfr-associated nf kappa b kinase-binding kinase host defense and endogenous interferon-gamma in the intestines during an oral infection with listeria monocytogenes gastroenteritis due to listeria monocytogenes innate recognition of bacteria by a macrophage cytosolic surveillance pathway the enhanceosome an atomic model of the interferon-beta enhanceosome the helicase ddx recognizes the bacterial secondary messengers cyclic di-gmp and cyclic di-amp to activate a type i interferon immune response opposing roles for interferon regulatory factor- (irf- ) and type i interferon signaling 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of the murine alpha interferon gene family listeria pathogenesis and molecular virulence determinants host-specific response to hcv infection in the chimeric scidbeige/alb-upa mouse model: role of the innate antiviral immune response nod contributes to mouse host defense against helicobacter pylori via induction of type i ifn and activation of the isgf signaling pathway circulating virus, interferon and antibody after vaccination with the -d strain of yellow-fever virus decoding the complexity of type i interferon to treat persistent viral infections epigenetic control of t-helpercell differentiation innate immune pathways triggered by listeria monocytogenes and their role in the induction of cell-mediated immunity c-di-amp secreted by intracellular listeria monocytogenes activates a host type i interferon response cyclic gmp-amp is an endogenous second messenger in innate immune signaling by cytosolic dna listeria monocytogenes strain-specific impairment of the tetr regulator underlies the drastic increase in cyclic di-amp secretion and beta interferoninducing ability the cytosolic nucleic acid sensor lrrfip mediates the production of type i interferon via a β-catenin-dependent pathway innate and adaptive immune responses to listeria monocytogenes: a short overview interferon-λs: special immunomodulatory agents and potential therapeutic targets stimulation of inducible nitric oxide synthase expression by beta interferon increases necrotic death of macrophages upon listeria monocytogenes infection conflict of interest statement: the authors declare that the research was con the authors declare no conflict of interest. the authors' work has been supported in part by the institut pasteur, inserm, inra, université paris-diderot, labex ibeid, anr (grant epilis), french ligue nationale contre le cancer, fondation louis-jeantet, fondation le roch, and european research council (advanced grant award to pascale cossart). pascale cossart is an international research scholar of the howard hughes medical institute. key: cord- - qtsj xv authors: heckman, carol a.; dalbey, walden e. title: pathogenesis of lesions induced in rat lung by chronic tobacco smoke inhalation date: - - journal: j natl cancer inst doi: . /jnci/ . . sha: doc_id: cord_uid: qtsj xv lesions were induced in the lungs of specific-pathogen-free f rats by chronic tobacco smoke exposure. animals exposed to cigarettes/day were killed after , . , or years of exposure. parallel lifetime exposures induced pulmonary tumors in % of the animals. in serially killed animals, four types of lesions were found: ) perivascular or peribronchiolar accumulation of lymphoreticular cells, ) fibrotic and cellular enlargement of peribronchiolar septa, ) type ii cell hyperplasia with septal fibrosis, and ) air-space enlargement (emphysema). however, emphysema occurred only in animals exposed to a higher ( cigarettes) dose of tobacco smoke. ultrastructural studies showed all of the focal lesions to be infiltrated by cells typical of the inflammatory response. the type ii hyperplastic and peribronchiolar alveolar lesions involved larger portions of the parenchyma in fibrotic changes but differed in structure, location, and frequency. the incidence of the peribronchiolar alveolar lesions was temporally related to tumor incidence. numerous previous reports have described the effects of tobacco smoke exposure on lung structure. in studies by roe's group ( , ) , the major lesions induced in the rat lung by lifetime exposure were columnar, cuboidal, and squamous metaplasias of the alveolar epithelium. all three types of lesions were significantly more frequent in smoke-exposed than in control animals ( ) and could be induced by intratracheal instillation of smoke condensate ( ) . however, neoplasms could be induced only by intratracheal instillation of the polycyclic aromatic hydrocarbon-containing fraction of smoke condensate ( ) . no statistically significant increase in pulmonary neoplasm incidence was found in animals exposed to smoke by inhalation, presumably because the animals died prematurely from chronic respiratory disease ( ). short-term studies conducted independently by walker et al. ( ) on rats exposed to tobacco smoke for weeks showed characteristic lesions at the level of the respiratory bronchiole, including peribronchiolar and perivascular infiltration by lymphocytes, focal pneumonitis, and alveolar cuboidal or columnar metaplasia. the metaplastic changes, along with increases in macrophage size and number, were thought to be dose-dependent. the other lesions were considered early manifestations of chronic pulmonary infection ( ) , morphologic changes have also been induced in the lungs of beagle dogs by - years of tobacco smoke exposure ( ) . marked fibrosis and emphysema were found, as were type ii cell hyperplasia and squamous metaplasia of the alveolar epithelium. at the epithelial-mesenchymal interface, projections of the epithelial cells into the stroma and thickening and reduplication of the basal lamina ( ) were also seen. despite the substantial number of experiments with chronic tobacco smoke inhalation, only two laboratories have reported significant induction of pulmonary tumors in exposed rodents. one report showed a twofold higher inci-dence of adenocarcinomas in smoke-exposed mice than in control mice, the gas phase of the smoke was found to induce an even higher tumor incidence than the whole smoke ( ) . more recently, a lifetime exposure of rats to tobacco smoke produced a % incidence of respiratory tract tumors, including adenomas, adenocarcinomas, and squamous cell carcinomas ( ) . in this experiment, animals exposed in parallel with those for the lifetime study were killed at earlier intervals for assessment of morphologic lesions induced by smoke inhalation. the present report characterizes those lesions. many of the alterations appeared to be similar to those described above. however, lesion was found that had not been found in previous experiments, i.e., fibrotic thickening of alveolar septa in peribronchiolar locations. exposure conditions.-spf female f rats were exposed to tobacco smoke in the maddox/ornl smoking machine as previously described ( ) . beginning at - weeks of age, the animals were gradually exposed to increasing dosages of tobacco smoke from standard -mm, nonfiltered experimental cigarettes (national cancer institute code ). two final dose levels were used, or cigarettes/day, and the animals were killed at time intervals from to years after exposure began. the exact number of animals killed at various times in each dose group is shown in table . since mortality was high in the lo-cigarette-per-day group, all the remaining animals in this group were killed at . years. both untreated and sham-exposed groups were killed in parallel with the exposed animals. additional data were tz"ssue preparation.-before they were killed, the animals were anesthetized with mg fentobarbital/kg body weight. for tissue fixation the trachea was cannalized; the airways and lungs were removed and fixed with buffered formaldehyde under cm of pressure for - hours. the lung lobes were cut along the bronchus, except for the left lobe, which was cut in cross section just anterior to the entry of the bronchus. the tissues were embedded in paraffin. sections from the right lobes were used to determine the number of fibrotic lesions in the lungs of smoke-exposed animals. all of the sections were examined, and the cumulative number of lesions, normalized to the number of animals killed, was used as an estimate of the frequency of each type of lesion. sections from the left lobes, stained with hematoxylin and eosin, were used for morphometric analysis. for each sample, fields were selected at random and viewed at x magnification through a weibel ocular. fields comprised of more than one-third conducting airways or vasculature were excluded. the mean linear intercept was determined, and point counts ( points/lobe) were made to obtain the volume percent of alveolar versus nonalveolar air space and respiratory versus nonrespiratory tissue. alveoli were defined as enclosed circles or as open semicircles with a radius greater than half the distance across the opening. additional sections of the left lobes were stained with snook's silver stain for reticulum, aldehyde fuchsin, and lushbaugh's stain. each time rats were killed, tissues were also processed for electron microscopy by airway perfusion of a % glutaraldehyde solution containing . % tyrode's solution and . m collidine buffer (ph . ). the tissues were fixed for hours under -cm pressure and retained in collidine buffer until further processing. after exposure to % osmium tetroxide in . m cacodylate for hours, selected blocks of tissue were dehydrated in acetone and embedded in a standard epoxy mixture ( ) . sections }.tm in thickness were cut on the sorvall jb- microtome and stained with . % toluidine blue in . % sodium carbonate. selected areas of the blocks were thin sectioned on the sorvall mt- ultramicrotome, mounted on formvar-coated grids, and stained with uranyl acetate and lead citrate ( ) . thin sections were examined with the siemens or hitachi hu- b electron microscope. for each pathologic change studied, at least four examples were examined ultrastructurally. some changes in the lung parenchyma following smoke exposure were readily apparent on a gross level. lungs from animals exposed to cigarettes/day for year had dark foci about mm in diameter scattered over their surfaces. with longer exposures or with exposure to cigarettes/day, there were increasing numbers of larger white nodules about - mm in diameter. four types oflesions could be distinguished microscopically in the pulmonary parenchyma: i) perivascular or peribronchiolar accumulation of lymphoreticular cells, ) fibrotic and cellular enlargement of peribronchiolar septa, ) type ii cell hyperplasia with septal fibrosis, and ) air-space enlargement (emphysema). the first type of lesion was most common in the lungs of animals exposed to cigarettes/day for years. the last lesion type was seen only in animals exposed to cigarettes/day. perivascular lesions occurred frequently in the lung parenchyma after years of smoke exposure, averaging lesions/section of approximately . cm . the frequency after a -year exposure was approximately double that after a i-year exposure. the lesions were characteristically associated with venules and arterioles in the parenchyma. they were most common in branches of the pulmonary vein located at or below the level of the terminal bronchiole (figs. la, b) and were also found in bronchial veins, but they were never seen in the adventitia of major arteries. perivascular accumulations of cells were also found rarely in unexposed rats and consisted of large lymphocytes and macrophages (fig. ic). the lesions of smoke-exposed lung tissues contained predominantly particulate-laden macrophages (figs. la, ib). an ultrastructural analysis of several lesions associated with venules showed that lymphocytes, plasma cells, and, occasionally, fibroblasts were closely apposed to macrophages in the adventitia ( fig. ). while macrophages and lymphocytes were usually seen adjacent to the basal lamina of the epithelium, the cell mass was separated from the epithelium by a basement membrane in some lesions. in addition to the adventitial lesions, a separate lesion was sometimes found around major veins, consisting of lymphocytes within the lumen of lymphatic vessels. the lymphatic endothelium in these lesions frequently contained smoke particulates (figs. a, b). however, lesions with characteristics intermediate between those of the lymphatic and adventitial lesions were not found, so a common etiology seemed unlikely. lesions similar to the other adventitial perivascular lesions were also found near the bronchiolar-alveolar junction, most frequently between the bronchiole and the bronchial vein and extending only part of the way around the bronchiole. therefore, most of these lesions \ ere also perivascular in location. septal enlargement was characteristic in peribronchiolar locations, but it was also found in type ii hyperplasias. the enlarged septa in both lesions had similar ultrastructural features. generally, both histologic types of lesions were larger than perivascular lesions and could be discerned readily in paraffin-embedded lung tissue. neither type of lesion was found in the lungs of control animals, and the frequency of both types increased with the number of cigarettes smoked and the cumulative time of smoke exposure (table ). the type ii hyperplastic lesions always constituted more than % of the total number oflarge lesions. however, the total number of peribronchiolar lesions increased markedly in the later stages of exposure at a dose of cigarettes/ day. in animals exposed at the higher dose rate of cigarettes/day for . years, the peribronchiolar lesions constituted only % of the large lesions, even though the cumulative dose of tobacco smoke was comparable to that delivered in the -year exposure at the lower dose rate. the peribronchiolar lesions constituted % of the large lesions under the latter conditions. in paraffin-embedded sections treated with lushbaugh's stain for collagen, the amount of collagen seen in lesions showing septal enlargement appeared to increase in relation to the dose of tobacco smoke. no obvious increases in reticular fibers (snook's silver stain) or elastic fibers (aldehyde fuchsin) were observed. although the two types of lesions exhibiting septal enlargement differed in location, frequency, and the type of lining epithelium present, the septal constituents were similar when examined ultrastructurally. peri bronchiolar lesions were characterized by an attenuated epithelium septal thickening may also have occurred in loci separate from larger lesions, this was evidently rare; a few areas where enlargement was seen in isolation from neighboring lesions were found by serial sectioning to be adjacent to lesions. type ii hyperplastic lesions were observed most frequently at the pleura (figs. a- c) but were also in the lung parenchyma adjacent to the distal airways ( fig. ib) . in animals exposed to a dose of cigarettes/day for i or years, about % of the lesions were located on the pleura. when pleural lesions embedded in epoxy resins were serially sectioned, nearly all of them were found to be adjacent to sites where one or more alveolar ducts terminated on the pleura; about half were also distal to or surrounding perivascular lesions (fig. b ). fibrosis was obvious at the light microscopic level in the alveolar septa of all type ii cell lesions. the septal enlargements studied by electron microscopy contained numerous cells of the lymphoreticular system, i.e., plasma cells, lymphocytes, polymorphonuclear leukocytes, macrophages, and mast cells. fibroblasts were also seen in all of the lesions and frequently appeared hypertrophic relative to those in unaffected septa ( fig. ). there were also extensive bundles of collagen fibers within the interstitium. capillaries within the septa had swollen or hypertrophic endothelial cells that frequently occluded the lumen. in other areas the basal lamina showed extensive thickening, infolding, or reduplication ( fig. ). the largest peri bronchiolar lesions exhibited an aberrant morphology in which lengths of the basal lamina were completely surrounded by lymphoreticular cells, indicating areas of probable fusion of adjacent septa. although the cellular constituents were similar to those in smaller lesions, they included fragments of cells embedded in an amorphous extracellular material resembling fibrin (fig. ) . these were thought to be derived from capillaries that were no longer patent. in densely fibrotic and cellular areas, there were relatively few capillaries of normal appearance. both the squamous and type ii epithelial cells in these areas sometimes contained an osmiophilic cytoplasm with an elaborate endoplasmic reticulum. both epithelial cells and fibroblasts in these areas frequently contained osmiophilic granules resembling lipid droplets ( fig. ) . the cellular and extracellular constituents involved in enlargement of the interstitium in type ii hyperplastic lesions were similar to those described in the preceding section. all of the type ii lesions studied showed some degeneration of basal lamina structure; in areas where marked degeneration occurred, the type i epithelium was separated from the underlying connective tissue (fig. ). degenerative changes were frequently, though not always, found in areas where luminal macrophages were in close contact with the type i epithelium. the presence of phagocytes (i.e., macrophages and polymorphonuclear leukocytes) in the interstitium had little relationship to the position of basal lamina degeneration. the lining epithelium in hyperplastic lesions consisted of cells with an undifferentiated appearance, which could be flattened, distorted in shape, or hypertrophic. some of the cells showed long microvilli on their luminal surfaces. in addition, most lesions contained cuboidal, type iii cells, with an extensive endoplasmic reticulum rather than the lamellar bodies typical of type ii cells ( fig. ). other type iii cells had a dense cytoplasm, abundant profiles of endoplasmic reticulum, and osmiophilic inclusions ( fig. ). lymphocytes were frequently found as cellular constituents of the epithelium. binucleate cells and evidence of stratification in the epithelium were seen in about half of the lesions. a few lesions also contained large cells with an exceptionally clear cytoplasm ( fig. ). in addition to the focal lesions described above, we detected a general tendency for enlargement of the air space in animals exposed to a high dose of tobacco smoke for year or more. this trend was indicated by an increase in the apparent nonalveolar air space. although a similar trend was seen in the surviving animals of the low-dose group, it fell short of statistical significance. exposure of animals to cigarettes/day for up to . years did not significantly influence the mean linear intercept (table ) . however, exposure to cigarettes/day had a more pronounced effect on lung architecture: the mean linear intercept was creased significantly above control levels at both and . years of exposure (table ) . we obtained the present results on tobacco smoke inhalation in parallel with a lifetime study that demonstrated the induction of several types of lung tumors by tobacco smoke ( ) . the animals were maintained under spf conditions; thus the histopathologic interpretation of pulmonary lesions was not complicated by chronic lung disease, and survival was not compromised by infection. in the lifetime experiment, the observation of tobacco smoke-induced tumors clearly was dependent on the long-term survival of the experimental animals. the mean survival time (± sd) was . ±o. years for rats exposed to cigarettes/ day, . ±o. years for untreated controls, and . ± . years for sham-exposed controls. the survival curves for these groups were not significantly different. the earliest of the adenomas found among the treated animals appeared at . years, and the average age at death of the animals with these tumors was . years. for the other lung and nasal tumors, which were adenocarcinomas and a squamous cell carincoma, the average age at death was . years. only alveologenic carcinoma was observed in the control animals over their life-span. the final incidences of respiratory tract tumors were and % in the experimental and control groups, respectively. the present studies have shown that the most common type of focal lesions to develop in the lungs of tobacco smoke-exposed animals consisted of accumulated lymphocytes and macrophages in the vascular adventitia. similar accumulations were also found in the lamina propria of terminal bronchioles, where some of them were continuous with lesions surrounding the bronchial veins or branches of the pulmonary veins. while all of the focal lesions were found to include cellular infiltrates typical of the inflam- "values are means ± sd. b significantly different from untreated controls (p< . ). c animals were exposed for mo, followed by mo without treatment. matory response, the cell types found in the vascular adventitia were typically restricted to lymphocytes and macrophages. similar accumulations of lymphoreticular cells in perivascular locations have been noted in the normal course of aging in the rat ( ) and in cases of multiple sclerosis in humans ( ). although perivascular lesions were often contained within some of the more extensive lesions, it is not clear whether they played an essential role in the pathogenesis of the larger lesions. the larger lesions could have been induced by infiltration of lymphoreticular cells into the surrounding parenchyma or could have been initiated independently. since these locations were probably subjected to tobacco smoke exposure under similar local conditions, inflammatory responses could have resulted from the same, presumably chemotactic, biochemical intermediaries in both cases. however, the larger lesions showed the full spectrum of inflammatory cells, including polymorphonuclear leukocytes and mast cells. another feature common to the more extensive lesions was degradation of the basal lamina, particularly under the type i or squamous epithelial cells. we considered this alteration a possible contributing factor in fibrogenesis and/or epithelial hyperplasia. the proximity of phagocytes, particularly macrophages, to these sites suggested that enzymes released from the cells during phagocytosis may affect the structure of the lamina. the presence of lymphocytes, monocytes, and macrophages in the interstitium has also been reported in human diffuse interstitial lung diseases ( ) . lesions of comparable structure to the type ii hyperplastic and peribronchiolar fibrotic lesions have been induced in many previous studies of lung pathology, but authors have tended to classify the cuboidal and columnar metaplastic lesions together. the exceptions that particularly note "bronchiolization" of the peribronchiolar alveoli have been reports on mice and hamsters infected with infll!enza virus ( ), mice exposed to synthetic smog or calcium chromate dust ( ) , and hamsters treated with chemical carcinogens ( ) . in experiments on rats treated with various agents, particularly intratracheal instillation of tobacco smoke condensate ( ) or of casein and carbon particulates (j ), or a single exposure to benzo[a)pyrene followed by lifetime tobacco smoke inhalation ( ), bronchiolization appeared to have occurred but to have been classified with other types of metaplasia. our studies, based on the improved structural detail obtained by the examination of tissues embedded in epoxy resins, suggest that the bronchiolar lesions differ in several respects from the more common cuboidal metaplasias, which were identified here as type ii hyperplasias. since only about half of the peribronchiolar lesions showed epithelial metaplasia, our studies are also the first performed on rodents to suggest that fibrotic and cellular enlargement of the peri bronchiolar septa precedes the replacement of the squamous, type i epithelium with a columnar epithelium. however, prominent areas of peribronchiolar fibrosis without columnar metaplasia have also been noted in the lungs of human smokers and of nonsmokers exposed to lung irritants ( ). in the present studies, hyperplasia of the type ii epithelium appeared concurrently with changes in the intersti- (ium. alveolar bronchiolization was rather infrequent and appeared to occur well after inflammatory and fibrotic changes in the interstitium. in addition, the two types of larger lesions differed in other respects: their locations were typically dissimilar, the type ii lesions being mainly at the pleura. more importantly, the frequency of the lesions was related to the duration of tobacco smoke exposure. increasing the time of exposure at the lower dose of tobacco smoke from to years led to approximately a tripling in the frequency of type ii hyperplasias but to a sixfold increase in the frequency of peribronchiolar lesions. while exposure to cigarettes/day for . years induced as many type ii hyperplasias as the -year exposure to cigarettes/day, it induced far fewer peri bronchiolar lesions. thus the peribronchiolar lesions were dependent on the duration of exposure and/or on the age of the exposed animals. in summary, the rat inhalation model offers a prototype of human syndromes induced by tobacco smoke exposure. however, the exposure level required for induction of emphysematous changes also leads to high mortality. the ultrastructure of the lesions appears to implicate the inflammatory response in their pathogenesis. the least common lesion seen, fibrosis of the peribronchiolar alveoli, is temporally related to tumor incidence and can be considered a precursor lesion. . note that several of the lymphatic endothelial cells contain aggregated smoke particulates (arrows). animal was exposed to cigarettes/day for yr. bar = /lm. x , . animals were exposed to cigarettes/day for yr. bar = /lm. x figure .-small pleural type ii hyperplastic lesion. sa) type ii hyperplasia distal to terminal bronchiole. bar = /lm. x . b) pulmonary vein with lymphoreticular cell accumulations (arrows) apical to type ii hyperplastic lesion. bar = /lm. x . c) high-magnification view of the epithelium, showing type ii cells (arrows). animal was exposed to cigarettes/day for yr. toluidine blue. bar = /lm. x figure .-minimal septal enlargement at the periphery of a peribronchiolar fibrotic lesion. plasma cells (p), lymphocytes (l), and hypertrophic fibroblasts (f) are present in interstitium. collagenous thickenings (c) are also found at irregular intervals within alveolar septa. thickening (arrows) and reduplication (double arrows) of basal lamina underlying the epithelium were also found. most capillaries contained a hypertrophied endothelium (e), which frequently occluded the lumen completely. a few hypertrophic type ii cells (ii) were present. bar = /lm. x , . inset: higher magnification view of occluded capillary at top of micrograph (e) with an adjacent plasma cell (p). capillary lumen is marked by arrowheads. note extensive infolding of basal lamina (double arrows). animal was exposed to cigarettes/day for yr. bar = /lm. x , figure .-portion of a peri bronchiolar fibrotic lesion that contains fibrotic septa showing collagenous thickenings (c), a fibroblast containing osmiophilic granules (f), and capillaries in which the endothelium appears to be degenerating (d). animal was exposed to cigarettes/day for yr. bar = /lm. x , figure s.-portion of a type ii hyperplastic lesion showing cuboidal epithelial cells with exceptionally clear cytoplasm and secretory inclusions (se) and collagenous areas in the interstitium (c). most of the cells comprising the cuboidal population are type ii cells (ii). degradation of the basal lamina was extensive, leading to detachment of type i epithelial cells from the septum in some areas (arrows). animal was exposed to cigarettes/day for yr. bar = mm. x , figure .-type ii hyperplastic lesion showing cuboidal and squamous (s) epithelial cells. a columnar cell with extensive endoplasmic reticulum represented a third type of epithelial cell (iii). basal lamina underlying the epithelium is degraded in several areas (arrows). lymphocytes (l) are present in both epithelium and interstitium. interstitium also contains plasma cells (p), mast cells (m), macrophages with ingested particulate matter, and extensive areas filled with collagen fibers (c). hypertrophic fibroblasts (f), were present. nearly all capillary endothelial cells (e) were hypertrophied and occluded the capillary lumen. profiles of linearly oriented acellular structures adjoined macrophages in the air space (a). animal was exposed to cigarettes/day for . yr. bar = /lm. x , figure o.-portion of a type ii hyperplastic lesion containing cuboidal cells (iii) that showed numerous profiles of endoplasmic reticulum and densely osmiophilic granules in the cytoplasm. basal lamina also was reduplicated (arrows). animal was exposed to cigarettes/day for yr. bar = /lm. x , response of rat lung to inhaled tobacco smoke with or without prior exposure to , -benzpyrene (bpl given by intratracheal instillation it administered repeatedly by intratracheal instillation inhalation toxicity studies on cigarette smoke. vi. -week comparative experiments using modified flue-cured cigarettes: histopathology of the lung electron microscopic observations on pulmonary fibrosis and emphysema in smoking dogs alveolar cell hyperplasia in the lungs of smoking dogs effects of chronic inhalation of whole fresh cigarette smoke and of its gas phase on pulmonary tumorigenesis in snell's mice chronic inhalation of cigarette smoke by f rats the laboratory diagnosis of mycoplasma infectioll rapid and improved methods for embedding tissues in epon and araldite the use of lead citrate at high ph as an electronopaque stain in electron microscopy pathology of aging rats multiple sclerosis: presence of lymphatic capillaries alld lymphoid tissue in the brain and spinal cord analysis of airspace and interstitial mononuclear cell populations in human diffuse inlerstitiallung disease histology and ultrastructure of metaplasia of alveolar epithelium following infection of mice and hamsters with influenza virus morphogenesis of alveolar bronchiolization morphology of experimentally induced respiratory tumors in syrian golden hamster response of rat lung to , -benzyprene administered by intratracheal instillation in infusine with or without carbon black pathological changes in the peripheral airways of young cigarette smokers animal was exposed to cigarettes/day for . yr. bar = j.lm. ib) perivascular lesion (arrow) associated with a small venule near the alveolar duct (ad) key: cord- -j cgsjt authors: tang, xiaolu; wu, changcheng; li, xiang; song, yuhe; yao, xinmin; wu, xinkai; duan, yuange; zhang, hong; wang, yirong; qian, zhaohui; cui, jie; lu, jian title: on the origin and continuing evolution of sars-cov- date: - - journal: natl sci rev doi: . /nsr/nwaa sha: doc_id: cord_uid: j cgsjt the sars-cov- epidemic started in late december in wuhan, china, and has since impacted a large portion of china and raised major global concern. herein, we investigated the extent of molecular divergence between sars-cov- and other related coronaviruses. although we found only % variability in genomic nucleotides between sars-cov- and a bat sars-related coronavirus (sarsr-cov; ratg ), the difference at neutral sites was %, suggesting the divergence between the two viruses is much larger than previously estimated. our results suggest that the development of new variations in functional sites in the receptor-binding domain (rbd) of the spike seen in sars-cov- and viruses from pangolin sarsr-covs are likely caused by mutations and natural selection besides recombination. population genetic analyses of sars-cov- genomes indicated that these viruses evolved into two major types (designated l and s), that are well defined by two different snps that show nearly complete linkage across the viral strains sequenced to date. although the l type (∼ %) is more prevalent than the s type (∼ %), the s type was found to be the ancestral version. whereas the l type was more prevalent in the early stages of the outbreak in wuhan, the frequency of the l type decreased after early january . human intervention may have placed more severe selective pressure on the l type, which might be more aggressive and spread more quickly. on the other hand, the s type, which is evolutionarily older and less aggressive, might have increased in relative frequency due to relatively weaker selective pressure. these findings strongly support an urgent need for further immediate, comprehensive studies that combine genomic data, epidemiological data, and chart records of the clinical symptoms of patients with coronavirus disease (covid- ). the coronavirus disease (covid- ) epidemic started in late december in wuhan, the capital of central china's hubei province. since then, it has rapidly spread across china and in other countries, raising major global concerns. the etiological agent is a novel coronavirus, sars-cov- , named for the similarity of its symptoms to those induced by the severe acute respiratory syndrome. as of february , , , cases of sars-cov- infection have been confirmed in china, with , deaths. worryingly, there have also been more than , confirmed cases outside of china in countries and areas (https://www.who.int/emergencies/diseases/novel-coronavirus- /situation-reports/), raising significant doubts about the likelihood of successful containment. further, the genomic sequences of sars-cov- viruses isolated from a number of patients share sequence identity higher than . %, suggesting a very recent host shift into humans [ ] [ ] [ ] . coronaviruses are naturally hosted and evolutionarily shaped by bats [ , ] . indeed, it has been postulated that most of the coronaviruses in humans are derived from the bat reservoir [ , ] . unsurprisingly, several teams have recently confirmed the genetic similarity between sars-cov- and a bat betacoronavirus of the sub-genus sarbecovirus [ ] [ ] [ ] [ ] [ ] [ ] . the whole-genome sequence identity of the novel virus has . % similarity to a bat sars-related coronavirus (sarsr-cov; ratg ) collected in yunnan province, china [ , ] , but is not very similar to the genomes of sars-cov (about %) or mers-cov (about %) [ , ] . it has also been confirmed that the sars-cov- uses the same receptor, the angiotensin converting enzyme ii (ace ), as the sars-cov [ ] . although the specific route of transmission from natural reservoirs to humans remains unclear [ , ] , several studies have shown that pangolins may have provided a partial spike gene to sars-cov- ; the critical functional sites in the spike protein of sar-cov- are nearly identical to one identified in a virus isolated from a pangolin [ ] [ ] [ ] . despite these recent discoveries, several fundamental issues related to the evolutionary patterns and driving forces behind this outbreak of sars-cov- remain unexplored [ ] . herein, we investigated the extent of molecular divergence between sars-cov- and other related coronaviruses and carried out population genetic analyses of sequenced genomes of sars-cov- . this work provides new insights into the factors driving the evolution of sars-cov- and its pattern of spread through the human population. for each annotated orf in the reference genome of sars-cov- (nc_ ), we extracted the orthologous sequences in human sars-cov, four bat sars-related coronaviruses (sarsr-cov: ratg , zxc , zc , and bm - ), one pangolin sarsr-cov from guangdong (gd) [ ] , and six pangolin sarsr-cov genomes from guangxi (gx) [ ] (table s ) . we aligned the coding sequences (cdss) based on the protein alignments (see materials and methods). most orfs annotated from sars-cov- were found to be conserved in other viruses, except for orf and orf (table ). the protein sequence of sars-cov- orf shared very low similarity with sequences in sars-cov and bm - , and orf had a premature stop codon in both sars-cov and bm - (fig. s ) . a one-base deletion caused a frame-shift mutation in orf of zxc ( fig. s ). to investigate the phylogenetic relationships between these viruses at the genomic scale, we concatenated coding regions (cdss) of the nine conserved orfs (orf ab, e, m, n, s, orf a, orf , orf a, and orf b) and reconstructed the phylogenetic tree using the synonymous sites ( fig. a) . we also used codeml in the paml [ ] a ). in parallel, we also calculated the pairwise dn, ds, and ω values between sars-cov- and another virus ( table ) . the genome-wide phylogenetic tree indicated that sars-cov- was closest to ratg , followed by gd pangolin sarsr-cov, then by gx pangolin sarsr-covs, then by zc and zxc , then by human sars-cov, and finally by bm - (fig. a) . notably, we found that the nucleotide divergence at synonymous sites between sars-cov- and other viruses was much higher than previously anticipated. for example, although the overall genomic nucleotides overall differ ~ % between sars-cov- and ratg , the genomic average ds was . , which means the divergence at the neutral sites is % between these two viruses (table ) . this is because the nonsynonymous sites are usually under stronger negative selection than synonymous sites, and calculating sequence differences without separating these two classes of sites may underestimate the extent of molecular divergence by several folds. notably, the ds value varied considerably across genes in sars-cov- and the other viruses analyzed. in particular, the spike gene (s) consistently exhibited larger ds values than other genes (table ). this pattern became clear when we calculated the ds value for each branch in fig. a for the spike gene versus the concatenated sequences of the remaining genes ( fig. s ). in each branch, the ds of spike was . ± . (mean ± sd) times as large as that of the other genes. this extremely elevated ds value of spike could be caused either by a high mutation rate or by natural selection that favors synonymous substitutions. synonymous substitutions may serve as another layer of genetic regulation, guiding the efficiency of mrna translation by changing codon usage [ ] . if positive selection is the driving force for the higher synonymous substation rate seen in spike, we expect the frequency of optimal codons (fop) of spike to be different from that of other genes. however, our codon usage bias analysis (table s ) suggests the fop of spike was only slightly higher than that of the genomic average ( . versus . , see materials and methods). thus, we believe that the elevated synonymous substitution rate measured in spike is more likely caused by higher mutational rates; however, the underlying molecular mechanism remains unclear. both sars-cov and sars-cov- bind to ace through the rbd of spike protein in order to initiate membrane fusion and enter human cells [ , , [ ] [ ] [ ] [ ] [ ] . five out of the six critical amino acid (aa) residues in rbd were different between sars-cov- and sars-cov (fig. b) , and a d structural analysis indicated that the spike of sars-cov- has a higher binding affinity to ace than sars-cov [ ] . intriguingly, these same six critical aas are identical between gd pangolin-cov and sars-cov- [ ] . in contrast, although the genomes of sars-cov- and ratg are more similar overall, only one out of the six functional sites are identical between the two viruses ( fig. b) . it has been proposed that the sars-cov- rbd region of the spike protein might have resulted from recent recombination events in pangolins [ ] [ ] [ ] . although several ancient recombination events have been described in spike [ , ] , it also seems likely that the identical functional sites in sars-cov- and gd pangolin-cov may actually the result of coincidental convergent evolution [ ] . if the functional aa residues in the sars-cov- rbd region were acquired from gd pangolin-cov in a very recent recombination event, we would expect the nucleotide sequences of this region to be nearly identical between the two viruses. however, for the cds sequences that span five critical aa sites in the sars-cov- spike (ranging from codon to , covering five adjacent functional sites: f , q , s , n , and y ; fig. s originated from the gd pangolin-cov due to a very recent recombination event. alternatively, it seems more likely that a high mutation rate in spike, coupled with strong natural selection, has shaped the identical functional aa residues between these two viruses, as proposed previously [ ] . although these sites are maintained in sars-cov- and gd pangolin-cov, mutations may have changed the residues in the ratg lineage after it diverged from sars-cov- (the blue arrow in fig. a ). in summary, it seems that the shared identity of critical aa sites between sars-cov- and gd pangolin-cov might be due to random mutations coupled with natural selection, and not necessarily recombination. the genome-wide ω value between sars-cov- and other viruses ranged from . to . (table ) we downloaded publicly available sars-cov- genomes, aligned the sequences, and identified the genetic variants. for ease of visualization, we marked each virus strain based on the location and date the virus was isolated with the format of "location_date" throughout this study (see table s for details; each id did not contain information of the patient's race or ethnicity). although sars-cov- is an rna virus, for simplicity, we presented our results based on dna sequencing results throughout this study (i.e., the nucleotide t ( / ) of nonsynonymous mutations), indicating either a recent origin [ ] or population growth [ ] . in general, the derived alleles of synonymous mutations were significantly skewed towards higher frequencies than those of nonsynonymous ones (p < . , wilcoxon rank-sum test; fig. ), suggesting the nonsynonymous mutations tended to be selected against. however, . % ( out of ) synonymous mutations, and one nonsynonymous (orf (l s, , )) mutation had a derived frequency of ≥ % across the sars-cov strains. the nonsynonymous mutations that had derived alleles in at least two sars-cov- strains affected six proteins: orf ab (a t, i v, l f, i t), s (h y, v f), orf a (g v), orf a (p s), orf (v l, s l), and n (s l, s n, p s). to detect the possible recombination among sars-cov viruses, we used haploview [ ] to analyze and visualize the patterns of linkage disequilibrium (ld) between variants with minor alleles in at least two sars-cov- strains (fig. a ). since most mutations were at very low frequencies, it is not surprising that many pairs had a very low r or lod value ( fig. b -c). consistent with another recent report [ ] , we did not find evidence of recombination between the sars-cov strains. however, we found that snps at location , (orf ab: t c, synonymous) and , (orf : c t, s l) showed significant linkage, with an r value of . (fig. b, red) and a lod value of . (fig. c, red) . among the sars-cov- virus strains, of them exhibited complete linkage between the two snps: strains exhibited a "ct" haplotype (defined as "l" type because t , is in the codon of leucine) and strains exhibited a "tc" haplotype (defined as "s" type because c , is in the codon of serine) at these two sites. thus, we categorized the sars-cov- viruses into two major types, with l being the major type (~ %) and s being the minor type (~ %). although we defined the l and s types based on two tightly linked snps, strikingly, the separation between the l (blue) and s (red) types was maintained when we reconstructed the haplotype networks using all the snps in the sars-cov- genomes ( fig. a ; the number of mutations between two neighboring haplotypes was inferred parsimoniously). this analysis further supports the idea that the two linked snps at sites , and , adequately define the l and s types of sars-cov- . to determine whether l or s type is ancestral, we examined the genomic alignments of sars-cov- and other highly related viruses. strikingly, nucleotides of the s type at sites , and , were identical to the orthologous sites in the most closely related viruses ( fig. b) . remarkably, both sites were highly conserved in other viruses as well. hence, although the l type (~ %) was more prevalent than the s type (~ %) in the sars-cov- viruses we examined, the s type is actually the ancestral version of sars-cov- . to further examine the relationship among the strains in the l and s types, we reconstructed a phylogenetic tree of all the sars-cov- viruses based on their whole-genome sequences. our phylogenetic tree also clearly shows the separation of the two types (fig. ) . viruses of the l type (blue) first clustered together, and likewise, viruses of the s type (red) were also more closely related to each other. therefore, our whole-genome comparisons further confirm the separation of the l and s types. thus far, we found that, although the l type is derived from the s type, l (~ %) is more prevalent than s (~ %) among the sequenced sars-cov- genomes we examined. this pattern suggests that l has a higher transmission rate than the s type. furthermore, our mutational load analysis indicated that the l type had accumulated a significantly higher number of derived mutations than s type (p < . , wilcoxon rank-sum test; fig. s ). we propose that, although the l type newly evolved from the ancient s type, it transmits faster or replicates faster in human populations, causing it to accumulate more mutations than the s type. thus, our results suggest the l might be more aggressive than the s type due to the potentially higher transmission and/or replication rates. to test whether the two types of sars-cov- had differences in temporal and spatial distributions, we stratified the viruses based on the locations and dates they were isolated ( fig. and table s ). if the l type is more aggressive than the s type, why did the relative frequency of the l type decrease compared to the s type in other places after the initial breakout in wuhan? one possible explanation is that, since january , the chinese central and local governments have taken rapid and comprehensive prevention and control measures. these human intervention efforts might have caused severe selective pressure against the l type, which might be more aggressive and spread more quickly. the s type, on the other hand, might have experienced weaker selective pressure by human intervention, leading to an increase in its relative abundance among the sars-cov- viruses. thus, we hypothesized that the two types of sars-cov- viruses might have experienced different selective pressures due to different epidemiological features. of note, the above analyses were based on very patchy sars-cov- genomes that were collected from different locations and time points. more comprehensive genomic data is required for further testing of our hypothesis. it is currently unclear how the l type specifically evolved from the s type during the development of sars-cov- . however, we found that the sequence of viruses isolated from to further investigate the heteroplasmy of sars-cov- viruses in patients, we searched deep-sequencing libraries of sars-cov- genomes that were deposited in the sequence read archive (sra) ( table s , materials and methods). we found genomic sites that showed evidence of heteroplasmy of sars-cov- virus in five patients, but we did not find any other instances of the co-existence of l and s types in any patient (table ) . these findings evince the developing complexity of the evolution of sars-cov- infections. further studies investigating how the different alleles of sars-cov- viruses compete with each other will be of significant value. in this study, we investigated the patterns of molecular divergence between sars-cov- and other related coronaviruses. although the genomic analyses suggested that sars-cov- was closest to ratg , their difference at neutral sites was much higher than previously realized. our results provide novel insights into tracing the intermediate natural host of sars-cov- . with population genetic analyses of genomes of sars-cov- , we found that sars-cov- viruses evolved into two major types (l and s types), and the two types were well defined by just two snps that show nearly complete linkage across sars-cov- strains. although the l type (~ %) was more prevalent than the s type (~ %) in the sars-cov- viruses we examined, our evolutionary analyses suggested the s type was most likely the more ancient version of sars-cov- . our results also support the idea that the l type is more aggressive than the s type. since nonsynonymous sites are usually under stronger negative selection than synonymous sites, calculating sequence differences without separating these two classes of sites could lead to a potentially significant underestimate of the degree of molecular divergence. for example, although the overall nucleotides only differed by ~ % between sars-cov- and ratg , the genomic average ds value, which is usually a neutral proxy, was . between these two viruses ( table ) . of note, the genome-wide ds value is . between humans and chimpanzees [ ] , and . between humans and rhesus macaques [ ] . thus, the neutral molecular divergence between sars-cov- and ratg is times larger than that between humans and chimpanzees, and twice as large as that between humans and macaques. the genomic average ds value between sars-cov- and gd pangolin-cov is . , which is comparable to that between humans and mice ( . ) [ ] , and the ds value between our analyses of molecular evolution and population genetics suggested that some amino acid changes might be favored by natural selection during the evolution of sars-cov- and other related viruses. however, negative selection appears to be the predominant force acting on these viruses. interestingly, the virus isolated from one patient in shenzhen on january , (sz_ / / .a, gisaid id: epi_isl_ ) had c at both positions , and , in the genome, belonging to neither l nor s type ( fig. a and ) . notably, this strain had one stop-gain mutation in orf ab and had accumulated silent and nonsynonymous mutations after diverging from the ancestor haplotype (fig. a ). thus, it is possible that functional constraints on the genomic sequence were weakened after the disruption of orf ab in this strain. notably, on viruses isolated from a patient living in south korean (skorea_ / .a, gisaid: epi_isl_ ), acquired six nonsynonymous mutations that were different from the most recent common ancestor of sars-cov- : orf ab (m i and t m), s (s w), orf a (w l and g v), and e (l h). if these changes are not due to sequencing errors, it would be interesting to test whether and how these mutations affect the transmission and pathogenesis of sars-cov- . in this work, we propose that sars-cov- can be divided into two major types (l and s types): the s type is ancestral, and the l type evolved from s type. intriguingly, the s and l types can be clearly defined by just two tightly linked snps at positions , (orf ab: t c, synonymous) and , (orf : c t, s l). however, it is currently unclear whether l type evolved from the s type in humans or in the intermediate hosts. it is also unclear whether the l type is more virulent than the s type. orf ab, which encodes replicase/transcriptase, is required for viral genome replication and might also be important for viral pathogenesis [ ] . although the t c mutation in orf ab does not change the protein sequence (it changes the codon agt (ser) to agc (ser)), we hypothesized this mutation might affect orf ab translation since agt is preferred while agc is unpreferred (table s ). orf promotes the expression of atf , the er unfolded protein response factor, in human cells [ ] . thus, it will be interesting to investigate the function of the s l aa change in orf , as well as the combinatory effect of these two mutations in sars-cov- pathogenesis. in summary, our analyses of sequenced sars-cov- genomes suggest that the l type is more aggressive than the s type and that human interference may have shifted the relative abundance of l and s type soon after the sars-cov- outbreak. as previously noted [ ] , the data examined in this study are still very limited, and follow-up analyses of a larger set of data are needed to have a better understanding of the evolution and epidemiology of sars-cov- . there is a strong need for further immediate, comprehensive studies that combine genomic data, epidemiological data, and chart records of the clinical symptoms of patients with sars-cov- . the set of complete genome sequences were downloaded from gisaid (global initiative on sharing all influenza data; https://www.gisaid.org/) with acknowledgment, genbank (https://www.ncbi.nlm.nih.gov/genbank), and nmdc (http://nmdc.cn/#/ncov). sequences and annotations of the reference genome of sars-cov- (nc_ ) and other related viruses were downloaded from genbank or gisaid (table s ). the genomic sequences of sars-cov- were aligned using muscle v . . [ ] . the annotated cdss of other viruses were downloaded from genbank. to avoid missing annotations in other viruses, we also annotated the orfs using cdss annotated in sars-cov- using exonerate (--model protein genome:bestfit --score -g y) [ ] . the protein sequences of sars-cov- and other related viruses were aligned with muscle v . . [ ] , and the codon alignments were made based on the protein alignment with revtrans [ ] . the codon alignments of the conserved orfs were further concatenated for down-stream evolutionary analysis. the phylogenetic tree was constructed by the neighbor-joining method in mega-x [ ] using the parameters of kimura -parameter model, and only the third positions of codons were considered. yn from paml v . a [ ] was used to calculate the pairwise divergence between sars-cov- and other viruses for each individual gene or for the concatenated sequences. the free-ratio model in codeml in the paml [ ] package was used to calculate the dn, ds, and ω values for each branch. positive selection was detected using easycodeml [ ] , a recently published wrapper of codeml [ ] . the m and m models were compared. in the m model, ω follows a beta distribution such that ⩽ω⩽ , and in the m model, a proportion p of sites have ω drawn from the beta distribution, and the remaining sites with proportion p are positively selected and have ω > . the lrts between m and m models were conducted by comparing twice the difference in log-likelihood values ( ln Δl) against a χ -distribution (df= ). the positively selected sites were identified with the bayes empirical bayes (beb) score larger than . . dnasp v . . [ ] was used to generate multi-sequence aligned haplotype data, and popart v . [ ] was used to draw haplotype networks based on the haplotypes generated by dnasp. raxml v . . [ ] was used to build the maximum likelihood phylogenetic tree of aligned sars-cov- genomes with theparameters "-p -m gtrcat". we downloaded sars-cov- metagenomic sequencing libraries (table s ) , and mapped the ngs reads to the reference genome of sars-cov- (nc_ ) using bwa ( . . -r ) [ ] with the default parameters. snp calling was done using bcftools mpileup (bcftools . ) [ ] . we calculated the rscu (relative synonymous codon usage) value of each codon in the sars-cov- reference genome (nc_ ). the rscu value for each codon was the observed frequency of this codon divided by its expected frequency under equal usage among the amino acid [ ] . the codons with rscu > were defined as preferred codons, and those with rscu < were defined as unpreferred codons. the fop (frequency of optimal codons) value of each gene was calculated as the number of preferred codons divided by the total number of preferred and unpreferred codons. the authors declare that they have no conflicts of interest. for each gene, the dn and ds values between sars-cov- and another virus are given, and the dn/ds (ω) ratio is given in the parenthesis. genomic characterisation and epidemiology of novel coronavirus: implications for virus origins and receptor binding a pneumonia outbreak associated with a new coronavirus of probable bat origin identification of a novel coronavirus causing severe pneumonia in human: a descriptive study origin and evolution of pathogenic coronaviruses bat origin of a new human coronavirus: there and back again. science china life sciences bats are natural reservoirs of sars-like coronaviruses detection of group coronaviruses in bats in north america genome composition and divergence of the novel coronavirus ( -ncov) originating in china evolution of the novel coronavirus from the ongoing wuhan outbreak and modeling of its spike protein for risk of human transmission. sci china life sci the -new coronavirus epidemic: evidence for virus evolution discovery of a novel coronavirus associated with the recent pneumonia outbreak in humans and its potential bat origin genomic characterization of the novel human-pathogenic coronavirus isolated from a patient with atypical pneumonia after visiting wuhan evolutionary perspectives on novel coronaviruses identified in pneumonia cases in china full-genome evolutionary analysis of the novel corona virus ( -ncov) rejects the hypothesis of emergence as a result of a recent recombination event return of the coronavirus: -ncov evidence of recombination in coronaviruses implicating pangolin origins of ncov- isolation and characterization of -ncov-like coronavirus from malayan pangolins identification of -ncov related coronaviruses in malayan pangolins in southern china moral imperative for the immediate release of -ncov sequence data. national science review paml : phylogenetic analysis by maximum likelihood codon optimality, bias and usage in translation and mrna decay receptor recognition by novel coronavirus from wuhan: an analysis based on decade-long structural studies of sars cryo-em structure of the -ncov spike in the prefusion conformation characterization of spike glycoprotein of -ncov on virus entry and its immune cross-reactivity with spike glycoprotein of sars-cov identification of two critical amino acid residues of the severe acute respiratory syndrome coronavirus spike protein for its variation in zoonotic tropism transition via a double substitution strategy difference in receptor usage between severe acute respiratory syndrome (sars) coronavirus and sars-like coronavirus of bat origin a new coronavirus associated with human respiratory disease in china homologous recombination within the spike glycoprotein of the newly identified coronavirus may boost cross-species transmission from snake to human moderate mutation rate in the sars coronavirus genome and its implications origin time and epidemic dynamics of the novel coronavirus decoding evolution and transmissions of novel pneumonia coronavirus using the whole genomic data haploview: analysis and visualization of ld and haplotype maps the chimpanzee s, analysis c. initial sequence of the chimpanzee genome and comparison with the human genome evolutionary and biomedical insights from the rhesus macaque genome initial sequencing and comparative analysis of the mouse genome sars coronavirus replicase proteins in pathogenesis discovery of a rich gene pool of bat sars-related coronaviruses provides new insights into the origin of sars coronavirus muscle: multiple sequence alignment with high accuracy and high throughput automated generation of heuristics for biological sequence comparison revtrans: multiple alignment of coding dna from aligned amino acid sequences molecular evolutionary genetics analysis across computing platforms easycodeml: a visual tool for analysis of selection using codeml dnasp : dna sequence polymorphism analysis of large data sets popart: full-feature software for haplotype network construction raxml version : a tool for phylogenetic analysis and post-analysis of large phylogenies fast and accurate short read alignment with burrows-wheeler transform the sequence alignment/map format and samtools codon usage in regulatory genes in escherichia coli does not reflect selection for 'rare' codons the authors thank the researchers who generated and shared the sequencing data from note the derived alleles of synonymous mutations are skewed towards higher frequencies than those of nonsynonymous mutations. a. ld plot of any two snp pairs among the sites that have minor alleles in at least two strains. the number near slashes at the top of the image shows the coordinate of sites in the genome. color in the square is given by standard (d'/lod), and the number in square is r value. b. the r of each pair of snps (y-axis) against the genomic distance between that pair (x-axis). c. the lod of each pair of snps (y-axis) against the genomic distance between that pair (x-axis). note that in both b and c, the red point represents the ld between snps at , and , . a. the haplotype networks of sars-cov- viruses. blue represents the l type, and red is the s type. the orange arrow indicates that the l type evolved from the s type. note that in this study, we marked each sample with a unique id that starting with the geological location, followed by the date the virus was isolated (see table s for details). each id did not contain information of the patient's race or ethnicity. b. evolution of the l and s types of sars-cov- viruses. genome sequence alignments with the seven most closely related viruses indicated that the s type was most likely the ancient version of sars-cov- . ".", the nucleotide sequence is identical; "-", gap. in our recent publication (https://doi.org/ . /nsr/nwaa ), we showed that among circulating sars-cov- (with genomes analyzed) two different viral genomes co-exist. we identified them as lineages l and s. the concerned amino acid we used to define the l and s lineages is located in orf (open reading frame ), which plays a yet undefined role in the viral life cycle. based on the finding that "l" lineage has a higher frequency than lineage s, we described the l lineage as aggressive. we now recognize that within the context of our study the term "aggressive" is misleading and should be replaced by a more precise term "a higher frequency". in short, while we have shown that the two lineages naturally co-exist, we provided no evidence supporting any epidemiological conclusion regarding the virulence or pathogenicity of sars-cov- . by saying so, corrections will be made in the print version of this paper to avoid being misleading. key: cord- - n vko authors: chardonnet, kostia; saurin, alexis; valiron, benoît title: toward a curry-howard equivalence for linear, reversible computation: work-in-progress date: - - journal: reversible computation doi: . / - - - - _ sha: doc_id: cord_uid: n vko in this paper, we present a linear and reversible language with inductive and coinductive types, together with a curry-howard correspondence with the logic [image: see text] : linear logic extended with least and greatest fixed points allowing inductive and coinductive statements. linear, reversible computation makes an important sub-class of quantum computation without measurement. in the latter, the notion of purely quantum recursive type is not yet well understood. moreover, models for reasoning about quantum algorithms only provide complex types for classical datatypes: there are usually no types for purely quantum objects beside tensors of quantum bits. this work is a first step towards understanding purely quantum recursive types. computation and logic are two faces of the same coin. for instance, consider a fig. features a graphical presentation of the corresponding proof. horizontal lines stand for deduction steps-they separate conclusions (below) and hypotheses (above). these deduction steps can be stacked vertically up to axioms in order to describe complete proofs. in fig. the proofs of a and a → b are symbolized with vertical ellipses. the ellipsis annotated with s indicates that s is a complete proof of a → b while t stands for a complete proof of a. this connection is known as the curry-howard correspondence [ , ] . in this general framework, types correspond to formulas and programs to proofs, while program evaluation is mirrored with proof simplification (the so-called cutelimination). the curry-howard correspondence formalizes the fact that the proof s of a → b can be regarded as a function-parametrized by an argument of type a-that produces a proof of b whenever it is fed with a proof of a. therefore, the computational interpretation of modus-ponens corresponds to the application of an argument (i.e. t) of type a to a function (i.e. s) of type a → b. when computing the corresponding program, one substitutes the parameter of the function with t and get a result of type b. on the logical side, this corresponds to substituting every axiom introducing a in the proof s with the full proof t of a. this yields a direct proof of b without any invocation of the "lemma" a → b. paving the way toward the verification of critical softwares, the curry-howard correspondence provides a versatile framework. it has been used to mirror first and second-order logics with dependent-type systems [ , ] , separation logics with memory-aware type systems [ , ] , resource-sensitive logics with differential privacy [ ] , logics with monads with reasoning on side-effects [ , ] , etc. this paper is concerned with the case of reversible computation, a sub-class of pure quantum computation. in general quantum computation, one has access to a co-processor holding a "quantum" memory. this memory consists of "quantum" bits having a peculiar property: their state cannot be duplicated, and the operations one can perform on them are unitary, reversible operations. the co-processor comes with an interface to which one can send instructions to allocate, update or read quantum registers. quantum memories can be used to solve classical problems faster than with purely conventional means. quantum programming languages are nowadays pervasive [ ] and several formal approaches based on logical systems have been proposed to relate to this model of computation [ , , ] . however, all of these languages rely on a purely classical controlflow: quantum computation is reduced to describing a list of instructions-a quantum circuit-to be sent to the co-processor. in particular, in this model operations performed on the quantum memory only act on quantum bits and tensors thereof, while the classical computer enjoys the manipulation of any kind of data with the help of rich type systems. this extended abstract aims at proposing a type system featuring inductive and coinductive types for a purely reversible language, first step towards a rich quantum type system. we base our study on the approach presented in [ ] . in this model, reversible computation is restricted to two main types: the tensor, written a ⊗ b and the co-product, written a ⊕ b. the former corresponds to the type of all pairs of elements of type a and elements of type b, while the latter represents the disjoint union of all elements of type a and elements of type b. for instance, a bit can be typed with ⊕ , where is a type with only one element. the language in [ ] offers the possibility to code isos-reversible maps-with pattern matching. an iso is for instance the swap operation, typed with a ⊗ b ↔ b ⊗ a. the language also permits higher-order operations on isos, so that an iso can be parametrized by another iso, and is extended with lists ). however, if [ ] hints at an extension toward pure quantum computation, the type system is not formally connected to any logical system. the main contribution of this work is a curry-howard correspondence for a purely reversible typed language in the style of [ ] . we capitalize on the logic [ , ] : an extension of the additive and multiplicative fragment of linear logic with least and greatest fixed points allowing inductive and coinductive statements. this logic contains both a tensor and a co-product, and its strict linearity makes it a good fit for a reversible type system. the logic [ , ] is an extension of the additive and multiplicative fragment of linear logic [ ] . the syntax of linear logic is extended with the formulas μx.a and its dual νx.a (where x is a type variable occuring in a), which can be understood at the least and greatest fixed points of the operator x → a. these permit inductive and coinductive statements. we are only interested in a fragment of which contains the tensor, the plus, the unit and the μ and ν connectives. note that our system only deals with closed formulas. our syntax of formulas is a, b :: the derivation rules are shown in fig. . they defined a binary relation Δ Γ on set of formulas defined inductively. for each rule the assumptions are above the line while the conclusion is under. in the rules, the comma stands for the disjoint union: observe that each formula has to be used exactly once and cannot be duplicated or erased. in one can for instance define the type of natural numbers as μx. ⊕ x, of lists of type a as μx. ⊕ (a ⊗ x) and of streams of type a as νx.a ⊗ x. we consider proofs to be potentially non-well-founded derivation trees: they are not necessarily finite as we can for instance consider the formula μx.x and apply the rule μ r an infinite number of times. among non well-founded proof-objects we distinguish the regular derivation trees that we call circular pre-proofs. these trees can then be represented in a compact manner, see fig. . one problem with such a proof-system is to determine whether or not infinite derivations are indeed proofs. indeed, if every infinite derivation is accepted as a proof, it would be possible to prove any formula f, as shown in fig. . to answer this problem, comes with a validity criterion for derivations. it roughly says that a derivation is valid if, in every infinite branch of the derivation, there exists an infinite number of rules μ l or an infinite number of rules ν r . the intuition is that since μx.a formulas represent least fixed points, their objects are finite. an infinite number of rule μ r would mean producing an infinite object, which is not possible. on the other hand, we can explore an arbitrarily large object as input with the rule μ l . for the other case, since νx.a formulas represent greatest fixed points, their object are infinite. we therefore want to ensure that we can produce infinite objects: hence the infinite number of rules ν r . this criterion can be understood in a more operational way as a requirement for productivity. our language is based on the one presented in [ ] . we build on the reversible part of the paper by extending the language to support both a more general rewriting system and inductive and coinductive types. the language is defined by layers. terms and types are presented in table , while typing derivations, based on ,can be found in tables and . the language consists of the following pieces. basic type. they are first-order and typed with base types. the constructors inj l and inj r represent the choice between either the left or right-hand side of a type of the form a ⊕ b; the constructor , builds pairs of elements (with the corresponding type constructor ⊗); fold and pack respectively represent inductive and coinductive structure for the types μx.a and νx.a. a value can serve both as a result and as a pattern in the clause of an iso. generalized patterns are used as special patterns: v g : a can match any value of type a. terms are expressions at "surface-level": applying an iso always gives a term, whereas it is an expression only when the argument is a generalized pattern. first-order isos. an iso of type α acts on terms of base types. an iso is a function of type a ↔ b, defined as a set of clauses of the form the tokens e i and e i in the clauses are expressions. compared to the original language in [ ] , we allow general expressions both on the left and on the right of a clause. in order to apply an iso to a term, the iso must be of type a ↔ b and the term of type a. in the typing rules of isos, the od predicate (taken from [ ] and not described in this paper) syntactically enforces the exhaustivity and non-overlapping conditions that the left-hand-side and right-hand-side of clauses should satisfy. exhaustivity for an iso {e ↔ e | . . . | e n ↔ e n } of type a ↔ b means that the expressions on the left (resp. on the right) of the clauses describe all possible values for the type a (resp. the type b). non-overlapping means that two expressions cannot match the same value. for instance, the left and right injections inj l e and inj r e are non-overlapping while a pattern v g is always exhaustive. higher-order isos. an iso of type t manipulate other isos as basic blocks. since isos represent closed computations, iso-variable are non-linear and can be duplicated at will while term-variable are linear. the constructions λf.ω and ω ω represent respectively the abstraction of a function and the application of an iso to another. the construction μg.ω represents the creation of a recursive function, rewritten as ω[g := μg.ω] by the operational semantics. the typing rule for μg.ω has a productivity criterion. indeed, since isos can be non-terminating (because of coinduction), productivity is important to ensure that we work with total functions. these checks are crucial to make sure that our isos are indeed bijections in the mathematical sense. the construction inv ω corresponds to the inversion of the iso ω. if ω is of type a ↔ b then inv ω is of type b ↔ a. finally, our language is equipped with a rewrite system (→) on terms. the evaluation of an iso applied to an argument works with pattern-matching. the non-overlapping and exhaustivity conditions guarantee subject-reduction (see proposition . ). we can define the iso of type : remark . . in our two examples, the left and right-hand side of the ↔ on each function respect both the criteria of exhaustivity-every-value of each type is being covered by at least one expression-and non-overlapping-no two expressions cover the same value. both isos are therefore bijections. t : a and t → t then we have t : a. moreover, it enjoys confluence: let → * be the reflexive, transitive closure of →. if t → * t and t → * t then there exists t such that t → * t and t → * t . we conjecture that well-typed isos are indeed isomorphisms: an iso ω : a ↔ b corresponds to both a computation sending a value of type a to a result of type b and a computation sending a value of type b to a result of type a. we can mechanically translate such an iso to a pair of derivations π, π ⊥ in , where π is a proof of a b and π ⊥ is a proof of b a. this mechanical translation constructs circular pre-proofs, as discussed in sect. . we however still need to show that the obtained derivations respect the validity criterion for circular proof. once proven, we would obtain a static correspondence between programs and proofs. we would however still need to show that this entails a dynamic correspondence between the evaluation procedure of our language and the cutelimination procedure of . for that, we would need to make sure that the proofs we obtain are indeed isomorphisms, meaning that if we cut the aforementioned proofs π and π ⊥ , performing the cut-elimination procedure would give either the identity on a or the identity on b. isomorphism of proofs. provided that the above holds, we moreover have simulation of evaluation. provided that t is a value and v is a normal form, if ω t → * v, if π is the proof corresponding to ω t, and if π is the proof corresponding to v, then π → * π with the cut-elimination procedure. we define the two mutually recursive proofs π and π by π = Π(ψ f , π ) and π = Π(ψ f ⊥ , π ) where ψ f and ψ f ⊥ correspond to the isos f and inv f . the proof associated with the iso in eq. ( ) is π . the proof Π(φ , φ ) is shown in fig. . we presented a higher-order, linear, reversible language with inductive and coinductive types together with an interpretation of programs into derivations in the logic . this work is still in progress: a number of proofs still need to be completed. after completing the proofs of our current conjectures, we want to extend our language to linear combinations of terms in order to study purely quantum recursive types and generalized quantum loops: in [ ] , lists are the only recursive type which is captured and recursion is terminating. the logic would help providing a finer understanding of termination and non-termination. infinitary proof theory: the multiplicative additive case least and greatest fixed points in linear logic interactive theorem proving and program development -coq'art functionality in combinatory logic open source software in quantum computing linear dependent types for differential privacy linear logic the formulae-as-types notion of construction rustbelt: securing the foundations of the rust programming language formal verification of a realistic compiler the next relational program logics. pacmpl (popl) qwire: a core language for quantum circuits separation logic: a logic for shared mutable data structures a categorical model for a quantum circuit description language from symmetric pattern-matching to quantum control a lambda calculus for quantum computation with classical control dependent types and multi-monadic effects in f key: cord- -z ifu d authors: lagaillardie, nicolas; neykova, rumyana; yoshida, nobuko title: implementing multiparty session types in rust date: - - journal: coordination models and languages doi: . / - - - - _ sha: doc_id: cord_uid: z ifu d multiparty session types (mpst) is a typing discipline for distributed protocols, which ensures communication safety and deadlock-freedom for more than two participants. this paper reports on our research project, implementing multiparty session types in rust. current rust implementations of session types are limited to binary (two-party communications). we extend an existing library for binary session types to mpst. we have implemented a simplified amazon prime video streaming protocol using our library for both shared and distributed communication transports. in the last decade, the software industry has seen a shift towards programming languages that promote the coordination of concurrent and/or distributed software components through the exchange of messages over communication channels. languages with native message-passing primitives (e.g., go, elixir and rust) are becoming increasingly popular. in particular, rust has been named the most loved programming language in the annual stack overflow survey for four consecutive years ( - ) . the advantage of message-passing concurrency is well-understood: it allows cheap horizontal scalability at a time when technology providers have to adapt and scale their tools and applications to various devices and platforms. messagepassing based software, however, is as vulnerable to errors as other concurrent programming techniques [ ] . much academic research has been done to develop rigorous theoretical frameworks for verification of message-passing programs. one such framework is multiparty session types (mpst) [ ] -a type-based discipline that ensures that concurrent and distributed systems are safe by design. it guarantees that message-passing processes following a predefined communication protocol, are free from communication errors and deadlocks. rust is a particularly appealing language for the practical embedding of session types. its affine type system allows for static typing of linear resourcesan essential requirement for the safety of session type systems. rust combines efficiency with message-passing abstractions, thread and memory safety [ ] , and has been used for the implementation of large-scale concurrent applications such as the mozilla browser, firefox, and the facebook blockchain platform, libra. despite the interest in the rust community for verification techniques handling multiple communicating processes , the existing rust implementations [ , ] are limited to binary (two-party) session types. in this short paper, we present our design and implementation for multiparty session types in rust. our design follows a state-of-the-art encoding of multiparty into binary session types [ ] . we generate local types in rust, utilising the scribble toolchain [ , ] . our library for mpst programming in rust, mpst-rust, is implemented as a thin wrapper over an existing binary session types library [ ] . differently from other mpst implementations that check the linear usage of channels at runtime (e.g. [ , ] ), we rely on the rust affine type system to type-check mpst programs. in addition, since we generate the local types from a readable global specification, errors caused by an affine (and not linear) usage of channels, a well-known limitation of the previous libraries [ , ] , are easily avoided. this paper is organised as follows: sect. gives an overview of our framework with a usecase; sect. shows our implementation and discusses the advantages of our approach; and sect. concludes with related and future work. our library is available from https://github.com/nicolaslagaillardie/mpst rust github. framework overview: mpst in rust. our design resembles the top-down methodology of multiparty session types, as illustrated in fig. . it follows three main steps [ , ] . first, a global type, also called a global protocol, is defined as a shared contract between communicating endpoint processes. a global protocol is then projected to each endpoint process, resulting in a local type. a local type involves only the interactions specific to a given endpoint. finally, each endpoint process is type-checked against its projected local type. the specific parts of our framework that distinguish it from other stateof-the-art mpst works are highlighted in red, which corresponds to our new library for mpst programming in rust, mpst-rust. it is realised as a thin wrapper on top of an existing rust library for validation of binary ( -party-only) session types. developers use the mpst primitives provided by mpst-rust to implement endpoint programs. also, our framework allows the types for each communication primitive to be either ( ) generated from the scribble toolchain; or ( ) written by the developers. the scribble toolchain [ ] provides facilities for writing, verifying and projecting global protocols. our framework guarantees that processes implemented using mpst-rust primitives with scribble-generated types are free from deadlocks, reception errors, and protocol deviations. next, we explain, via an example, how the framework of mpst can be applied to rust. example: amazon prime video streaming. the amazon prime video streaming service is a usecase which can take full advantage of multiparty session types. each streaming application connects to servers, and possibly other devices, to access services, and follows some specific protocol. to present our design, we use a simplified version of the protocol, illustrated in the diagram in fig. (right). the diagram should be read from top to bottom. the protocol involves three services -an authenticator service, a server and a client. at first, client connects to authenticator by providing an identifying id. if the id is accepted, the session continues with a choice on client to either request a video or end the session. the first branch is, a priori, the main service provided by amazon prime video. client cannot directly request videos from server, and has to go through authenticator instead. on the diagram, the choice is denoted as the frame alt and the choices are separated with the horizontal dotted line. the protocol is recursive, and client can request new videos as many times as needed. the arrow going back on client side in fig. represents this recursive behaviour. to end the session, client first sends close message to authenticator, which then subsequently sends a close message to server. implementing the authenticator role using mpst-rust. due to space limitations, we only show the implementation of the authenticator role (hereafter role a), the implementations of the other roles (role b for the server and role c for the client) are similar. the rust code for role a using the mpst-rust library is given in fig. (left). it closely follows the local protocol in fig. (right), that is projected from the global protocol by the scribble toolchain. first, line declares a function authenticator that is parametric in a multiparty channel s of type videop_a. the type videop_a specifies which operations are allowed on s. this type can either be written by the developer, or generated by scribble (cf. listing ). on line , a receives an identifying id from c. the function recv_mpst_a_to_c, provided by mpst-rust library returns the received value (the id) and the new multiparty channel, to be used in subsequent communications. line rebinds ok the multiparty channel s with the new channel that is returned. then, on line , we send back the answer to c, by utilising another mpst-rust communication primitive, send_mpst_a_to_c. the variable s is rebound again to the newly returned multiparty channel. note that although the name of the function, send_mpst_a_to_c, suggests a binary communication, the function operates on a multiparty channel s. our implementation follows the encoding, presented in [ ] , which encodes a multiparty channel as an indexed tuple of binary channels. internally, send_mpst_a_to_c extracts from s the binary channel established between a and c and uses it for sending. lines - proceeds by implementing the recursive part of the protocol. the implementation of authenticator_recurs realises an internal choice -a can either receive a videorequest or a close. this behaviour is realised by the mpst-rust macro offer_mpst_a_to_c! (line ), which is applied to a multiparty channel s of a sum type between choicea::video and choicea::end. the behaviour of each branch in the protocol is implemented as an anonymous function. for example, code in lines - supplies an anonymous function that implements the behaviour when c sends a videorequest, while lines - handle the close request. finally, close_mpst(s) closes all binary channels stored inside s. the types of the multiparty channel, as well as the generic types in the declaration of the mpst-rust communication functions, enable compile-time detection of protocol violations, such as swapping line and line , using another communication primitive or using the wrong payload type. fig. (left) , are given in listing . these types can be either written by the developer or generated from a global protocol, written in scribble. reception error safety is ensured since the underlying mpst-rust library checks statically that all pairs of binary types are dual to each other. deadlock-freedom is ensured only if types are generated from scribble since this guarantees that types are projected from a well-formed global protocol. next, we explain a type declaration for the authenticator role. lines - specify the three sessionmpst types which correspond to the types of the session channels used in fig. (left) -types videop_a (line ), video_prec_a (line ), and the types used inside the offer construct -choicea::video (line ), and choicea::end (line ). in the encoding of [ ] , which underpins mpst-rust, a multiparty channel is represented as an indexed tuple of binary channels. this is reflected in the implementation of sessionmpst, which is parameterised on the required binary session types. for example, the videop_a<n> takes as a parameter the binary types between a and c, and between a and b. at the beginning of the protocol (lines - in fig. (left) ) b and a do not interact, hence the binary type for b is end. the type inita<n> (line in listing ) specifies the behaviour between a and c, notably that a first receives a message, then it sends a message, and later it continues as the type recvchoice<n>. the binary session types between a and b, and between a and c are given in lines - and lines - respectively; we use the primitives declared in the existing binary session types library [ ] . the generic parameter n refers to a trait such as i . the third parameter for videop_a<n> (line ) is a queue-like data structure, queueainit (line ), that codifies the order of usage of each binary channel inside a multiparty session channel. this is needed to preserve the causality, imposed by the global protocol. the queues for the other sessionmpst types are given in lines - . for instance, the queue for the choicea:video branch of the protocol is queueavideo. note that, according to the protocol, a first has to receive a videorequest message from c, and then it has to forward that message to b hence, swapping of lines and from fig. is a protocol violation error. we can detect such violations since the queue for the type choicea::video, queueavideo (line ), is specified as roleatoc<roleatob ...>, which codifies that first the channel for c and then the channel for b should be used. note that none of the defined queues is recursive. recursion is implicitly specified on binary types, while each queue is related to a sessionmpst type. distributed execution environment. the default transport of mpst-rust is the built-in rust communication channels (crossbeam channel). also, to test our example in a more realistic distributed environment, we have also connected each process through mqtt (mq telemetry transport) [ ] . mqtt is a messaging middleware for exchanging messages between devices, predominantly used in iot networks. at the start of the protocol, each process connects to a public mqtt channel, and a session is established. therefore, we have mapped binary channels to mqtt sockets, in addition to the built-in rust channels. multiparty channels as an ordered tuple of binary channels. the main idea of the design of our framework is that a multiparty session can be realised with two ingredients: ( ) a list of separate binary sessions (one session for each pair of participants) and ( ) a queue that imposes the ordering between the binary channels. listing (lines - ) shows the implementation of a multiparty channel in a -party protocol. the sessionmpst structure holds two fields, session and session , that are of a binary session type. for an illustration purpose, we show only the implementation of a multiparty channel for three processes. the same approach can be generalised, using our code generation tool, to any number of communicating processes. for example, in case of a protocol with four roles, each multiparty session will have four fields -a field for the binary session between each pair of participants and a field for the queue. the order of usage of the binary channels of a sessionmpst object is stored inside the queue field. for instance, the behaviour that role a has to communicate first with role b, then with a role c, and then the session ends can be specified using a queue of type roleatob<roleatoc<roleend>>. note that all queue types, such as roleatob, roleatoc, are generated. as explained in sect. , programming with mpst-rust relies on communication primitives, such as send_mpst_a_to_b, that have the sender and receiver roles baked into their name. to ensure that the binary channels are used as specified by the global protocol, each communication function is parametric on a generic quadruple type <t, s , s , r> where t is a payload type, s and s are binary session types and r is a type for a queue (mpst-queue type) that imposes the order in which the binary sessions inside a multiparty session must be used. // basic structure for mpst pub struct sessionmpst< s : session, s : session, r: role> { pub session : s , pub session : s, pub queue: r } // implementation of a communication function from the mpst-rust library pub fn send_mpst_a_to_b<t, s , s , r>(x: t, s: sessionmpst<send<t, s >, s , roleatob<r>>,) -> sessionmpst<s , s , r> where t: ..., s : listing (lines - ) shows the implementation for send_mpst_a_to_b(). as clear from the type parameters, the client of the function should supply a mpstqueue type roleatob<r>. the binary session type s should be encapsulated in a send<t, s >. the body of the function sends the message of type t on the binary channel stored in the first field, session (corresponding to the binary session with role b), of the multiparty session s. since the communication is on a binary channel, we reuse the binary send primitive from [ ] . external and internal choices are implemented as macros that require an argument of type sessionmpst. the implementation of offer_mpst_a_to_c is given in lines - . in essence, a choice is implemented as a broadcast from one role to the others. in our usecase, the active role that makes the choice is c. hence, the macro offer_mpst_a_to_c explicitly performs a receive (recv_mpst_a_-to_c(s)) on the session channel s. the received value is pattern matched and passed to any of the functions given as arguments to offer_mpst_a_to_c. similarly, choose_mpst_c_to_all in lines - is a macro that performs a select operation. the active role c sends the selected label to all roles in the protocol. in our particular example, c sends the selected label l to a and b. discussions. our implementation, although intuitive, does not resolve the inherent conflict between rust, which is affine, and session types, which are linear. the implementation suffers from the same drawback as [ ] . however, the mpst methodology is a step forward in terms of usability. differently than the rust local types which can get convoluted, the syntax of global protocols is user-friendly and readable. developers can use the global protocol as guidance, and hence avoid errors such as prematurely ending of a session. moreover, as observed in kokke's library [ ] , most of the errors are caused by misuse of methods and functions. since we are code-generating the local types, the chance of misspelling is significantly reduced. another viable option for our framework is to take the bottom-up approach: to check directly whether a set of manuallywritten rust local types satisfy safety/liveness properties by a model checker [ ] or the multiparty compatibility (a property which guarantees deadlock-freedom of communicating automata, which are equivalent to local session types) [ , ] . the rust library in [ ] implements binary session types, following [ ] . it checks at compile-time that the behaviours of two endpoint processes are dual, i.e the processes are compatible. the library in [ ] , based on the egv calculus by fowler et al. [ ] , provides constructs for writing and checking binary session types, and additionally supports exception handling constructs. we build on top of the library in [ ] since it offers several improvements in comparison to [ ] . most importantly, the treatment of closing a channel prematurely in [ ] may lead to memory leaks. both libraries suffer from a well-known limitation of binary session types . notably, since deadlock-freedom is ensured only inside a session, a rust endpoint process, that communicates with more than one other process, is prone to deadlocks and communication errors. our framework solves that limitation by expanding the scope of a session to multiple participants. our proposed design follows the methodology given by [ ] , which generates java communicating apis from scribble. this, and other multiparty session types implementations, exploit the equivalence between local session types and communicating automata to generate session types apis for mainstream programming languages (e.g., java [ , ] , go [ ] , f# [ ] ). each state from state automata is implemented as a class, or in the case of [ ] , as a type state. to ensure safety, state automata have to be derived from the same global specification. all of the works in this category use the scribble toolchain to generate the state classes from a global specification and detect linearity violations at runtime. this paper proposes the generation of protocol-specific apis, which promotes type checking of protocols at compile-time. this is done by projecting the endpoints' state space in those protocols to groups of channel types in the desired language. in the future, we plan to implement the bottom-up approach, in addition to the top-down approach outlined in this paper, as to compare their productivity and scalability. distributed programming using role parametric session types in go multiparty compatibility in communicating automata: characterisation and synthesis of global session types exceptional asynchronous session types: session types without tiers language primitives and type discipline for structured communication-based programming multiparty asynchronous session types hybrid session verification through endpoint api generation mqtt-s-a publish/subscribe protocol for wireless sensor networks session types for rust rusty variation: deadlock-free sessions with failure in rust typechecking protocols with mungo and stmungo verifying asynchronous interactions via communicating session automata session types for rust a linear decomposition of multiparty sessions for safe distributed programming less is more: multiparty session types revisited the rust programming language. . . edn understanding real-world concurrency bugs in go a very gentle introduction to multiparty session types the scribble protocol language acknowledgement. the work has been partially supported by the following funding schemes vetss, epsrc ep/k / , ep/k / , ep/l x/ , ep/n / , ep/n / , ep/t / and, ep/t / . key: cord- -vmze mdx authors: vanheer, lotte; schiavo, andrea alex; van haele, matthias; haesen, tine; janiszewski, adrian; chappell, joel; roskams, tania; cnop, miriam; pasque, vincent title: revealing the key regulators of cell identity in the human adult pancreas date: - - journal: biorxiv doi: . / . . . sha: doc_id: cord_uid: vmze mdx cellular identity during development is under the control of transcription factors that form gene regulatory networks. however, the transcription factors and gene regulatory networks underlying cellular identity in the human adult pancreas remain largely unexplored. here, we integrate multiple single-cell rna sequencing datasets of the human adult pancreas, totaling cells, and comprehensively reconstruct gene regulatory networks. we show that a network of transcription factors forms distinct regulatory modules that characterize pancreatic cell types. we present evidence that our approach identifies key regulators of cell identity in the human adult pancreas. we predict that heyl and jund are active in acinar and alpha cells, respectively, and show that these proteins are present in the human adult pancreas as well as in human induced pluripotent stem cell-derived pancreatic cells. the comprehensive gene regulatory network atlas can be explored interactively online. we anticipate our analysis to be the starting point for a more sophisticated dissection of how transcription factors regulate cell identity in the human adult pancreas. furthermore, given that transcription factors are major regulators of embryo development and are often perturbed in diseases, a comprehensive understanding of how transcription factors work will be relevant in development and disease biology. highlights - reconstruction of gene regulatory networks for human adult pancreatic cell types - an interactive resource to explore and visualize gene expression and regulatory states - predicting putative transcription factors driving pancreatic cell identity - heyl and jund as candidate regulators of acinar and alpha cell identity, respectively a fundamental question in biology is how a single genome gives rise to the great diversity of cell types that make up organs and tissues. a key goal is to map all cell types of developing and mature organs such as the pancreas, an essential organ at the basis of multiple human disorders including diabetes and cancer (kahn, cooper and del prato, ; kamisawa et al., ; han et al., ) . single-cell rna sequencing (scrna-seq) provides a powerful tool to resolve cellular heterogeneity, identify cell types and capture highresolution snapshots of gene expression in individual cells (grün et al., ) . with the advent of singlecell transcriptomics, great progress has been made toward the creation of a reference cell atlas of the pancreas (baron et al., ; segerstolpe et al., ; wang et al., ; xin et al., ; enge et al., ; lawlor et al., ; augsornworawat and millman, ; han et al., ) . work from several groups provided cellular atlases of the pancreas during mouse development (stanescu et al., ; byrnes et al., ; scavuzzo et al., ) , in adult mice (muraro et al., ) and in human fetal and adult pancreas (liu et al., ; baron et al., ; muraro et al., ; segerstolpe et al., ; wang et al., ; enge et al., ; han et al., ) . efforts have also been made to map cellular identity during pancreas development starting from human pluripotent stem cells hrvatin et al., ; zhu et al., ; han et al., ; hogrebe et al., ; peterson et al., ) . taken together, these studies provide an opportunity to better understand the maintenance and establishment of cellular identity among different pancreatic cell types. work over the past decades indicated that cellular identity is established by combinations of transcription factors (tfs) that recognize and interact with cis-regulatory elements in the genome. these transcription factors, together with chromatin modifiers, give rise to gene expression programs. a small number of core tfs are thought to be sufficient for the establishment and maintenance of gene expression programs that define cellular identity during and after development (ohno, ) . studies conducted in both mouse and human have successfully identified tfs that are pivotal for the acquisition and maintenance of pancreatic cell fates (dassaye, naidoo and cerf, ) . these include pdx (zhou et al., ; shih et al., ) , mafa (nishimura, takahashi and yasuda, ) , ngn (gradwohl et al., ) , nkx . (sussel et al., ) , pax (sosa-pineda et al., ) , nkx . , neurod (mastracci et al., ) , arx (collombat et al., ) , mafb (artner et al., ) , rfx (smith et al., ) , gata (ketola et al., ) , foxa and sox (shroff et al., ; shih et al., ) . conditional deletion of tfs such as foxa and pdx in adult beta cells results in the loss of cellular identity and function (sund et al., ; gao et al., ) . genetic evidence for the role of these tfs in establishing human pancreatic cell identity is provided by the identification of tf loss-of-function mutations that cause pancreatic agenesis (stoffers et al., ; sellick et al., ; allen et al., ; shaw-smith et al., ; de franco et al., ) or neonatal or young-onset diabetes (senée et al., ; solomon et al., ; rubio-cabezas et al., smith et al., ; bonnefond et al., ; flanagan et al., ) . in addition, tf overexpression can reprogram somatic cells to adopt alternative identities (takahashi and yamanaka, ; zhou et al., ; vierbuchen et al., ; lima et al., ) . for example, the induced expression of ngn , pdx , and mafa was shown to reprogram mouse alpha cells into beta-like cells in vivo (zhou et al., ) . however, how key tfs underlie the maintenance of cellular identity in the human pancreas remains incompletely understood. over the past decade, multiple approaches to reconstruct gene regulatory networks (grns) from bulk and single-cell omics data have been developed (ghazanfar et al., ; lim et al., ; matsumoto et al., ; fiers et al., ) . in particular, it is now possible to combine single-cell transcriptomic data with cisregulatory information to infer grns (janky et al., ; aibar et al., ; van de sande et al., ) . because tfs recognize dna motifs in the genome, one can measure if inferred target genes are expressed within single cells, and therefore quantify the activity of tfs. such approaches have revealed the regulatory programs in distinct systems including the drosophila brain (davie et al , ) , cancer (wouters et al., ) , during early mouse embryonic development (peng et al , ) , in a mouse cell atlas (suo et al., ) and a human cell atlas (han et al., ) . analysis of grns in the human adult pancreas has identified distinct endocrine and exocrine regulatory states with multiple stable cell states for alpha, beta and ductal cells (kumar and vinod, ) . type diabetes and body mass index (bmi) were shown not to impact grn activity of alpha and beta cells (kumar and vinod, ) . previous data shows that type diabetic (faerch et al., ; dennis et al., ; dybala and hara, ; zaharia et al., ) and non-diabetic human islet preparations vary greatly depending on age (enge et al., ; westacott et al., ) and bmi (henquin, ) warranting the exploration of grns in larger cohorts. hence, it remains unclear whether previous grn findings can be extrapolated to a broader, highly heterogeneous non-diabetic and type diabetes patient population. the development of integration methods provides an opportunity to analyze multiple scrna-seq studies from multiple laboratories and patients (butler et al., ; luecken et al., ) . additional knowledge on how grns maintain cellular identity in the human adult pancreas may further the understanding of disease states as well as improve efforts to convert patient cells into functional, mature beta cells for diabetes treatment. here, we build an integrated human pancreas gene regulatory atlas. in this resource, we use single-cell transcriptomes of the human adult pancreas, taking advantage of integration strategies and computational tools to reconstruct grns. our analysis identifies the grn landscape and candidate regulators that are critical for cellular identity in the human adult pancreas. integrating multiple human adult pancreas scrna-seq datasets can further improve the power of scrnaseq analyses to create a human adult pancreas cell atlas. we set out to analyse and integrate five publicly available datasets covering a total of non-diabetic, type diabetic and type diabetic individuals using seurat v . cca integration tools ( figure a , detailed donor information can be found in table s ) (segerstolpe et al., ; wang et al., ; xin et al., ; enge et al., ; lawlor et al., ; hafemeister and satija, ; stuart et al., ) . after filtering out low quality transcriptomes and data integration, uniform manifold approximation and projection for dimension reduction (umap) visualisation revealed that cells localize into distinct clusters ( figure b) . cells from each original dataset localize together suggesting that the location of cells on the umap is not driven by the dataset of origin ( figure c ). we next sought to identify pancreatic cell types ( figure d ). clustering analyses based on the expression of well-established cell type specific markers led to the identification of eight cell types in the human adult pancreas: beta, alpha, gamma, delta, acinar, ductal, stellate and endothelial cells ( figure b , table s ). umap visualization allowed for the segregation of endocrine, exocrine and other lineages ( figure s a ). beta cells grouped together, away from other clusters and were marked by ins expression (figure e ). other distinct clusters corresponded to alpha, gamma and delta cells based on global transcriptional similarity and elevated expression of gcg, ppy and sst, respectively, and other markers ( figure e , table s ). using a similar approach, we reliably detected other, previously described, major pancreatic cell types (acinar, ductal, endothelial and stellate, figure b ). all cell types were detected in both non-diabetic and type diabetic pancreases ( figure f ). an additional four rare cell populations, that cannot be robustly identified through clustering analyses, were identified manually by assessing the expression of ghrl (epsilon cells), tps ab (schwann cells), cd (mast cells) and sox (major histocompatibility complex (mhc) class cells) (wierup et al., ; segerstolpe et al., ) (figure g ). these rare cell types often cluster with other common cell types. importantly, our annotation recapitulated previous annotations to a large extent (figure s b-c). in summary, we reconstructed an integrated single-cell atlas of the human adult pancreas, and annotated pancreatic cell types. next, we set out to comprehensively reconstruct grns for all pancreatic cell types from single-cell transcriptomic data, applying single-cell regulatory network inference and clustering (pyscenic) (aibar et al., ; van de sande et al., ) . pyscenic links cis-regulatory sequence information together with single-cell transcriptomes in three sequential steps by ) co-expression analysis, ) target gene motif and chip-seq track enrichment analysis, and ) regulon activity evaluation (figure a) . each regulon consists of a tf with its predicted target genes (co-expressed genes with an enriched tf motif), altogether forming a regulon. pyscenic identified regulons that characterize the grns of the human adult pancreas ( figure b /c, table s ). multiple regulons identified here as active in the pancreas correspond to tf binding motifs enriched in accessible chromatin in the pancreas (assessed by atac-seq in facs-purified pancreatic cells, (arda et al., ) ), supporting the validity of the approach ( figure s a ). umap visualization based on the activity of regulons in non-diabetic and type diabetic pancreata revealed groups of cells that differ from one another based on their regulatory activity ( figure b -c). in particular, there are distinct regulatory states for exocrine and endocrine pancreatic lineages, stellate and endothelial cells (figure b /c). endocrine cell types clustered together, indicating shared regulatory states, while exocrine cell types formed two distinct clusters. stellate and endothelial cells differed most from other cell types in their regulatory states. these results are consistent with previous analyses (baron, veres, samuel l. wolock, et al., ; lawlor et al., ; kumar and vinod, ) and are also in line with our findings above based on gene expression analysis ( figure d ). as expected, regulons active in endocrine cell types include rfx , pax and neurod ( figure d -g). these tfs have reported roles in endocrine cell fate commitment and maintenance of cell identity throughout adult life (smith et al., ; hart et al., ; mastracci et al., ) . using iregulon for visualization, many of the neurod target genes identified here have been previously linked to beta cell survival and function, such as snap , tspan , (gierl et al., ; haumaitre et al., ; hart et al., ; churchill et al., ) . clustering all cells based on the activity of all regulons identifies regulatory modules ( figure d , red squares). in the exocrine pancreas, one regulatory module, containing nr a , was shared between acinar and ductal cells, although with a tendency for increased regulon activity in ductal cells ( figure d /h). other exocrine regulons included onecut , rest and hnf b with reported roles in exocrine development (nissim et al., ; kropp, zhu and gannon, ) and the adult exocrine pancreas (quilichini et al., ; bray et al., ) ( figure d ). in summary, this analysis confirms the expected separation of exocrine and endocrine cells with distinct gene regulatory programs, and identifies known and novel candidate regulators of pancreas cell states. several regulatory modules are shared between different cell types within the endocrine and exocrine pancreas. additionally, each cell type is defined by cell-type specific regulatory modules ( figure d ). in the endocrine pancreas, alpha and beta cells shared endocrine regulons (mafb, meis ), whereas we observed distinct activities for arx and irx regulons in alpha cells and rxrg and pdx in beta cells ( figure d /h), expanding previous findings (kumar and vinod, ) . using iregulon for visualization, pdx target genes include slc a , pdia and abhd , which have been reported to control insulin release (thomas, brown and brown, ; eletto et al., ; rorsman and ashcroft, ) ( figure s c ). interestingly, gamma and delta cells overlapped with alpha and beta cells, respectively, suggesting a shared regulatory state ( figure c/d) . this includes shared regulon activity for arx in gamma and alpha and pdx in beta and delta cells ( figure d /h), consistent with their reported expression in published scrna-seq studies (baron et al., ; segerstolpe et al., ; lawlor et al., ) . gata and rbpjl, known acinar-specific tfs, were highly active in acinar cells (masui et al., ; carrasco et al., ) ( figure h) . similarly, ductal cells were characterised by highly active sox and pou f regulons, in line with previous literature (shroff et al., ; yamashita et al., ) (figure h ). in sum, this analysis confirms that alpha, beta, acinar and ductal cells are defined by the activity of distinct tf combinations that form gene regulatory modules. in conclusion, the network approach recovers many of the expected regulators of pancreatic cellular identity allowing for the comprehensive characterisation of the gene regulatory state of all major human adult pancreatic cell types. a comprehensive network analysis provides an opportunity to predict and identify critical regulators of cell identity. to identify regulons with highly cell type-specific activities within the human adult non-diabetic pancreas, we calculated regulon specificity scores (rss) (a complete list of rsss can be found in table s ) (suo et al., ) . the rss utilises jensen-shannon divergence to measure the similarity between the probability distribution of the regulon's enrichment score and cell type annotation wherein outliers receive a higher rss and are therefore considered cell type-specific (suo et al., ) . it can therefore be used to rank the activity of tfs within specific cell types. among the top regulons identified in alpha cells, we recover well known regulators of alpha and endocrine cell fate such as arx, irx , pax , mafb, neurod and rfx ( figure a /b) (collombat et al., ; artner et al., ; delporte et al., ; smith et al., ; dorrell et al., ; mastracci et al., ) . in addition, we identified jund, egr , srebf and stat , which have not yet been implicated in alpha cell identity. egr (but not egr ) has been shown to transcriptionally regulate glucagon expression (leung- theung-long et al., ) as well as the pdx promoter in beta cells (eto, kaur and thomas, ) . stat and jund have been described in pancreatic tissue in general and beta cells, respectively, but not in alpha cells (yu and kim, ; good et al., ) (figure b /c). these tfs respond to the jnk and egfr signalling pathways and may have important physiological functions. both jund and the jund/jnk signaling pathway have been implicated in pancreatic cancer (shin et al., ; recio-boiles et al., ) . immunocytochemistry of the human adult pancreas confirmed the presence of nuclear jund in islets ( figure di ). we also detected nuclear jund protein in a subset of human induced pluripotent stem cells (ipscs) subjected to beta cell differentiation (figure e/f) . surprisingly, we also detected jund protein in ductal cells, despite lower jund regulon activity in this cell type (figures c, dii) . thus, protein expression does not always predict regulatory activity. nevertheless, these results show that jund is present and active in a subset of pancreatic cell types in the human adult pancreas and human pluripotent stem cell derived islet cells. altogether, this analysis predicts tfs active in human alpha cells, recovering known as well as new candidate tfs. among the top regulons identified in beta cells, we retrieved well-known as well as new candidate regulators of beta and endocrine cell identity. known tfs include rxrg, pdx , neurod , pax and rfx (zhou et al., ; miyazaki et al., ; smith et al., ; mastracci and sussel, ; hart et al., ) (figure g /h). in addition, we found that znf d, ascl and hoxd were highly ranked regulons ( figure h /i). hoxd and bhlhe have been shown to be present in the exocrine pancreas (cantile et al., ; sato et al., ) . interestingly, ascl has been reported to interact with β-catenin of the wnt pathway, the latter has an established role in endocrine fate specification during in vitro differentiation (schuijers et al., ; sharon et al., ; vethe et al., ) . many putative target genes of ascl including pdx , ins, abcc , foxa , kcnk , fxyd are directly related to glucose sensing and beta cell identity, in line with the beta cell-specific regulatory activity of ascl ( figure s a ) (gao et al., ; arystarkhova et al., ; vierra et al., ; park, lee and park, ) . fxyd γa, a regulatory subunit of the na + -k + -atpase, is a transcript exclusively expressed in human beta cells (flamez et al., ) . immunohistochemistry of human adult pancreas sections showed that ascl is expressed in ins + beta and islet cells ( figure j) . surprisingly, ascl was mainly localized to the cytoplasm ( figure j) , which is unexpected for tfs which tend to localize to the nucleus (baranek, sock and wegner, ) . cytoplasmic localization of ascl has been reported in the context of colon and breast cancer (zhu et al., ; xu et al., ) . these results implicate additional tfs including ascl in the regulation of beta cell identity. they also illustrate the value of network analyses to increase our understanding of the biology of the human pancreas. in summary, grn analysis and regulon ranking allowed us to pinpoint both known and novel candidate regulators of pancreatic endocrine cell identity, providing a resource for further investigation of their roles in cellular identity and function. similarly, the comprehensive network analysis provides an opportunity to predict and identify critical regulators of exocrine cell identity. we also identify known and new tfs in acinar cells. among the top acinar-specific regulons, we recovered well known regulators of acinar and exocrine cell identity such as ptf a, rbpjl, gata and nr a (ketola et al., ; masui et al., ; nissim et al., ; sakikubo et al., ) (figure a/b) . these findings are in line with a recent study that used single-nucleus rna-seq on pancreatic acinar tissue (tosti et al., ) . furthermore, we identified mecom, heyl and tgif as highly ranked regulons ( figure b /c). interestingly, aberrant mecom expression has been linked to the induction of gastric genes in acinar cells, which disrupts acinar cell identity and increases susceptibility to malignancy (hoang et al., ) . the loss of tgif has been linked to pancreatic ductal adenocarcinoma progression making further exploration of these regulons interesting in the context of cancer biology (weng et al., ) . ectopic expression of tgif (but not tgif ) reprograms mouse liver cells towards a pancreas progenitor state (cerdá-esteban et al., ) . heyl is a reported notch signalling target gene in ngn + exocrine cells (gomez et al, ) . we confirmed nuclear expression of heyl in human acinar and islet cells (figure di /ii, detailed donor information can be found in table s ) by immunohistochemistry, in agreement with elevated heyl regulon activity in acinar cells ( figure c ). further functional studies in the healthy pancreatic context (matsumoto et al., ; coleman et al., ) . in summary, grn analysis and regulon ranking allowed us to pinpoint both known and novel candidate regulators of pancreatic exocrine cell identity. specifically, we identified heyl as a candidate tf that might be important for acinar cell identity, warranting further investigation. to enable users to easily navigate the human pancreatic cell network atlas, we provide a loom file that allows for the visualisation and exploration of the data using the web-based portal scope (davie et al., ) (.loom file and tutorial available at http://scope.aertslab.org/#/pancreasatlas/*/welcome and https://github.com/pasquelab/scpancreasatlas). features such as cell type annotation as defined in this paper, gene expression and regulon activity can be explored on the regulon and gene expression based umap. this resource enables users to select and visualize up to three genes or regulons simultaneously and select subsets of cells for downstream analyses. for example, the expression of covid- related genes can be interactively explored (yang et al., ) . target genes of a specific regulon can be downloaded to facilitate further exploration, for example in iregulon or gene ontology analysis (janky et al., ) . a list of predicted target genes of all regulons can also be found in table s . furthermore, a list of target genes can be manually defined to compute the activity of a custom regulon. this resource can be used to further study cell identity and gene regulation in the context of the pancreas, diabetes and cancer. in this resource, we take advantage of integration strategies and new computational tools to reconstruct an integrated cell and grn atlas of the human adult pancreas from single-cell transcriptome data. this approach provides a comprehensive analysis of the gene regulatory logic underlying cellular identity in the human adult pancreas in a broad range of individuals, limiting the influence of inter-donor variability. we recovered known regulators of pancreatic cell identity and uncovered novel candidate regulators of cell identity that can be further investigated for their roles in cellular identity and function. by validating regulon analyses and creating an easily accessible interactive online resource which allows for the exploration of the gene regulatory state of cells from individuals, this approach extends beyond previous gene regulatory studies in the human adult pancreas (augsornworawat and millman, ) . the present analysis identified regulators of pancreatic development, function and survival that are known to be critical in humans because loss-of-gene function causes pancreatic agenesis or young onset diabetes. for example, ptf a (sellick et al., ; weedon et al., ) and gata (shaw-smith et al., ) , whose loss of function are linked to pancreatic agenesis and neonatal diabetes, were among the top acinarspecific regulons (figure ). in addition, monogenic diabetes related genes pdx (nicolino et al., ), neurod (rubio-cabezas et al., , pax (solomon et al., ) , rfx (smith et al., ; patel et al., ) and glis (senée et al., ) were among the top beta cell-specific regulons (figure and table s ). stress signalling (table s ) . creb and creb l are non-canonical er stress transducers that are induced in human islets and clonal beta cells upon exposure to the saturated fatty acid palmitate (cnop et al., ) . interestingly, srebf and - undergo similar er exit and proteolytic processing in the golgi as these er stress transducers, but they do so in response to changes in er cholesterol content; both also have high regulon activity in alpha and beta cells. xbp is abundantly expressed in the exocrine and endocrine pancreas (cnop et al., ) , but the xbp regulon has its highest specificity in beta cells. atf and atf are tfs that are activated upon eif α phosphorylation, an er stress response pathway to which no less than monogenic forms of diabetes belong (eizirik, pasquali and cnop, ) . our data underscores the importance of these tfs for endocrine pancreatic cell identity. given that we predict novel regulators of cell identity in the human pancreas, it will be interesting to also expand this analysis to pancreas embryonic development. our work may also be beneficial in guiding improvements of and better understanding the in vitro derivation of pancreatic cell types. for example, the emergence of sst-positive cells together with beta-like cells at the end of in vitro differentiation could be explained by the overlap in regulatory states between beta and delta cells (baron et al., ) . grn analyses are particularly interesting for in vitro derived beta cells since a better understanding of the regulatory logic underlying control of beta cell fate may improve or facilitate future applications in regenerative medicine (pagliuca et al., ; rezania et al., ; nostro et al., ; russ et al., ; baeyens et al., ) . alternatively, many observed grns such as ascl , mecom, ppard, gata and cdx are linked to pancreatic cancer making the additional exploration of grns interesting in the context of cancer biology (matsumoto et al., ; zhu et al., ; coleman et al., ; hoang et al., ; xu et al., ; weng et al., ; brunton et al., ) . finally, recent reports have stratified type diabetes patients based on age at diagnosis, bmi, hba c and insulin secretion and sensitivity, and identified subtypes with different genetic predisposition, treatment response, disease progression and complication rates (ahlqvist et al., ) . hence, it would be interesting to assess differences in gene regulatory state and gene expression profiles of alpha and beta cells between different type diabetic subgroups. it is important to note that pyscenic is a stochastic algorithm that does not produce precisely the same regulons for repeated applications, limiting reproducibility when comparing different datasets (huynh-thu et al., ; van de sande et al., ) . to mitigate this uncertainty, we ran the full pyscenic pipeline five times and only kept consistent regulons with the highest regulon activity. the performance of pyscenic, and other grn inference methods, suffers due to the large amount of drop-out events in scrna-seq data warranting caution when interpreting results (chen and mar, ) . this could explain the absence of wellestablished pancreas tfs such as mafa (olbrot et al., ) , mnx (flanagan et al., ) , neurog (krentz et al., ) , foxa (lee et al., ) and nkx - (mastracci et al., ) in this analysis. nevertheless, in support of the validity of our findings, atac-seq, literature and immunohistochemistry of human pancreas sections corroborate several pyscenic predictions. chen and colleagues underline the importance of using large sample sizes to derive the most accurate network inference possible (chen and mar, ) , highlighting the importance of dataset integration to increase the number of cells analysed. in the future, it will be interesting to extend our analyses to include many more cells and patients. in spite of current caveats, grn analysis has enabled the capture of biological relevant information (butte et al., ) . one additional limitation of this study is the assumption that all tfs bind their binding motifs in the promoters of expressed genes. however, tf binding can be restricted to a subset of tf motifs in the genome due to influence of chromatin processes including the presence of nucleosomes as well as dna methylation. therefore, additional approaches such as single cell multi-omics that capture additional layers of genome regulation will be helpful to increase our understanding of gene regulation in the context of the human pancreas. taken together, our grn atlas, containing individuals, provides a valuable resource for future studies in the human pancreas development, donor variability, homeostasis and disease including type diabetes and pancreatic cancer. finally, our results provide new insights into the activity of tfs and gene regulation in the human adult pancreas from a gene regulatory perspective. questions about data analysis should be directed to the lead contact, vincent pasque (vincent.pasque@kuleuven.be). the reviewer tokens for this geo repository is cfsjciumfferjkd. motif discovery of bulk atac-seq data paired-end raw reads for bulk atac-seq (see key resource table) were downloaded from sra using sra toolkit (v . . ). reads were aligned and further analyzed using the encode atac-seq pipeline with default parameters using the encode human reference genome grch . (lee, ) . bed files containing the global open chromatin landscape of adult alpha (alpha_ ; ea and alpha_ ; ea ), beta (beta_ ; ea and beta_ ; ea ), acinar (acinar_ ; ea and acinar_ ; ea ) and ductal (ea ) cells or cell type specific differentially accessible regions were used as input for motif discovery by homer (v . . ) using the 'findmotifsgenome.pl' with options using hg with size given (heinz et al., ) . the tfs whose motifs identified by homer correspond with tfs identified by pyscenic are visualized in figure s a . analysis of publicly available scrna-seq data raw reads for five publicly available scrna-seq datasets (see key resource table) were downloaded from sra using sra toolkit (v . . ). afterwards, reads were aligned to the human reference genome grch . using star (v . . a) with default parameters followed by the conversion to the coordinate sorted bam format. next, the featurecounts command from the "rsubread" (v . . ) package in r (v . . ) was used to assign mapped reads to genomic features. low quality transcriptomes with a mitochondrial contamination greater than % and less than expressed genes per cell were excluded from subsequent analyses. the resulting raw count matrix was batch corrected using the findintegrationanchors and integratedata functions from the "seurat" package (v . . ) after which subsequent analyses were carried out in the r package "seurat" (v . . ). gene expression was used to cluster all cells with umap, using seurat's function runumap. clusters for cell type annotation were defined using seurat's shared nearest neighbour algorithm findclusters function after which differential expression analysis was performed using wilcoxon's rank sum test with a minimum cutoff of . average log fold change and min.pct of . . pyscenic grns were inferred using pyscenic (python implementation of scenic, v . . ) in python version . . (aibar et al., ) . integrated read counts were used as input to run genie (huynh-thu et al., ) which is part of arboreto (v . . ). grns were subsequently inferred using pyscenic with the hg _refseq-r motif database and default settings. to control for the stochasticity, which is inherent to pyscenic, a consensus grn was generated by merging results from five repeat pyscenic runs. if regulons were identified in multiple pyscenic runs, only the regulon with the highest auc value was retained. regulon activity represented by aucell values was used to cluster all cells with umap, using seurat's runumap function. all regulons within non-diabetic cell types were visualized using the 'clustermap' function of the python package "seaborn" (v . . ). the z-score for each regulon across all cells was calculated using the z-score parameter of the seaborn 'clustermap' function. extended analysis of the target genes of specific regulons was conducted in cytoscape (v . . ) using the iregulon application (v . ). the list of target genes of a specific regulon was downloaded from the loom file through the scope platform (https://github.com/pasquelab/scpancreasatlas) (davie et al., ) . to quantify the cell-type specificity of a regulon, we utilized an entropy-based strategy as described previously (suo et al., ) using the aucell matrix as input in matlab r b. the top most specific regulons were subsequently visualized using the r package ggplot (v . . ). the complete regulon ranking list is available in table s . control ipsc line hel . (cosentino et al., ) was differentiated into beta cells using a previously published -step protocol (cosentino et al., ) . at the end of the stage , cells were seeded into -well aggrewell microwell plates (stem cell technologies) at a density of . · cells per well after which differentiation was carried out as described previously (cosentino et al., ) . stage differentiated beta cells were washed twice with pbs containing . mm edta and incubated in accumax (sigma #a ) for min at °c after which % volume of knockout serum replacement (thermofisher # ) was added to stop the reaction. after centrifugation at g for min at room temperature, cells were resuspended in ml ham's f- medium, supplemented as indicated before (demine et al., ) . , cells in μl medium were seeded per square of a nunc lab-tek ii icc chamber (thermofisher). immunohistochemistry analyses were carried out largely as described previously (demine et al., ) , immunohistochemistry analyses were carried out largely as described previously (ceulemans et al., ) , using primary antibodies against the following proteins: ascl (merck, mab , clone e , / ), jund (atlas antibodies, hpa , / ), heyl (atlas antibodies, hpa , / ) and ins (agilent, ir , / ). pictures were taken using a leica dmlb (leica microsystems). the integrated single-cell rna-seq data and pyscenic results can be explored interactively in scope (davie et al., ) . loompy (v . . ) (linnarsson lab., ) was used to create the loom files which were uploaded to scope. the embedding of the regulon and integrated gene expression based umap clustering, as seen in this article, were added to the loom file. this table is related to figure , , and . this table is related to figure . this table is related to figure , and . list of regulon specificity scores for all regulons of non-diabetic alpha, beta, acinar and ductal cells. this table is related to figure and . list of putative target genes for each regulon. this table is related to figure , and . 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permanent scope link, shengbao suo for sharing the matlab script for calculating the regulon specificity score, the key: cord- - pfxrhbc authors: petrie, john r title: sglt inhibitors and renal complications in type diabetes date: - - journal: lancet diabetes endocrinol doi: . /s - ( ) - sha: doc_id: cord_uid: pfxrhbc nan adding in non-insulin agents is one of several promising strategies under investigation to improve glycaemic control in type diabetes. unlike uptitration of insulin, the ideal so-called adjunct drug would not cause increased hypoglycaemia and weight gain. it would also reduce rates of cardiovascular, renal, and other adverse outcomes by improving glycaemic control or other mechanisms. these complications still result in an average reduction in life expectancy of - years among people with type diabetes. of several drug classes repurposed from type to type diabetes, sglt inhibitors have made the most progress. the concept behind these drugs is that inhibiting reabsorption of glucose (and sodium) in the proximal renal tubules reduces blood glucose only when it is above a reduced renal threshold, so hypoglycaemia is not increased, while weight is reduced due to urinary loss of glucose equivalent to approximately kcal per day. because adverse cardiovascular events are reduced in patients with type diabetes with these drugs (as has been found in the empareg, canvas, declare-timi trials), the hypothesis that this might also happen in type diabetes is not unreasonable. in this issue of the lancet diabetes & endocrinology, per-henrik groop and colleagues report the effect of the sglt inhibitor dapagliflozin on albuminuria in adults with type diabetes in a post-hoc subgroup analysis of the depict- and depict- phase trials. they report that in the ( %) of participants with albuminuria at baseline, dapagliflozin reduced urinary albumin excretion (the urinary albumin to creatinine ratio [uacr]) compared with placebo with a mean change from baseline at weeks of − · % ( % ci − · to · ) for dapagliflozin mg and − · % (− · to − · ) for dapagliflozin mg. this finding is biologically plausible given compelling recent evidence that dapagliflozin and see articles page in pregnancy have noted that some patients will still require insulin in the third trimester. for future consideration, metformin clearance is increased in pregnancy, but the effects on dose selection or efficacy are unknown. metformin is excreted into the milk during lactation, and although caution is advised, no adverse effects have become evident. initial studies indicated that infants who were exposed to metformin in utero subsequently gained weight normally and generally achieve slightly above average growth, but more data on child development would be welcome. other potential adjuncts to insulin treatment in pregnancy include sulfonylureas such as glibenclamide. this drug has been shown to improve maternal glycaemic control but crosses the placenta and stimulates fetal insulin secretion, with consequent macrosomia and risk of hypoglycaemia to the fetus and neonate. no adequate clinical data exist regarding the use of dpp- inhibitors, glp- receptor agonists, or sglt inhibitors in pregnancy, but these medications are not recommended on the basis of preclinical studies that suggest possible adverse effects during the late stages of fetal development. in conclusion, mity has provided prospective, controlled evidence to support the low-cost, potentially beneficial, metabolic effects of metformin with insulin in the management of pregnancy for type diabetes and gestational diabetes, and substantiated a favourable safety profile for neonates. i report personal fees from abbott diabetes care, boehringer ingelheim, astrazeneca, lexicon-sanofi, merck, msd, napp, and novo, outside of the submitted work. other sglt inhibitors reduce rates of end-stage kidney disease in patients with chronic kidney disease, whether or not associated with type diabetes. mechanistically, sglt inhibitors are thought to protect the glomerulus by reflex constriction of the afferent arteriole in response to renal tubular sodium loss rather than by relaxation of the efferent arteriole as with renin-angiotensin system blocking drugs. despite the many beneficial effects of sglt inhibition in several conditions, including heart failure, their promotion of ketosis has been the major barrier to widespread uptake in type diabetes, in which diabetic ketoacidosis still accounts for more than % of deaths. this adverse effect is unlikely to be eliminated because many of the positive effects, particularly on heart failure outcomes, are thought to be mediated by increased availability of free fatty acids and ketone bodies for metabolism. nevertheless, of the sglt inhibitors that have entered phase trials in type diabetes, dapagliflozin (depict programme) and sotagliflozin (intandem programme) have relatively favourable therapeutic profiles. , in pooled analyses of dapagliflozin, a dose of mg per day on average reduced hba c by · % versus placebo at weeks in the context of a three times increase in adjudicated diabetic ketoacidosis risk versus placebo ( · events per patient years in the dapagliflozin group vs · events per patient years in the placebo group), decreasing substantially in those with a bmi of kg/m² or higher ( · vs · per patient years). based on such therapeutic profiles, both dapagliflozin and sotagliflozin were granted a european (but not us) license for adjunct therapy in type diabetes in the first half of for those with a bmi of kg/m² or higher. the uk national institute for healthcare excellence (nice) subsequently recommended both drugs (dapagliflozin in august, , and sotagliflozin in february, ) to be cost-effective for use within the uk national health service for individuals who additionally had a relatively high insulin requirement (≥ · units/kg per day) and had completed an evidence-based qualityassured structured education programme. notably, as of august, , dapagliflozin is widely available but sotagliflozin has yet to be launched in many countries. nice estimated that around of the estimated adults with type diabetes in the uk might be eligible for sglt inhibition. however, more than year on from approval, uptake has been much lower (petrie jr, unpublished) . although many diabetologists have cared for people with type diabetes who have derived great benefit from dapagliflozin (usually in the context of regular blood ketone monitoring; petrie jr, unpublished), their enthusiasm has been tempered by small numbers of patients who have been admitted to hospital for severe treatment-resistant diabetic ketoacidosis attributed to sglt inhibitor therapy, in some cases presenting late due to relative euglycaemia. the strength of this perception was reinforced during the early months of the covid- pandemic by guidance from the association of british clinical diabetologists that sglt inhibitors should be stopped in all people with diabetes (even those with type ) who had been admitted to hospital. some uk centres and clinics went even further and proactively contacted stable patients with type diabetes to advise discontinuation of the drug, even those who were able to skip doses on sick days as recommended. as sglt inhibitors are restarted now that the uk is coming out of the covid- lockdown, can re-introduction now be informed by the knowledge that renoprotection is an additional and previously unrecognised benefit of dapagliflozin? the analysis by groop and colleagues has some limitations. because it was based on a non-prespecified surrogate measure in small numbers of individuals during relatively short-term follow-up, they could not report on clinical renal outcomes. the · % (se · ) reduction in mean percentage change uacr from baseline to week observed in those allocated to the placebo group indicated considerable regression to the mean. additionally, despite evidence suggesting dose-dependence of dapagliflozin, reduction in uacr with the recommended dose of dapagliflozin ( mg per day) was not significant. this finding is in contrast with significant reduction in uacr with sotagliflozin ( mg per day), but no significant reduction with the higher dose ( mg per day). despite these considerations, when taken in the context of evidence with sglt inhibition in other conditions and data for sotagliflozin, the dapagliflozin analysis by groop and colleagues contributes to proof of concept for renoprotection for this class of drug in type diabetes and helps support the benefit rather than the for a press release from astrazeneca about dapagliflozin in people with and without diabetes see https://www.astrazeneca.com/ media-centre/pressreleases/ /farxiga-phase-iiidapa-ckd-trial-will-be-stoppedearly-after-overwhelmingefficacy-in-patients-withchronic-kidney-disease.html for more on european license of dapagliflozin see https://www. astrazeneca.com/media-centre/ press-releases/ /forxigaapproved-in-europe-for-type- sglt inhibitors in type diabetes: knocked down, but up again? estimated life expectancy in a scottish cohort with type diabetes the effects of sglt inhibitors on cardiovascular and renal outcomes in diabetic patients: a systematic review and metaanalysis effect of dapagliflozin as an adjunct to insulin over weeks in individuals with type diabetes: post-hoc renal analysis of the depict randomised controlled trials benefit:risk profile of dapagliflozin mg in the depict- and - trials in individuals with type diabetes and bmi ≥ kg/m² canagliflozin and renal outcomes in type diabetes and nephropathy time trends in deaths before age years in people with type diabetes: a nationwide analysis from scotland sotagliflozin: a review in type diabetes nice recommends innovative treatment for type diabetes. national institute for health and care excellence optimising the benefits of sglt inhibitors for type diabetes key: cord- -nxeg xj authors: gerba, charles p.; goyal, sagar m. title: pathogen removal from wastewater during groundwater recharge date: - - journal: artificial recharge of groundwater doi: . /b - - - - . - sha: doc_id: cord_uid: nxeg xj nan solid wastes, and sewage oxidation ponds. additional sources of pathogens in groundwater may involve artificial recharge of groundwater aquifers with renovated wastewater including deep well injection, spray irrigation of crops and landscape, basin recharge, and land application of sewage effluent and sludges. leakage of sewage into the groundwater from septic tanks, treat ment lagoons, and leaky sewers is estimated to be over a trillion gallons a year in the united states [ ] . it should be realized that, as opposed to surface water pollution, con tamination of groundwater is much more persistent and is difficult to erad icate. because restoration of groundwater quality is difficult, time-consuming, and expensive, efforts should be made for the protection of groundwater quality rather than only for its restoration after degradation. secondary sewage treatment including disinfection by chlorination may not be able to remove all of the pathogens present in sewage. thus, intentional or unintentional recharge of groundwater with treated sewage effluent may be potentially hazardous to human and animal life. soil is considered a living filter, capable of removing pathogenic mi croorganisms from applied wastewater. the extent to which soil can remove these microorganisms depends on several factors such as the nature of the soil, the nature of the pathogen concerned, temperature, and antagonism from native microflora. because of their large size, parasitic protozoa and helminths may be efficiently removed by filtration through soil and may not be able to gain entrance into the groundwater. bacterial removal by soils also occurs largely by filtration, although adsorption is also involved. viruses, on the other hand, are thought to be removed by the process of adsorption only [ ] . unfortunately, however, viruses cannot be considered as permanently immobilized because they have been shown to elute and migrate further in soil following rainfall events [ ] [ ] [ ] . several investigators have reported on the isolation of viruses from groundwater [ ] and several outbreaks of viral hepatitis, yersiniosis, ty phoid, and shigellosis have also been attributed to contaminated groundwater [ ] . documented evidence of health problems associated with groundwater recharge is, however, lacking. the paucity of information on health problems associated with groundwater recharge programs may reflect either the absence of a problem, lack of intensive surveillance, or the insensitivity of present epidemiologic tools to detect recurrent small-scale incidents of disease. often the low fecal car riage rates of agents of infectious disease and the low background of enteric disease in the united states has been cited as further evidence that the potential of public health hazard as a result of direct or indirect reuse of wastewater is minimal. it should be realized, however, that levels of enteric disease in the united states are low primarily because of good sanitation, personal hygiene, and a network of sanitary engineering works. as a result of this low exposure to pathogens, the population at large may have become highly susceptible to even small numbers of pathogens. waterborne outbreaks of disease are no longer on the decline in this country (figure . ). a total of waterborne outbreaks occurred during , increasing the annual average of outbreaks to for the -year period from - . this number represents more than a percent increase over the - average of . the -year averages have steadily in creased from an annual average of ten during [ ] [ ] [ ] [ ] [ ] . before that pe riod, the trend was declining [ ] . it should be emphasized that reporting of waterborne disease out breaks, particularly in individual systems, is notoriously poor. according to craun [ ] outbreaks in municipal water systems, which number , and serve about million people, are probably the most likely to be reported. outbreaks in semipublic systems, which number about , and serve numerous transients, are the next most likely to be reported. the least likely to be reported are the outbreaks in individual water systems, which number about , , . in fact, one third of the individual groundwater supplies in a rural neighborhood of oregon were found to be fecally contaminated in a recent survey [ ] . waterborne hypothesis cannot be proved in all instances because epidemiologic investigations are sometimes incomplete or conducted long after the outbreak has subsided. also, the surveillance of waterborne diseases by the centers for disease control (cdc) is largely passive and clearly rep resents a fraction of the total number that occur. according to cdc, "the likelihood of an outbreak coming to the attention of health authorities varies considerably from one locale to another depending largely upon consumer awareness, physician interest, and disease surveillance activities of state and local health and environmental agencies. large interstate-outbreaks and outbreaks of serious illness are more likely to come to the attention of health authorities." of documented outbreaks of waterborne disease from to , ( %) were attributed to illness of probable viral etiology (e.g., hepatitis a, poliomyelitis, gastroenteritis). this number probably represents only a fraction of the actual number of virus-caused outbreaks, because of the difficulties involved in proving a viral etiology of a waterborne outbreak. in fact, direct evidence of virus involvement in waterborne outbreaks is limited to hepatitis a, adenovirus, and recently to norwalk agent and rotavirus [ ] . the lack of documentation of waterborne viral disease outbreaks may be ascribed to limitations in methodology for the detection of viruses in water and relative insensitivity of epidemiologic techniques to detect lowlevel transmission of viral diseases through water. it is easy to recognize the outbreaks of infectious hepatitis by the water route because of their explo sive nature and characteristic symptomatology. most enteric viruses, how ever, cause a wide variety of symptoms so that scattered cases of acute illness would probably have too varied symptoms to be attributed to a single etiologic agent. also, the presence of small numbers of viruses in water may result only in an inapparent infection in a person coming in contact with contaminated water. the virus may then multiply in the respiratory and gastrointestinal tract of that person who may, in turn, act as an effective carrier and transmit the virus to others. the development of acute disease in these contact persons will be epidemiologically classified as "transmitted by direct contact" rather than being waterborne. intensive surveillance is, therefore, necessary to determine the "real" cause of an outbreak. a discussion on epidemiology is incomplete without consideration of minimum infective dosage of various microorganisms. infective doses of most bacterial pathogens are relatively high. for instance, approximately enteropathogenic escherichia coli or vibrio cholerae cells must be con-sumed by healthy male volunteers to produce disease in a significant pro portion of subjects. in case of shigella, however, - cells are enough to cause dysentery. similarly, the infectious dose of protozoan cysts and helminth ova is very low, perhaps . the symptoms of helminth infections are dose-related, however. currently available information suggests that even a single virus particle may produce infection under favorable condi tions. after reviewing infective dose data for various microorganisms in human subjects, akin [ ] reached the conclusion that infective dose for some members of bacterial, viral, and parasitic groups may be as low as =^ detectable units. pathogenic microorganisms such as bacteria, viruses, protozoa, and parasitic worms are almost always present in domestic sewage. the number and types of organisms present in sewage, however, vary from community to com munity depending on urbanization, population density, sanitary habits, sea son of the year, and rates of disease in the contributing community [ ] . the most common bacterial pathogens associated with sewage are salmonella, shigella, vibrio, and campylobacter (table . ). salmonella occurs [ ] . since , however, cases of cholera have been documented along the gulf coast. the strains from all these cases appear essentially identical, suggesting that the toxigenic v. cholerae has persisted in that region for at least years. extra efforts should, therefore, be made to keep track of this potential problem. more than different virus types may be present in raw sewage (table . ). they range in size from about nm for polio virus to nm for rotavirus and up to nm for enteric coronavirus. all virus groups found in sewage contain single-or double-stranded rna except adenoviruses, which consist of double-stranded dna. these viruses are capable of causing a variety of illnesses at very low dosage levels [ ] . the amount of virus present in raw sewage is highly variable but as high as , infec tious virus particles per liter have been detected [ ] . studies indicate that bacteria and viruses are not removed effectively from wastewaters during primary treatment [ ] ; removal of viruses during secondary treatment (active sludge) is dependent largely on virus adsorption to solids. since rotavirus adsorbs poorly to activated sludge floes, it can be speculated that wastewater treatment processes that are highly effective in the removal of enteroviruses may not be as effective in removing rota and reoviruses. even within the enterovirus group, virus adsorption to activated sludge was found to be both type-and strain-dependent [ ] . it stands to reason, therefore, that different viruses will have different removal char acteristics during activated sludge treatment. an average of to percent of the enteric bacteria in sewage are reported to be removed by activated sludge process (table . ). coagulation with alum or lime is considered to be generally efficient for virus removal. in laboratory studies, - log reduction of viruses is common following lime treatment at ph . in field studies, however, viruses were isolated from lime sludge and lime-treated effluent [ ] . other tertiary treatments such as ferric chloride-polyelectrolyte flocculation, sand or granular filtration, re verse osmosis, and carbon adsorption have been found to significantly re duce the level of pathogens. feachem et al. [ ] reviewed the literature on pathogen removal by various sewage treatment processes. removal, but to look at orders of magnitude." they further stated that to talk of percent removal is misleading because a percent removal of path ogens from raw sewage containing pathogens per liter will produce an effluent that still contains pathogens per liter. this level may still be of great public health concern, depending on how the effluent is going to be used. as efficient as it may be, sewage treatment processes cannot be ex pected to remove/inactivate all of the pathogens present. disinfection of treated wastewater is, therefore, practiced to ensure further inactivation of microorganisms. in the united states, chlorination is practically the only process used for disinfection of wastewater. unfortunately, however, there is a great variability in resistance to chlorine among different microorga nisms. it is generally agreed that bacteria are much more susceptible to chlorine than are viruses and protozoan cysts. also, chlorine may be very [ ] . effective against mircoorganisms cultivated in the laboratory under artificial conditions, but it may not be as effective on naturally occurring strains of bacteria and viruses. the fate of pathogenic bacteria and viruses in the subsurface will be deter mined by their survival and their retention by soil particles. both survival and retention are largely determined by the three factors shown in figure . . climate will control two important factors in determining viral and bacterial survival: temperature and rainfall. the survival of microorganisms is greatly prolonged at low temperature; below ° c they can survive for months or even years [ ] . at higher temperatures, inactivation or dieoff is fairly rapid. in the case of bacteria, and probably viruses, the dieoff rate is approximately doubled with each ° c rise in temperature between ° c and ° c [ ] . above ° c temperature is probably the dominant factor determining virus survival time. rainfall mobilizes previously retained bac teria and viruses and greatly promotes their transport in groundwater. sev eral studies have shown that the greatest degree of drinking water well contamination occurs after periods of heavy rainfall [ ] [ ] [ ] . the nature of the soil will also play a major role in determining survival and retention. soil properties influence moisture-holding capacity, ph and organic matter-all of which will control the survival of bacteria and virus in the soil. other soil properties such as particle size, cation exchange ca pacity, and clay content will influence retention. resistance of microorga nisms to environmental factors will vary among different species as well as strains. bacteria are believed to be removed largely by filtration processes while adsorption is the major factor controlling virus retention [ ] . the following sections are a summary of the recent state of knowledge on factors currently believed to influence microbial persistence and transport in the subsurface. the straining or filtration of bacteria at the soil surface is a major limitation in their travel through soils. when suspended particles, including bacteria, accumulate on the soil surface, as water passes through the soil these par ticles themselves become the filter [ ] . such a filter is capable of removing even finer particles, by bridging or sedimentation, before they reach and clog the original soil surface. this phenomenon will in fact largely be dom inant if only a portion of the suspended particles are larger than the pore openings. as soon as a few such particles have accumulated, they become the straining surface for finer particles [ ] . in studies in which e. coli suspended in distilled water was allowed to percolate into sand columns, krone [ ] found that after the first arrival of bacteria the concentration in column effluents continued to rise until a max imum was reached, after which it fell, suggesting that accumulating bacteria at the soil surface enhances the straining removal. this same effect is seen during the land application of domestic sewage when repeated cycles of flooding and drying of infiltration basins is practiced [ ] . for example, at the flushing meadows project near phoenix, arizona, treated sewage effluent is spread into basins underlaid with loamy sand. the greatest numbers of coliforms and fecal coliforms are observed after the start of each new inundation period when newly infiltrated water arrives at the bottom of sampling wells, after which time a general decrease in values occurs. a similar phenomenon occurs when water containing microorga nisms is pumped into recharge wells. studies using sandy soils of various effective porosities indicate removal of bacteria from a liquid percolating through a given depth of soil is inversely proportional to the particle size of the soil. the greatest removal of bacteria occurs on the surface mat (top - mm) that forms on the soil. adsorption is the major factor in the removal of viruses by soil and also plays a role in bacterial removal. factors that reduce the repulsive forces between the two surfaces, such as the presence of cations, would be ex pected to allow closer interaction between them and allow adsorption to proceed. the very small size of clays, their generally platy shapes, the oc currence of large surface area per given volume, make them ideal adsorption sites for bacteria and viruses in soils. thus, adsorption phenomena will play a more important role in the removal of microorganisms in soils that contain clays [ ] . many factors are known to control microbial adsorption to soils and these are listed in tables . and . . soils differ considerably in their textural, chemical, and mineralogical prop erties and hundreds of soil types have been classified in this country [ ] . furthermore, both vertical and horizontal variability is a normal character istic of many soils. it is generally agreed that fine-textured soils retain mi croorganisms more effectively than sandy soils since the soil clay mineral fraction displays a high sorptive capacity toward viruses as a result of its high surface area and ion-exchange capacity. following examination of nine [ ] . soils from arkansas and california, it was shown that virus adsorption in creased with the clay content and the specific surface area of the soil [ ] . iron oxides, particularly magnetite, also display a high affinity toward viruses [ ] . hori et al. [ ] found that polio virus removals from distilled water in -in columns of three hawaiian soils, including two low-humic latosols (lahaina and wahiawa) and a volcanic cinder (tantalus), averaged > , > , and percent, respectively. with the two low-humic latosols, there was a trend of decreased retention over the -day test period. goyal and gerba [ ] noted considerable differences in the abilities of nine different soils to adsorb a number of enteric viruses. statistical analysis indicated that ph was the most important soil characteristic influencing virus retention, with soils having a ph < giving consistently high retention. exchangeable alu minum was another factor that correlated with the adsorption efficiency of in contrast to these findings, wang et al. [ ] and lance et al. [ ] found greater removal of poliovirus in sandy soils than total and fecal coliforms, and fecal streptococcus. but the lowest removal was observed with coliphage f [ ] , indicating that virus type plays a significant role in the extent of virus removal. additional studies are needed on the relative re moval of bacteria and viruses by soil types. moore et al. [ ] recently reported that poliovirus type adsorption to different soil materials suspended in a synthetic freshwater was neg atively correlated with soil organic matter content and with available neg ative surface charge as measured by adsorption capacity for a cationic polyelectrolyte. soil ph, surface area, and elemental composition were not significantly correlated with virus adsorption. furthermore, additional stud-ies by this same group indicated that the two poorest adsorbents for both poliovirus and reovirus among different soil materials were a muck soil and a silt loam, both of which had high organic matter content [ ] . these authors were also able to show a highly negative correlation between virus adsorption and the capacity of soils to bind a cationic polymer, pdadm (polydiallyldimethyl ammonium chloride). it was suggested that the ability to bind the polymer could serve as an indicator of the extent of viral ad sorption [ ] . the results of these studies indicate that soil type greatly influences the extent of virus transport or retention. it may be possible to distinguish soils by general class with respect to virus retention, based on their textural, mineralogic, and chemical properties. however, further studies with a wide range of soil types and viruses are needed to determine if such classifications are possible and to identify the soil characteristics that most influence virus retention. the effects of ph on virus adsorption to soils are explainable on the basis of electrochemical features of virus and soil surfaces. the surface charge of a virus is influenced primarily by ionization of the carboxyl and amino groups on the outer surface of the virion protein capsid; and at neutral ph, most viruses are negatively charged. soils also tend to be generally electronega tive at neutral ph; therefore, virus adsorption is not favored due to repul sion of the two negatively charged surfaces. however, if the ph of the surrounding medium is lowered, protonation causes decreased ionization of virion carboxyl groups and increased ionization of amino groups. as a re sult, viruses become less electronegative or even electropositive at lower ph levels. although soil particles will also tend to become more electropositive at lower ph levels, the isoelectric points of soil particles are generally lower than those of viruses. for example, electrophoretic mobility studies have shown that a common soil clay mineral, montmorillonite, is negatively charged at ph . to . . muck soils also have a high negative charge [ ] . at lower ph levels, the viruses may be electropositive but the soils are still electronegative, thereby resulting in electrostatic attraction and increased adsorption. the relationship between virus adsorption and ph is not clearcut, however, because of many complicating factors. the ph of the soil, as conventionally measured, does not reflect necessarily the ph at the surface of soil colloidal particles such as clays. various soil components (clay, sand, oxides of aluminum and iron) display different isoelectric points. there is also a lack of information on the isoelectric points of more than viruses that occur in wastewater or groundwater. so far, we know that the isoelectric point varies with virus type and strain [ ] [ ] . the results of a number of studies indicate that virus retention by soils generally increases at lower ph levels. in an early report drewry and eliassen [ ] found decreased bacteriophage tl, t , and f adsorption to ar kansas and california soils at higher ph levels. more recently, burge and enkiri [ ] found that the rates of bacteriophage x adsorption to five soils were significantly correlated with soil ph. in batch adsorption studies with a variety of viruses and nine soils by goyal and gerba [ ] , ph was found to be the single most important soil factor influencing adsorption. soils having a saturated ph less than were the best adsorbers. studies by sobsey et al. [ ] showed that poliovirus type and reovirus type adsorp tion to eight different soil materials suspended in settled sewage at ph levels between . and . was generally greater at the lower ph levels. in studies by duboise et al. [ ] with cores of sandy forest soil receiving poliovirus in sewage effluent at various ph levels between . and . , virus retention was best at ph . , and the release and migration of retained viruses by subsequent distilled water applications was lower from the cores that re ceived sewage effluent having lower ph values. similar observations have been made for bacteria [ ] . the types and concentrations of ionizable salts in the soil-water environment greatly influence the extent of bacteria and virus transport. in general, in creasing concentrations of ionic salts and increasing cation valencies enhance virus adsorption. divalent cations (e.g., ca + , mg + ) are very efficient in promoting virus adsorption to a sandy soil [ ] . cations are necessary to reduce the repulsive forces on both the virus and soil particles and allow adsorption to take place. viral and bacterial retention by soils is generally greater in the presence of sewage effluents than in distilled water [ ] [ ] . wastewater effluents have indeed higher conductivity ( - fxmhos/ cm) than distilled water ( - fxmhos/cm) or rainwater ( - |xmhos/cm). rainwater, being of lower conductivity than sewage effluents, may thus lead to reduced viral and bacterial adsorption or to desorption with the subse quent redistribution of these organisms within the soil profile. this phenom enon was well demonstrated via soil core studies under controlled laboratory conditions [ , , , ] . landry et al. [ ] showed that virus penetration was more extensive in rainwater-rinsed cores than in wastewater-rinsed cores. moreover, the desorbed viruses may readsorb at greater depths. heavy rainfall might then remobilize soil-bound viruses with the potential contam ination of groundwater supplies [ ] . however, it now appears that the ability of rainwater to release viruses depends on the soil type, the release being more pronounced in sandy than in clay soils [ ] . the elution pattern also depends on the virus type and strain. for example, poliovirus and echovirus were mobilized by artificial rainwater, whereas echovirus was not affected. the elution pattern of the reference strain of poliovirus differed from that of field and mutant strains [ ] . rainfall will also effect bacterial retention by lowering ionic concen tration and increasing infiltration rates. several surveys have indicated that rainfall and well depth are related to microbial groundwater quality. studies in washington indicated that shallow drinking water wells average medium coliform values of mpn per ml with an average depth of . m ( ft), while deep wells with an average depth of . m ( ft) average mpn per ml [ ] . it was also observed that virtually all bacterial con tamination coincided with the periods of heaviest rainfall. brooks and cech [ ] observed in rural eastern texas that practically all dug wells with depths of ft ( m) or less were positive for either fecal coliforms or fecal strep tococci. while presence of fecal bacteria was much less common in deeper wells, some wells as deep as ft ( m) were positive. increased levels of bacterial contamination of drinking well water after periods of rain have been noted in several studies [ ] [ ] [ ] ] . in one study, it was noted that while an increase in coliform bacteria appears almost immediately after periods of heavy rainfall in shallow wells, in deeper wells the increase did not occur until weeks later [ ] . thus, any satisfactory study of well water quality should include sampling during periods of highest rainfall. soluble organic materials are known to compete with viruses and bacteria for adsorption sites. it may then be possible that organics present in sewage may interfere with virus sorption to soils. however, several studies have shown that viruses are well adsorbed to various types of soils in the presence of secondary and even primary wastewater effluents. as discussed above, wastewater effluents contain enough salts to overcome any interference by soluble organic matter. humic and fulvic acids are highly colored organic compounds that are naturally present in both water and soils. recent studies indicate that these compounds can cause increased virus transport through soils not only by interfering with virus adsorption but also by causing desorption. bitton et al. [ ] found that poliovirus retention by columns of sandy soil was exten sively reduced when applied in highly colored (high concentrations of humic and fulvic acids) cypress dome water compared to its retention from tap water. more recently, scheuerman et al. [ ] reported extensive interfer ence by humic and fulvic acids with poliovirus type retention in columns of organic sediment, muck soil, and brown-red sand. soils that were capable of retaining all or most of the applied virus in the absence of these organics retained considerably less virus in their presence. the extent of virus trans port through the columns correlated with the color of the column effluents. this phenomenon was confirmed by bixby and o'brien [ ] , who re ported that fulvic acids complex ms phage and prevent its adsorption to soil. more recently, such soils were found to display a lower adsorption capacity than other mineral soils [ , ] . the results of a number of studies suggest that organic soils and other soils or waters with high concentrations of humic and fulvic acids may not be suitable for land application of wastewater. the effects of other organics in waters and soils on virus retention remain uncertain. additional studies are needed to further understand and quantify the effects of humic and fulvic acids in water and soil on the infectivity and retention of a variety of viruses in different soils. such studies are also needed for other classes of water, wastewater, and soil organics. hydraulic conditions in soils receiving wastewater appear to have a consid erable effect on virus transport for at least some soils. such conditions as flow rate, hydraulic loading, and application frequency may all influence the extent of virus migration through soils. vaughn et al. [ ] reported that infiltration rate greatly influenced poliovirus removal in a groundwater re charge system where tertiary effluent was applied to a coarse sand-fine gravel soil. recharge at to cm per hour resulted in considerable virus movement into groundwater while at two lower recharge rates, and . - . cm per hour, there was considerably less virus movement. at the lower infiltration rates, the surface mat of sewage solids that formed on the soil surface may have contributed to the greater virus removals observed. lance et al. [ ] found that poliovirus type removal was not affected by infiltration rates in the range of to cm per day. more recently lance and gerba [ ] found that increasing flow rates from . to . m per day resulted in increased movement of viruses down the column. however, there was no further increase in virus movement at flow rates up to m per day. in comparative studies of several soils it was found that by linear regression analyses, the rate of virus removal in soil columns was negatively correlated with the flow rate of the percolating sewage effluent [ ] . the authors suggested that flow rate of water through the soil may be the most important factor in predicting the potential virus movement into groundwater. little virus movement has been observed in unsaturated soil columns [ ] . although the results of at least some studies suggest that virus migra tion increases with increasing hydraulic loads and flow rates and under con ditions of saturated flow, further studies are needed with a wide range of soil types and field conditions to quantify the extent of virus movement through soils under different hydraulic conditions. recent studies have shown that different types and strains of viruses are not equally retained by soils. these virus-specific differences in adsorption to soils are probably related to physicochemical differences in virus capsid surfaces. although all enteric viruses possess outer capsids comprised of polypeptide subunits and generally behave as charged, amphoteric, colloidal particles, the surfaces of the virions differ in the details of their configura tion, charge density and distribution, and other features. in fact, even the same virus can display different surface properties that will influence its physicochemical behavior as a result of conformational changes brought about by ph effects and interactions with soluble chemicals and particulate surfaces [ ] . goyal and gerba [ ] found that different enteric virus types and strains varied in their ability to adsorb to soils. for example, adsorption efficiencies of six different strains of echovirus type in suspensions of sandy soil in deionized water ranged from to . percent. type and strain dependence of enterovirus adsorption to a sandy loam soil suspended in distilled water was also reported in another study from the same laboratory [ ] . adsorp tion efficiencies of ten different virus types and strains ranged from percent for echovirus type , strain v , and coxsackie virus b , strain v , to . percent for echovirus type , wallace strain, and poliovirus type , strain lsc. landry et al. [ ] reported type and strain differences in enter ovirus adsorption to sandy soil cores. vaccine strain poliovirus type (lsc), a widely employed enterovirus model in soil and other environmental stud ies, was efficiently adsorbed but not readily eluted with either distilled water or sewage effluent. some of the other enteroviruses tested, including field strains, were less efficiently adsorbed and more easily eluted. it was con cluded that vaccine strain poliovirus type may be an inappropriate model for studying the nature and extent of virus transport in soils. in contrast to the findings from batch laboratory studies by the same group, hurst et al. [ ] found that under field conditions at a rapid infiltra tion site, echovirus type , farouk strain, did not migrate as far down in the soil as poliovirus type , strain lsc. they suggested that the adsorptive behavior of viruses in laboratory batch studies may not be totally reflective of their behavior under field conditions, possibly because of virus adsorption to soil particles prior to infiltration. it is now agreed that poliovirus type adsorbs well to most soils. it was recently concluded that viruses may be grouped into three categories according to their adsorptive behavior [ ] . category contains the poorly adsorbed viruses (echovirus , echovirus , coxsackie virus b , x , ms ) and category includes the highly adsorbed viruses (poliovirus , echovirus , coxsackie virus b , t , and t ). phage f was placed in a third category exhibiting the lowest adsorption of all viruses tested. at the turn of the century, it was found that the eating of raw vegetables grown on soil fertilized with raw sewage resulted in outbreaks of typhoid fever. as a result, the survival of enteric bacteria in soil systems has been extensively studied. there are several major reviews on the survival of en teric bacteria in soil [ ] [ ] [ ] , and we will only consider herein factors that affect the length of survival of these bacteria. less is known about virus survival. most enteric bacterial pathogens dieoff very rapidly outside of the human gut, whereas indicator bacteria such as e. coli will persist for longer periods of time. survival times among different types of bacteria and viruses vary greatly and are difficult to assess without studying each type individ ually. in most cases, it appears that to months is sufficient for reduction of pathogenic to negligible numbers once they have been applied to the soil, although survival times as long as years have been reported [ ] . factors known to influence bacterial and viral survival in the soil are listed in tables . and . . a major factor determining the survival of bacteria in soil is moisture. young and greenfield [ ] showed that moisture was a factor in the viability of e. coli in soils. beard [ ] stated that moisture was the most important deter mining factor in the survival of salmonella typhosa. bacterial survival was determined in various types of soil exposed outdoors in clay flowerpots. the survival in all types of soil tested was found to be greatest during the rainy season. in sand, where drying was rapid due to its low moisture-retaining power, survival time was short-between and days during dry weather. in soils that retain a high amount of moisture such as loam and adobe peat, the organisms persisted longer than days. bouma et al. [ ] have suggested that survival data for fecal organisms could be compared with soil-moisture characteristic curves, and hence the distance of soil filteration necessary for removal be defined as a function of moisture content. soil moisture also influences virus survival in soil. bagdasar'yan [ ] reported that enteroviruses survived three to six times longer in soils with percent moisture content than in air-dried soils. duboise et al. [ ] found that poliovirus type was inactivated considerably more rapidly in drying soil, as the moisture content decreased from to . percent, than in the same soil type maintained at or percent moisture content. inactivation of percent of the initial viruses occurred within week in drying soil but took - and - weeks in soils with to percent moisture content, respectively. yaeger and o'brien [ ] compared the degree of poliovirus inactiva- ? tion in eight different soils saturated with riverwater, groundwater, or septic wastewater and in the same soils that were allowed to dry out during the course of the experiment. upon drying, none of the initial viruses was de tectable in any of the dried soils (> . % inactivation), but considerable quantities were still present in the same types of saturated soils. in experi ments on the rate of poliovirus inactivation at different soil moisture levels, there was a sharp increase in the inactivation rate at . percent soil moisture compared to that at . percent. hurst et al. [ ] also observed differences in poliovirus inactivation rates at different soil moisture levels, with the greatest inactivation rate at a moisture level of percent. inactivation proceeded more slowly at both higher and lower moisture levels, but the slowest inactivation rates were at and percent. in a field study on virus survival in a rapid-infiltration system for wastewater, hurst et al. [ ] found that virus inactivation rates were greater in more rapidly drying soils. allowing soils in rapid-infiltration systems to pe riodically dry and become aerated between wastewater applications en hances virus inactivation. the effects of both drying and aerobic microbial activity may contribute to virus inactivation under these conditions. in stud ies on the mechanisms of virus inactivation in soils, yeager and o'brien [ ] found that the loss of poliovirus infectivity in moist and dried soils resulted from irreversible damage to the virus particles, including ( ) dis sociation of viral genomes and capsids, and ( ) degradation of viral rna. in both moist and dried nonsterile soils, viral rna was released from cap sids and found in a degraded form. in dried, sterile soils, viral rna was released but remained largely as intact molecules. viral capsid components were not readily recoverable from drying soils due to irreversible binding, but they could be recovered as empty capsids from moist soils. further experiments with dried viruses showed that their capsids became isoelectrically altered. the results of these studies suggest that poliovirus and perhaps other viruses are inactivated by different mechanisms in moist and drying soils. temperature is a major factor in the survival of enteric organisms in soil and other environments. temperature affects chemical and biologic pro cesses in soils, which may indirectly affect the survival of enteric viruses and bacteria. s. typhosa may survive as long as months at freezing temper atures [ ] . mirzoev [ ] pointed out that in areas with prolonged winterse.g., the russian arctic-the processes of soil self-disinfection are slowed down or suspended. he showed that low temperatures (down to - ° c) were very favorable for the survival of dysentery bacilli, which he was able to detect days after it had been added to the soil. van donsel et al. [ ] found that a percent reduction in the number of fecal coliforms took . days in the summer and . days in the winter in exposed soil plots. bagdasar'yan [ ] observed that viruses could survive up to days in soil at to ° c and that survival was higher at to ° c than at to ° c. similar observations were made by lefler and kott [ ] with regard to poliovirus type and bacteriophage f survival in a sandy soil in israel. yeager and o'brien [ ] found that coxsackie virus bl inactivation rates in sandy loam soils suspended in riverwater, groundwater, and septic wastewater increased as temperatures were increased from to ° c. in pilotscale outdoor studies on poliovirus persistence on vegetables and in soils irrigated with sewage effluent in cincinnati, ohio, larkin et al. [ ] and tierney et al. [ ] found that percent inactivation in soils took about months during the winter months and only to days in the warm summer months of june and july. in a field study by hurst et al. [ ] on virus survival and movement in a rapid-infiltration system for wastewater, the rate of inactivation of indigenous viruses was greater in the fall than in the winter, possibly due in part to the effects of higher temperatures in the former season. the direct effects of ionic salts and ph on microbial survival in soils have been less extensively investigated than their effects on virus retention by soils. hurst et al. [ ] determined that virus inactivation in soils correlated with soil levels of resin-extractable phosphorous, exchangeable aluminum, and soil ph. because these same factors also influence virus adsorption to soils, the observed differences in survival rates may be related to changes in the extent of virus adsorption to the soil material and, therefore, changes in the extent of virus protection from inactivation in the adsorbed state. beard [ ] also found that the death of s. typhosa was very rapid in peat soil with a ph between and . kligler [ ] found that moist, slightly alkaline soils were the most favorable for the survival of s. typhosa. cuthbert et al. [ ] inoculated various peat (ph . - . ) and limestone (ph . - . ) soils held in the laboratory with e. coli and strep, faecalis. they found that both organisms could persist for several weeks in the limestone soils, but would die out in a few days in acid peat soils. they felt that the low ph could act to adversely affect not only the viability of the organism but also the availability of nutrients or to interfere with the action of inhibiting agents. the frequent addition of broth culture fluid to soil has been found to in crease the survival of s. typhosa [ ] . under field conditions, it has been found that some aftergrowth of e. coli and strep, faecalis can occur, partic ularly after wet weather [ ] . the survival of fecal coliforms is greatly ex tended in organic soils over that observed in mineral soils [ ] . the extended survival and growth in organic soils may be due not only to the presence of organics but to the high moisture-holding capacity of these soils [ ] . the effects of organic matter on enteric virus survival in soils have not been established, but recent findings suggest that fulvic and humic acids may mask virus infectivity by a reversible process. bixby and o'brien [ ] found that fulvic acid complexation of bacteriophage ms caused consid erable loss of infectivity and prevented adsorption to soil. the infectivity of the complexed phage could be restored by treating with percent beef extract solution at ph . soil moisture, temperature, ph, and the availability of organic matter can also indirectly influence the survival of enteric bacteria by regulating the growth of antagonistic organisms [ ] . bryanskaya [ ] showed that actinomyces in soil were capable of suppressing the growth of salmonella and dysentery bacilli. in addition, the longer survival time of enteric organisms after inoculation into sterilized soil as compared to unsterilized soil found by a number of workers [ ] indicates that antagonism is an important fac tor. tate [ ] observed that the protozoan population of a muck soil in creased dramatically after addition of e. coli and suggested that soil protozoa could play a significant role in the decline of these organisms in these soils. since it is evident that enteric bacteria are capable of utilizing nutrients found in nature, it could be argued that competition by the natural soil microflora is in large part responsible for their eventual disappearance from the soil. bagdasar'yan [ ] noted greater enterovirus inactivation in nonsterile than in sterile sandy and loamy soils, incubated at - and - ° c. in more recent studies by sobsey et al. [ ] on rates of poliovirus and reovirus inactivation in eight different soil suspensions in settled sewage at ° c, the time required for percent inactivation was almost always shorter in nonsterile than in sterile suspensions. hurst et al. [ ] observed increased inactivation of poliovirus and echovirus in nonsterile sandy soil wetted with distilled water and incubated under aerobic conditions at ° and ° c, compared to sterile control samples. however, inactivation rates in sterile and nonsterile samples were similar at ° c under aerobic conditions and at °, °, and ° c under anaerobic conditions. thus, appreciable virus in activation due to microbial activity in soils appears to occur only under aerobic conditions and at moderate to high temperatures. although the mechanisms of microbially mediated antiviral activity in soils have not been fully elucidated, yaeger and o'brien [ ] have reported differences in poliovirus structural changes during inactivation in sterile and nonsterile soils depending on soil moisture level. in both sterile and non sterile soils under moist conditions, viral rna was probably damaged be fore release from capsids. in sterile, dried soils released rna genomes remained largely intact, but in nonsterile, dried soils the released rna was degraded. the role of microbially produced nucleases in these findings is uncertain. data available indicate that viruses survive longer than bacteria in soil (ta ble . ) [ ] . field and laboratory studies using mcfeters's-type survival chambers indicate that enteric bacteria can survive from a few days to more [ ] . than a month [ ] [ ] . it is also possible that under some conditions they could regrow in groundwater if sufficient nutrients are present. e. coli bac teria have been found to survive and even multiply on organic matter filtered out from lake water during underground recharge projects in israel [ ] . in some areas of israel surface water during the rainy season is used to recharge the underground water supply. during those parts of the year when there is an increased need for water the same wells transformed to pumping wells. during such projects it was found that although the water pumped under ground contained less than coliforms per ml after chlorination, the repumped water contained counts as high as - per ml, which persisted for long periods of time after the initiation of pumping. subsequent studies showed that organic matter that had accumulated in the sand around the well casing enabled the regrowth of the few remaining coliforms. also of interest was the finding that so long as recharge continued, the bacteria did not multiply; it was only during the period between recharge and pump ing that growth occurred [ ] . enteroviruses have been detected at the surface of soils irrigated with sewage in the united states [ ] . a field study revealed virus survival for at least days in soil following application of a package treatment plant effluent in a cypress dome in gainesville, florida [ ] . other field studies confirmed the important role played by temperature and soil moisture in virus persistence in soils [ , ] . similarly, it appears that virus survival in sludge-amended soils is controlled primarily by desiccation and soil tem perature [ , ] . during surface application of digested sludge on soils in pensacola, florida, it was shown that indigenous enteroviruses were able to survive only days after sludge application [ ] . a simple conceptual model based on the current state of knowledge on indicator and pathogen dieoff has been described by reddy et al. [ ] . microbial dieoff was described by assuming first-order kinetics. first-order dieoff rate constants (k) were calculated from the literature for enteric mi crobial dieoff in soil-water systems. correction factors were presented to adjust constants for changes in temperature, moisture, and ph of the soil. average dieoff rate constants (log () /day _ ) for selected microorganisms are shown in table . . in the article by reddy et al. [ ] , data on dieoff of viruses during anaerobic digestion were used. only data on virus dieoff in soil systems is shown in table . . these values were obtained from various experiments and represent an average value of several soil and environ mental variables. such an approach could prove useful for estimating mi crobial survival in soil-water systems, but a greater database is needed especially for viruses and other pathogenic bacteria. also, most of our da tabase on microbial survival is in soil-water systems and not in groundwater. even though there have been no reports of disease outbreaks associated with land treatment of wastewater, there are a growing number of studies concerning the detection of viruses in groundwater after wastewater appli cation to land or direct groundwater recharge. these studies are summarized in table . . wellings et al. [ ] demonstrated vertical and lateral movement of virus in secondary effluent discharged into a cyprus dome (a wetland eco system). poliovirus , coxsackievirus b , and echoviruses , , and were recovered from m-deep wells in three of samples, at concentrations ranging from to pfu. viruses migrated to m laterally from the application point and survived at least days. the soil at this site ranged from a top . m layer of black organic soil ( - % clay) to a sandy clay and a solid blue clay with a permeability of x " cm per minute to x ~ cm per minute. thus, the viruses moved horizontally as well as vertically and survived many days under ambient conditions, indicating a necessity to evaluate such sites for their aquifer movement and transmission of viruses to drinking water sources. in an earlier study, wellings et al. [ ] recovered viruses from groundwater after spray irrigation of secondary sewage effluent onto an imolokee sand (little or no silt or clay). of particular interest in this study was that viruses survived chlorination, sunlight, spraying, and percolation through to m of sandy soil; furthermore, after a period of heavy rains, a burst of viruses was detected in samples that had previously been negative. these studies demonstrate that soil type, rainfall, and other factors can affect viral movement into groundwater, and that viruses are capable of surviving long periods-which, when combined with the ability to move long distances laterally, could lead to wide dispersal through an aquifer. vaughn and landry [ ] and vaughn et al. [ ] reported isolations of viruses from four groundwater recharge sites, from a stormwater recharge basin, and from groundwater under a sanitary landfill in new york. these sites have soils of coarse sand, fine gravel, and to percent silt. at the groundwater recharge sites, viruses were recovered at depths up to . m and at distances up to . m from the injection point of secondary or tertiary chlorinated effluent. as much as to percent of the -gal samples at the four sites were positive for viruses, with concentrations of . to . pfu per gallon. in addition, total coliforms were found in these samples. in order to reach the groundwater, viruses moved through . to m of the overlying soil. moreover, at the pines site, viruses were discovered in groundwater under basins where effluent seeded with viruses was applied at rates of to cm per hour. the slower infiltration rates were more effective in re moving the viruses, suggesting that site management is important. both the landfill and stormwater recharge basin also yielded viruses. at the landfill stie, viruses were detected at depths of . m and up to m downstream. coxsackievirus b and other unidentified viruses were de tected. at the stormwater recharge site, samples taken at -m depths directly in the basin were positive for echoviruses and and for coxsackievirus a . this contamination may have originated from runoff from cesspools in the area. schaub and sorber [ ] reported on a study of viruses in groundwater under rapid infiltration cells at ft. devens, massachusetts. the soil consisted of silty sand and gravel underlaid by bedrock. the groundwater contained viruses at depths of m and lateral distances of m, with concentrations of about . percent of the applied effluent. fecal streptococcal bacteria were also found in the . m-deep well. marker f virus was applied at this same site; only about percent of the virus was removed, and it was detectable for at least days. this site was deemed to have poor filtration properties, which points out the need for site-specific evaluation. at the vineland, new jersey, rapid-infiltration site [ ] primary ef fluent was applied to cohansey sand and coarse gravel. viruses were found at . m depths and up to m lateral distances in of samples. polio-, echo-, and coxsackie viruses were identified. total coliforms and fecal coliforms were found consistently at depths up to m beneath the recharge basins. total coliforms also occasionally occurred at greater depths and downstream. in contrast, no fecal coliforms were found in samples taken below . m and coliforms occurred only once in a shallow well m downgradient. thus, viruses penetrated deeper into the ground and moved longer distances than did the coliforms. the potential for viruses to migrate great distances, as in the previous study, was further demonstrated by noonan and mcnabb [ ] , who used the phages x and t to demonstrate lateral movements of m and m, respectively, in new zealand groundwater in just hours. the viruses moved at greater than m per day and survived for at least days. in laboratory studies, . days were necessary for a percent reduc tion in liter; so in this case, the viruses could theoretically travel at least . km in groundwater before a percent reduction could be effected under these conditions. viruses in groundwater at other recharge sites have been studied with varying success. at the flushing meadows site near phoenix, arizona [ ] , it was found that a fine loamy sand over coarse sand and gravel effectively removed viruses. laboratory studies confirmed that this soil was an excellent adsorber. no viruses were detected in any of the samples of renovated water, even though levels of to pfu per liter were detected in the effluent applied. however, coliform organisms were detected in the reno vated water, suggesting that the removal mechanisms must have been dif ferent for viruses and bacteria, and that viruses may have been present. since this site is no longer in existence, these findings cannot be confirmed. however, since then, virus has been detected in a sample from an . mdeep well at a nearby land application site. the isolate was identified as coxsackievirus b . at two land treatment sites where sewage is used to irrigate cropland, both positive and negative virus isolations have been made [ , ] . at the lubbock, texas, site, coxsackievirus b was isolated from a depth of . m; at roswell, new mexico, no virus isolates were detected in samples taken from to m depths. in the latter case, irrigation is seasonal and inter mittent, whereas application at the lubbock site is continuous. at an operational land application site in kerrville, texas [ ] , no viruses were detected in the monitoring wells at depths of . to . m even though viruses could be detected in . m-deep lysimeters. in one often-cited report [ ] on the santee project, no viruses were detected in renovated water. this is not surprising, since the detection meth ods available at that time were not quantitative. these negative results must therefore be considered highly questionable, as should the results obtained at the whittier narrows, california [ ] , projects, which did not employ techniques sensitive enough to detect low levels of virus. this situation reiterates the need for careful evaluation of methods used in any report before negative conclusions are accepted. summaries of data on the soil penetration of bacteria at some of the most important rapid-infiltration systems land treatment sites are presented in table . . the data suggest that bacteria at rapid-infiltration sites may pen etrate about m vertically and variable distances laterally. these distances are, of course, highly site-specific, and the vertical distance may be more than m but is usually much less. to prevent the entry of enteric bacteria into groundwater, it would thus be advisable (unless an underdrain system is installed) not to site land treatment systems where the water table is shallow, particularly if the soil is sandy or gravelly, large cracks or root tunnels are present, or a thin soil mantle overlies rock with solution channels or fissures. this is especially true for rapid-infiltration systems. once in the groundwater, the bacteria may travel long distances in situations where coarse soils or solution channels are present, but normally the filtering action of the matrix should restrict horizontal travel to only a few hundred feet. the actual distance travelled also depends on the rate of movement of the groundwater and the survival time of the bacteria. the rate of movement of groundwater is highly site-specific but often is ex tremely slow. from the foregoing discussion, it is apparent that many factors control the removal of pathogenic bacteria and viruses during the percolation of sewage through the soil. most of this chapter has dealt with the fate of viruses in soil because of their apparent greater potential for health problems associ ated with land treatment. although the presence of viruses in groundwater has been demonstrated, it would appear that with proper site selection and management the presence of viruses could be minimized or eliminated. the key is to define the processes involved in the survival and transport of pathogens in groundwater. with proper design, land treatment could be used as an effective method for reducing the number of pathogens in wastewater. with the proper soil type, viruses and bacteria can be reduced to levels as effectively as by chlorination as currently practiced, after the travel of wastewater through only a few centimeters of soil. as we have shown, high removals by soil can be achieved from even raw wastewater. in the soil natural processes will eventually destroy the pathogens. thus, in groundwater recharge operations, the soil should be considered as part of the treatment process and not simply as a final disposal source. the key to operating such systems for pathogen removal is to gain an understanding of the processes involved and methods by which they can be quantified and controlled. based on both field and laboratory experiments, several potential treatment practices may be useful in enhancing virus removal during land application of sewage, and these are summarized in table . . [ ] . waterborne disease-a status report emphasizing outbreaks in groundwater review of the causes of waterborne disease outbreaks out breaks of waterborne disease in the united states viruses in soil systems magnitude of the groundwater contamination problem wastewater bacteria and viruses in soil virus movement in soil col umns flooded with secondary sewage effluent adsorp tion of enterovirus to soil cores and their subsequent elution by artificial rainwater demon stration of virus in groundwater after effluent discharge into soil viruses in groundwater waterborne disease: occurrence is on the upswing bacterial contamination of drinking water supplies in a mod ern rural neighborhood a review of infective dose data for enteroviruses and other enteric microorganisms in human subjects public health implications of the appli cation of wastewaters to land gastroenteritis association with a sewage leak health effects of land treatment; microbiological viruses in water: the problem, some solutions virus survival in wastewater treatment viruses in sewage: effect of phosphate removal with calcium hydroxide (lime) appropriate technology for water supply and sanitation: health aspects of excreta and sillage management-a state of the art review behavior and transport of microbial pathogens and indicator organisms in soils treated with organic wastes well-water quality deteri oration in central pierce county, washington the relationship between rainfall and well water pollution in a west african (gambian) village the pollution hazard to village water supplies in eastern botswana the movement of disease producing organisms through soils high-rate land treatment. ii. water quality and economic aspects of the flushing meadows project fundamentals of soil science virus movement in groundwater adsorption of viruses onto surfaces in soil and water migration of poliovirus type in percolating water through selected oahu soils comparative adsorption of human enteroviruses, simian rotavirus, and selected bacteriophages to soils virus adsorption by five soils interactions and survival of enteric viruses in soil materials comparative movement of dif ferent enteroviruses in soil poliovirus adsorption by minerals and soils influence of ph and electro lyte composition on adsorption of poliovirus by soils and minerals quantitative assess ment of the adsorptive behavior of viruses to soils poliovirus survival and move ment in a sandy forest soil effect of dissolved salts on the filtration of coliform bacteria in sand dunes virus survival in water and wastewater systems poliovirus retention in -cm soil cores after sewage and rainwater application nitrates and bacterial distribution in rural do mestic water supplies bacterial contamina tion of drinking water supplies in a modern rural neighborhood hohe nitratgehalte in einem landlichen gebiet in nigeria verursacht durch ungeordnete ablagerung hauslicher alofalle and exkremente poliovirus removal from primary and sec ondary sewage effluent by soil filtration effect of secondary treated effluent on the movement of viruses through a cypress dome soil transport of viruses through organic soils and sediments influence of fulvic acid on bacteriophage adsorption and complexation in soil virus re moval during groundwater recharge: effects of infiltration rate on adsorp tion of poliovirus to soil poliovirus movement during high rate land filtration of sewage water effect of soil permeability on virus removal through soil columns enterovirus inactivation in soil type and strain dependence of enterovirus adsorption to activated sludge, soils, and estuarine sediments survival of enteroviruses in rapid-infiltration basins during the land application of wastewater the use of sewage for irrigation: a literature review literature review on the occurrence and survival of enteric, pathogenic and relative organisms in soil, water, sewage and sludges, and on vegetation recycling treated municipal wastewater and sludge through forest and cropland observations on the viability of the bact. coli group under natural and artificial conditions longevity of eberthella tyhosus in various soils university of wiscon sin-extension geological and natural history survey survival of viruses of the enterovirus group (poliomy elitis, echo, coxsackie) in soil and on vegetables infectious disease potential of land application of wastewater effects of environmental variables and soil characteristics on virus survival in soil extent of survival of dysentery bacilli at low temperatures and self-disinfection of soil and water in the far north seasonal variations in survival of indicator bacteria in soil and their contribution to storm-water pollution persistence of virus on sewage-irrigated vegetables persistence of poliovirus in soil and on vegetables grown in soil previously flooded with inoculated sewage sludge or effluent investigations of soil pollution and the relation of the various types of privies to the spread of intestinal infections survival of bacterium coli type and streptococcus faecalis in soil cultural and environmental factors affecting the longevity of escherichia coli in histosols antagonistic effect of actinomyces on pathogenic bac teria in soil virus survival in receiving water comparative survival of indicator bacteria and enteric pathogens in well water survival of enteric viruses and indicator bacteria in groundwater water quality aspects of groundwater recharge in israel clogging and contamination processes in re charge wells viruses in groundwater be neath sewage irrigated cropland high levels of microbial contamination of vegetables irrigated with wastewater by the drip method fate of vi ruses following land application of sewage sludge. i. survival and transport patterns in core studies under natural condition survival of enteroviruses and coliform bacteria in a sludge lagoon virus survival following wastewater spray irrigation of sandy soils an assessment of the occurrence of hu man viruses in long island aquatic systems survey of human virus occurrence in wastewater-recharged groundwater on long island virus and bacteria removal from wastewater by rapid infiltration through soil long-term effects of land application of domestic wastewater the quality and movement of groundwater in alluvial aquifers of virus and bacteria removal from wastewater by land treatment viral transport to groundwater at a wastewater land application site virologic assess ment of sewage treatment at water reclamation at whittier nar rows groundwater pollution microbiology virus removal following wastewater spray irrigation of sandy soils long-term recharge of trickling filter effluent into sand dan region project, groundwater recharge with municipal effluent virus removal with land filtration key: cord- -f khcjdy authors: lópez, alfonso; martinson, shannon a. title: respiratory system, mediastinum, and pleurae date: - - journal: pathologic basis of veterinary disease doi: . /b - - - - . - sha: doc_id: cord_uid: f khcjdy nan diseases of the respiratory system (respiratory apparatus) are some of the leading causes of morbidity and mortality in animals and a major source of economic losses. thus veterinarians are routinely called to diagnose, treat, and implement health management practices to reduce the impact of these diseases. in companion animals, diseases of the respiratory tract are also common and, although of little economic significance, are important to the health of the animals and thus to clinicians and pet owners. in the past few years, animal shelters have been recognized as a major risk factor for respiratory diseases in dogs and cats, a comparable situation to what is reported in human beings with nosocomial infections. to facilitate the understanding of the structure and function, it is convenient to arbitrarily divide the respiratory system into conducting, transitional, and gas exchange systems ( fig. - ). the conducting system includes nostrils, nasal cavity, paranasal sinuses, nasopharynx, larynx, trachea, and extrapulmonary and intrapulmonary bronchi, all of which are largely lined by pseudostratified, ciliated columnar cells, plus a variable proportion of secretory goblet (mucous) and serous cells (figs. - and - and e- fig. - ). the transitional system of the respiratory tract is composed of bronchioles, which are microscopic structures that serve as a transition zone between the conducting system (ciliated) and the gas exchange (alveolar) system (see fig. - ). the disappearance of cilia in the transitional system is not abrupt; the ciliated cells in the proximal bronchiolar region become scarce and progressively attenuated, until the point where distal bronchioles no longer have ciliated cells. normal bronchioles also lack goblet cells but instead have other types of secretory cells, notably club cells (formerly clara cells) and neuroendocrine cells. club cells, also referred to as secretory bronchiolar cells, contain numerous biosynthetic organelles that play an active role in detoxification of xenobiotics (foreign substances), similar to the role of hepatocytes ( fig. - ) . club cells are also critical stem cells in the repair and remodeling of not only the bronchioles but also of most of the respiratory tract. in addition, club cells contribute to the innate immunity of the lung by secreting protective proteins (collectins) and pulmonary surfactant (see b) . in carnivores and monkeys, and to a much lesser extent in horses and human beings, the terminal portions of bronchioles are lined not only by cuboidal epithelium but also by segments of alveolar capillaries. these unique bronchioloalveolar structures are known as respiratory bronchioles ; also see fig. - ). the gas exchange system of the respiratory tract in all mammals is formed by alveolar ducts and millions of alveoli ( fig. - ; also see fig. - ). the surface of the alveoli is lined by two distinct types of epithelial cells known as type i (membranous) pneumonocytes and type ii (granular) pneumonocytes ( fig. - ) . all three-the conducting, transitional, and exchange systems of the respiratory system-are vulnerable to injury because of constant exposure to a myriad of microbes, particles and fibers, and toxic gases and vapors present in the air. vulnerability of the respiratory system to aerogenous (airborne) injury is primarily because of ( ) the extensive area of the alveoli, which are the interface between the blood in alveolar capillaries and inspired air; ( ) the large volume of air passing continuously into the lungs; and ( ) the high concentration of noxious elements that can be present in the air (table - ). for human beings, it has been estimated that the surface of the pulmonary alveoli is approximately m , roughly the area animal and cultured for microbes, yeasts, and fungi, many species of bacteria are recovered, such as mannheimia (pasteurella) haemolytica in cattle; pasteurella multocida in cats, cattle, and pigs; and bordetella bronchiseptica in dogs and pigs. the organisms that constitute the normal flora of the respiratory tract are restricted to the most proximal (rostral) region of the conducting system (nasal cavity, pharynx, and larynx). the thoracic portions of the trachea, bronchi, and lungs are considered to be essentially sterile. the types of bacteria present in the nasal flora vary considerably among animal species and in different geographic regions of the world. some present in the nasal flora are pathogens that can cause important respiratory infections under some circumstances. for instance, mannheimia (pasteurella) haemolytica is part of the bovine nasal flora, yet this bacterium causes a devastating disease in cattle-pneumonic mannheimiosis (shipping fever). experimental studies have established that microorganisms from the nasal flora are continuously carried into the lungs via tracheal air. despite this constant bacterial bombardment from the nasal flora and from contaminated air, normal lungs remain sterile because of their remarkably effective defense mechanisms. the conducting portion of the respiratory system is lined by pseudostratified columnar ciliated epithelium (most of the nasal cavity, paranasal sinuses, part of the larynx, and all of the trachea and bronchi), olfactory epithelium (part of the nasal cavity, particularly ethmoidal conchae), and squamous epithelium (nasal vestibulum and parts of the larynx). the pattern of injury, inflammation, and host response (wound healing) are characteristic for each of these three types of epithelium independent of its anatomic location. pseudostratified ciliated epithelium, which lines most of the nasal cavity and nasopharynx, part of the larynx, and all of the trachea and bronchi, is exquisitely sensitive to injury. when these cells are irreversibly injured, whether caused by viral infection, trauma, or inhalation of toxic gases, the ciliated cells swell, typically lose their attachment to underlying basement membrane, and rapidly exfoliate ( fig. - ) . a transient and mild exudate of fluid, plasma proteins, and neutrophils covers the ulcer. in the absence of of a tennis court. the alveolar surface of the equine lung is estimated to be approximately m . it has also been estimated that the volume of air reaching the human lung every day is approximately l. lungs are also susceptible to blood-borne (hematogenous) microbes, toxins, and emboli. this fact is not surprising because the entire cardiac output of the right ventricle goes into the lungs, and approximately % of the total blood volume is within the pulmonary vasculature. the pulmonary capillary bed is the largest in the body, with a surface area of m in the adult human; this area is equivalent to a length of km of capillaries, with ml of blood occupying up to km of capillary bed. the respiratory system has its own normal flora (microbiota), as does any other body system in contact with the external environment. if a sterile swab is passed deep into the nasal cavity of any healthy (rhinoviruses), infectious bovine rhinotracheitis (bovine herpesvirus ), feline rhinotracheitis (felid herpesvirus ), and viruses of the canine infectious respiratory disease (cird) group such as canine adenovirus (cav- ) and canine parainfluenza virus (cpiv) . if damage to the mucociliary blanket becomes chronic, goblet cell hyperplasia takes place, leading to excessive mucus production (hypersecretion) and reduced mucociliary clearance, and when there is loss of basement membrane, repair is by fibrosis and granulation tissue (scarring). in the most severe cases, prolonged injury causes squamous metaplasia, which together with scarring causes airway obstruction and an impediment to mucociliary clearance. in laboratory rodents, hyperplastic and metaplastic changes, such as those seen in nasal polyps and squamous metaplasia, are considered a prelude to neoplasia. the second type of epithelium lining the conducting system is the sensory olfactory epithelium, present in parts of the nasal mucosa, notably in the ethmoidal conchae. the patterns of degeneration, exfoliation, and inflammation in the olfactory epithelium are similar to those of the ciliated epithelium, except that olfactory complications or secondary bacterial infections, a specific type of progenitor cells known as basal cells or nonciliated secretory cells (preciliated cells) , which are normally present in the mucosa, migrate to cover the denuded basement membrane and undergoes mitosis, eventually differentiating into new ciliated epithelial cells (see fig. - ). cellular migration, proliferation, and attachment are regulated by locally released interleukins (il- β, il- , il- , and il- ), growth factors, integrins and extracellular matrix (ecm) proteins such as collagen, and fibronectin. the capacity of ciliated epithelium to repair itself is remarkably effective. for example, epithelial healing in an uncomplicated ulcer of the tracheal mucosa can be completed in only days. this sequence of cell degeneration, exfoliation, ulceration, mitosis, and repair is typically present in many viral infections in which viruses replicate in nasal, tracheal, and bronchial epithelium, causing extensive mucosal ulceration. examples of transient infections of this type include human colds epithelium. neurons in the olfactory mucosa have the unique ability to regenerate, a fact that is being explored as a potential source of new neurons in the treatment of spinal cord injury. squamous epithelium, located in the vestibular region of the nose (mucocutaneous junction), is the third type of epithelium present in the nasal passages. compared with ciliated and olfactory epithelia, nasal squamous epithelium is quite resistant to all forms of injury. the pharyngeal mucosa, composed of squamous epithelium, has similar patterns of necrosis and inflammation as the oral mucosa (see chapter ). the patterns of necrosis, inflammation, and repair in intrapulmonary bronchi are similar to those previously described for the nasal and tracheal epithelium. in brief, injury to ciliated bronchial epithelium may result in degeneration, detachment, and exfoliation of necrotic cells. under normal circumstances, cellular exfoliation is promptly followed by inflammation, mitosis, cell proliferation, cell differentiation, and finally by repair ( fig. - and see . depending on the type of exudate, bronchitis can be fibrinous, catarrhal, purulent, fibrinonecrotic (diphtheritic), and sometimes granulomatous. when epithelial injury becomes chronic, production of mucus is increased via goblet cell hyperplasia (chronic catarrhal inflammation). this form of chronic bronchitis is well illustrated in habitual smokers who continually need to cough out excessive mucus secretions (sputum). unfortunately, in some cases, excessive mucus cannot be effectively cleared from airways, which of the bronchial wall or cylindrical when destruction involves a large segment of a bronchus. grossly, bronchiectasis is manifested by prominent lumps in the lungs (bosselated appearance or having rounded eminences) resulting from distention of bronchi with exudate, which results in a concurrent obstructive atelectasis of surrounding parenchyma ( fig. - ). the cut surfaces of dilated bronchi are filled with purulent exudates; for this reason, bronchiectasis is often mistaken for pulmonary abscesses. careful inspection, usually requiring microscopic examination, confirms that exudate is contained and surrounded by remnants of a bronchial wall lined by squamous epithelium and not by a pyogenic membrane (connective tissue) as it is in the case of a pulmonary abscess. the squamous metaplasia further interferes with the normal function of the mucociliary escalator. the epithelial lining of the bronchiolar region (transitional zone) is exquisitely susceptible to injury, particularly to that caused by some respiratory viruses (bovine parainfluenza virus , bovine respiratory syncytial virus, adenovirus, or canine distemper virus), oxidant gases (nitrogen dioxide [no ], sulfur dioxide [so ], or ozone [o ]), and toxic substances ( -methylindole or paraquat). the precise explanation as to why bronchiolar epithelium is so prone to injury is still not clear, but it is presumably due in part to ( ) its high vulnerability to oxidants and free radicals; ( ) the presence of leads to chronic obstructive bronchitis and emphysema (see . chronic bronchial irritation causes squamous metaplasia of highly functional but vulnerable ciliated epithelium to nonfunctional, but more resistant, squamous epithelium. squamous metaplasia has a calamitous effect on pulmonary clearance because it causes a structural loss and functional breakdown of portions of the mucociliary escalator. hyperplasia of bronchial glands occurs frequently in chronic bronchitis, which translates to an increase of the reid index (bronchial-gland to bronchial-wall ratio) (e- fig. - ). this index is less than % in the healthy human lung and in the lungs of most domestic species, except for cats, which generally have an index higher than %. the term airway remodeling encompasses all the structural changes that accompany chronic bronchitis such as hypertrophy and hyperplasia of smooth muscle, submucosal glands, and goblet cells; fibrosis; and increased bronchial vascularity. bronchiectasis is one of the most devastating sequelae to chronic remodeling of the bronchi. it consists of a pathologic and permanent dilation of a bronchus with rupture of the bronchial wall as a result of obstruction or chronic inflammation. destruction of walls occurs in part when proteolytic enzymes and oxygen radicals released from phagocytic cells during chronic inflammation degrade and weaken the smooth muscle and cartilage (chondromalacia) that help to maintain normal bronchial diameter ( fig. - ). bronchiectasis may be saccular when destruction affects only a small localized portion . this same type of lesion is seen in viral or mechanical injury to the mucosa of the conducting system. two days after exposure, the basement membrane is lined by rapidly dividing preciliated cells, some of which exhibit mitotic activity (inset). ten days after injury, the nasal epithelium is completely repaired. h&e stain. b, schematic representation of the events of injury and repair in the respiratory mucosa of the conducting system. blue cell, ciliated mucosal epithelial cell; pink cell, goblet cell; red cell, neutrophil. (a from lópez a, prior m, yong s, et al: am j vet res : - , into well-organized, microscopic polyps inside the bronchiolar lumen. the external surface of the exudate eventually becomes covered by ciliated cells. this lesion is referred to as bronchiolitis obliterans, and the polyps may become so large as to cause airflow impairment ( fig. - and see fig. - ). in mild but persistent bronchiolar injury, goblet cells normally absent from bronchioles proliferate from basal cells, resulting in goblet cell metaplasia and causing a profound alteration in the physicochemical properties of bronchiolar secretions ( fig. - ). the normally serous bronchiolar fluid released by club (clara) cells becomes a tenacious material when mucus produced by goblet cells is added. as a result of increased viscoelasticity of the mucus, bronchiolar secretions cannot be removed effectively by ciliary action, leading to plugging and obstruction of distal airways. under such conditions, often grouped as chronic obstructive pulmonary disease, coughing is required to clear mucus from obstructed bronchioles. pulmonary emphysema and atelectasis are further sequelae to bronchiolar metaplasia and mucous hypersecretion blocking or partially blocking the lumens of these bronchioles. these two inflation abnormalities are characteristically present in chronic obstructive pulmonary disease (copd), which is called "recurrent airway obstruction (rao or "heaves") in horses (see recurrent airway obstruction, under disorders of horses). peribronchiolar club (clara) cells rich in mixed function oxidases, which locally generate toxic metabolites (see fig. - ); and ( ) the tendency for pulmonary alveolar macrophages and leukocytes to accumulate in this region of the lungs. depending on the types of injury and inflammatory response, bronchiolitis is classified as necrotizing, suppurative, catarrhal (mucous metaplasia), or granulomatous. once injury to bronchiolar ciliated cells becomes irreversible, the cells degenerate and exfoliate into the bronchiolar lumen, leaving a denuded basement membrane. repair in the bronchiolar region is similar to, but less effective than, that in the tracheal or nasal mucosa. under normal circumstances, recruited phagocytic cells remove exudate and cell debris from the lumina of affected bronchioles, thus preparing the basement membrane to be repopulated with new, undifferentiated cells originating from a rapidly dividing pool of club (clara) cells. after several days, these proliferating cells fully differentiate into normal bronchiolar cells. in severe acute injury, such as that caused by aspiration pneumonia or by highly pathogenic microorganisms, exudate attaches and cannot be removed from the basement membrane of bronchioles. the exudate becomes infiltrated by fibroblasts, which form small nodular masses of fibrovascular tissue that develop postviral bronchiolitis is associated with increased expression of tlrs and unusual susceptibility to inhaled endotoxin. hyperreactive animals typically have an increased number of mast cells, eosinophils, and t lymphocytes in the airway mucosa. clinically, airway hyperresponsiveness is characterized by an exaggerated bronchoconstriction after natural exposure to mild stimuli, such as cold air, or after animals are experimentally exposed to aerosols of histamine or methacholine. because of their extremely delicate structure, alveoli are quite vulnerable to injury once the local defense mechanisms have been overwhelmed. the alveolar wall is a thin membrane formed by a core of interstitium supporting an extensive network of alveolar capillaries. fibroblasts (septal cells), myofibroblasts, collagen, elastic fibers, and few interstitial macrophages and mast cells constitute the alveolar interstitium. the wall of the alveolar capillaries facing the airspace is remarkably thin and has three layers composed of vascular endothelium, basal lamina, and alveolar epithelium. these three layers of the alveolar capillaries constitute what is customarily referred to as the blood-air barrier (see fig. - ). the epithelial side of the alveolus is primarily lined by rather thin type i proliferation of lymphocytes (balt hyperplasia) is also a common microscopic lesion seen in chronic bronchiolitis. airway hyperresponsiveness, or hyperreactive airway disease, is another sequela of bronchiolar injury arising from gene-environment interactions. it develops in human beings and animals (experimentally) after a transient and often innocuous viral infection of the lower respiratory tract or from exposure to certain allergens. experimental work has shown that airway hyperreactivity in club (clara) cell edema. alveolar repair is possible as long as the basement membrane remains intact and lesions are not complicated by further injury or infection. within days, cuboidal type ii (granular) pneumonocytes, which are the precursor cells and more resistant to injury, undergo mitosis and provide a large pool of new undifferentiated cells . these new cells repave the denuded alveolar basement membrane and finally differentiate into type i pneumonocytes. when alveolar injury is diffuse, proliferation pneumonocytes, which are arranged as a very delicate continuous membrane extending along the alveolar surface (see fig. - ). type i pneumonocytes are particularly susceptible to noxious agents that reach the alveolar region either aerogenously or hematogenously. injury to type i pneumonocytes rapidly causes swelling and vacuolation of these cells . when cellular damage has become irreversible, type i cells detach, resulting in denudation of the basement membrane, increased alveolar permeability, and alveolar figure - hyperplasia of type ii pneumonocytes. a, acute alveolar injury, crude oil aspiration, cow. note proliferation of cuboidal epithelial cells (type ii pneumonocytes) (arrows) along the luminal surface of the alveolar wall. during alveolar repair, type ii pneumonocytes are the precursor cell for necrotic and lost type i pneumonocytes. h&e stain. b, chronic alveolar injury, interstitial pneumonia, horse. note entire alveolar membrane lined with cuboidal type ii pneumonocytes (arrowheads). the alveolar interstitium is expanded with inflammatory cells, and the alveolar lumens contain cell debris mixed with leukocytes. h&e stain. ( ( ). necrosis of these cells leads to transient alveolar edema (area that is pink) ( ), which is followed by hyperplasia of type ii pneumonocytes ( ), stem cells that differentiate ( ) into type i pneumonocytes as part of alveolar repair and healing ( ). (courtesy dr. a. lópez, atlantic veterinary college.) more information on postmortem examination of the lung can be found at www.expertconsult.com. information on this topic is available at www.expertconsult.com. information on this topic is available at www.expertconsult.com. microbes, toxins, and pneumotoxicants can gain access into the respiratory system by the following routes (table - ; also see table - ): aerogenous, hematogenous, direct extension, and by local production of free radicals and toxic metabolites. pathogens, such as bacteria, mycoplasmas, and viruses, along with toxic gases and foreign particles, including food, can gain access to the respiratory system via inspired air. this is the most common route in the transmission of most respiratory infections in domestic animals. some viruses, bacteria, parasites, and toxins can enter the respiratory system via the circulating blood. this portal of entry is commonly seen in septicemias, bacteremias, and with protozoa and viruses that target endothelial cells. also, circulating leukocytes may release infectious organisms such as retroviruses and listeria monocytogenes while traveling through the lungs. of type ii pneumonocytes becomes so spectacular that the microscopic appearance of the alveolus resembles that of a gland or fetal lung; this lesion has been termed epithelialization or fetalization. although it is part of the normal alveolar repair, hyperplasia of type ii pneumonocytes can interfere in gas exchange and cause hypoxemia. in uncomplicated cases, type ii pneumonocytes eventually differentiate into type i pneumonocytes, thus completing the last stage of alveolar repair (see fig. - ). in some forms of chronic interstitial lung injury, the surface of the alveolar basement membrane could become populated with migrating bronchiolar cells, a process known as alveolar bronchiolization or lambertosis. in severe cases, lambertosis, a metaplastic change, can be mistaken microscopically with alveolar adenomas. type i pneumonocytes are one of the three structural components of the blood-air barrier, so when these epithelial cells are damaged, there is an increase in alveolar capillary permeability and transient leakage of plasma fluid, proteins, and fibrin into the alveolar lumen (see fig. - ) . under normal circumstances, these fluids are rapidly cleared from the alveolus by alveolar and lymphatic absorption, and necrotic pneumonocytes (type i) and fibrin strands are phagocytosed and removed by pulmonary alveolar macrophages. when there is persistent and severe injury, fibroblasts and myofibroblasts may proliferate in the alveolar walls (alveolar interstitium), causing alveolar septal fibrosis, whereas in other forms of severe injury, fibroblasts and myofibroblasts actively migrate from the interstitium into the alveolar spaces, causing intraalveolar fibrosis. these two types of alveolar fibrosis are most commonly seen in toxic and allergic pulmonary diseases and have a devastating effect on lung function. endothelial cells are also major players in the normal and abnormal physiology of the alveolus (see . these cells trap and share circulating antigens with intravascular and interstitial macrophages. the junction between alveolar endothelial cells is not as tight as that of the type i pneumonocytes, allowing some movement of fluid and small-size molecular weight proteins into the alveolar interstitium. endothelial cells maintain an intimate cell contact with erythrocytes and leukocytes passing through the lung, since the lumen of alveolar capillaries is slightly smaller ( . µm) than the diameter of red and white blood cells. erythrocytes are easily deformable, so their transit time through the alveolar capillaries is shorter than that of leukocytes, which are less deformable cells. this longer transit time of leukocytes and their close cellular contact with alveolar endothelial cells have major impacts in lung inflammation and acute respiratory distress syndrome (ards). on a minute-to-minute basis, the pulmonary defense mechanisms deal effectively with noxious stimuli and mild tissue injury without the need for an inflammatory response. however, if normal defense mechanisms are ineffective or insufficient (overwhelmed), the inflammatory process is rapidly turned on as a second line of defense. postmortem examination of the respiratory tract should always be conducted in a thorough and systematic manner and include the conducting system (trachea, bronchi, and bronchioles), the lungs, and the thoracic cavity and pleura. detailed record keeping and photographic documentation are essential elements of a thorough examination. normal lungs typically have a homogeneous pink color and are slightly deflated from loss of negative intrathoracic pressure. the e-sections that follow describe a systematic approach to this process. .e chapter respiratory system, mediastinum, and pleurae the respiratory tract should always be examined in a systematic manner. to determine whether negative pressure is present in the thoracic cavity, the diaphragm is punctured through the abdominal cavity before the thoracic cavity has been opened. when the diaphragm is punctured in a fresh carcass, the loss of negative pressure in the thorax causes the diaphragmatic cupola to drop back caudally toward the abdominal cavity, and at the same time, there is an audible sound caused by the inrush of air into the thorax. lack of this movement may be an indication of advanced pneumothorax, pleural effusion, or the presence of uncollapsed lungs caused by pulmonary edema, pneumonia, fibrosis, or emphysema. in carcasses that have been dead for a long time, pulmonary air and gas produced by saprophytic bacteria leak into the pleural cavity, reducing the negative thoracic pressure and collapsing the lung. the rib cage must be removed by cutting along the costosternal joints and along the neck of the ribs (close to the costovertebral joints) in such a way that pleural adhesions and abnormal thoracic contents can be observed and grossly quantified (e.g., ml of clear, yellow fluid). the tongue, pharynx, esophagus, larynx, trachea, and thoracic viscera (lungs, heart, and thymus) should be removed as a unit (often called the pluck) and placed on the necropsy table. the pharynx and esophagus are opened starting at the pharynx by a single cut with scissors along the dorsal midline and are inspected for ulcers, foreign bodies, and neoplasms. the larynx and trachea must be examined by opening both along the dorsal midline from cranial to caudal ends and then extending the incision into the large bronchi of the caudal lung lobes. normal tracheobronchial mucosa has a smooth and glistening pearl-colored surface with empty lumina in airways. the presence of foamy fluid in airways indicates pulmonary edema. feed particles may suggest aspiration; however, careful examination of the mucosa is required because aspiration of ingesta from stomach or rumen into the lungs commonly takes place agonally or can be displaced into these areas when the carcass is moved. the lungs should be examined before incision. normal lungs typically have a homogeneous pink color (see fig. - ). external changes include the presence of rib imprints on the pleural surface when lungs fail to collapse. in addition, the lungs should be inspected for changes in color and texture and distribution of lesions. color changes can be various shades of red, indicating hypostatic congestion, hyperemia (acute pneumonia), and hemorrhage; dark blue collapsed lobules or areas are indicative of atelectasis; pale pink to white lungs indicate notable anemia, fibrosis, or emphysema; and uniformly or patchy yellow-brown lungs indicate chronic passive congestion and pulmonary fibrosis likely secondary to chronic heart failure. lungs from exsanguinated animals are generally paler than the normal pink color because of reduced blood in the pulmonary tissue. lungs with postmortem autolysis show green discoloration, a change that is also seen in other organs (e- fig. - ). a covering of yellowish material on the pleural surface indicates accumulation of fibrin. because it is impossible to describe the texture of normal lungs, experience in palpation is required to appreciate the actual texture of a normal lung. texture is determined by gently palpating the surface and parenchyma of the lungs. normal texture can change to firm, hard, elastic (rubbery), or crepitus (with a crackling sound or feeling). for a detailed description of lung texture, see the section on classification of pneumonias in domestic animals. palpation of the lungs, which should be gentle, also permits detection of nonvisible nodules or abscesses in the parenchyma. knowing the distribution of a lesion in the lungs also facilitates diagnosis because particular etiologic agents cause lesions with specific distribution. distribution of lesions is generally described as focal, multifocal, locally extensive, or diffuse. according to their topography, pulmo-nary lesions can also be classified as cranioventral, dorsocaudal, and so on. necropsy reports must also contain an estimate of the extent of the pulmonary lesions, preferably expressed as a percentage of the volume of the lungs affected. for instance, a report may read "cranioventral consolidation involving % of the lungs." if the lungs have focal lesions, a rough estimate of the number should also be included in the report. for instance, "numerous (approximately ), small ( to cm in diameter), hard nodules were randomly distributed in all lung lobes." two methods are used to examine the nasal structures. the first is making a midsagittal cut through the head and removing the nasal septum; the second is making several transverse sections of the nose at the level of the second premolar teeth. this latter method is preferred when examining pigs suspected of having atrophic rhinitis or animals suspected of having nasal neoplasms. microscopic examination of pulmonary tissue is routinely done in diagnostic laboratories. samples of normal and abnormal lungs, along with other appropriate tissue, should always be submitted in % buffered-neutral formalin for histopathologic evaluation. a minimum of four lung samples (left cranial, left caudal, right cranial, and right caudal) should be taken for histopathologic examination in animals with a history of respiratory signs. to improve fixation, a paper towel can be placed over the samples of lung floating in fixative. when detailed evaluation of the alveolar walls is required, lungs can be fixed by a gentle intratracheal injection of fixative; however, this technique displaces transudates and exudates and can artificially cause distention of the perivascular and peribronchial spaces. lung biopsy specimens are taken only sporadically because complications often outweigh the diagnostic value. however, the use of new techniques, such as endoscopic-directed biopsies, has notably reduced some of these complications. biopsies of the lungs are recommended in cases of chronic persistent pulmonary disease unresponsive to treatment or intrathoracic masses of undetermined origin. endoscopic-directed biopsies of the nasal and bronchial mucosa are routinely used in clinical practice and generally have a much better diagnostic value. two valuable diagnostic tools in human medicine, bronchoalveolar lavage (bal) and transtracheal wash (ttw), have in recent years become more widely used in veterinary clinical diagnosis of respiratory ailments, particularly in horses, dogs, and cats. the basis of bal and ttw is sampling the lung or trachea of a living animal by infusing sterile fluid into the trachea or deep lung (respectively) and retrieving it to determine the cellular and biochemical composition of this fluid. in other words, the composition of the fluid reflects what is present in the bronchioloalveolar spaces and trachea. these procedures are performed by inserting a tube directly through the larynx into the trachea or bronchus, or transtracheally by inserting a tube through a needle percutaneously into the cervical trachea. microscopic examination of properly collected, stored, and processed samples may reveal many erythrocytes and siderophages in pulmonary hemorrhage or left-sided heart failure; inclusion bodies or syncytial cells in viral pneumonias; increased number of leukocytes in pulmonary inflammation; abundant mucus in asthma or equine recurrent airway obstruction (rao); the presence of pulmonary pathogens, such as parasites, fungi, and bacteria; or tumor cells in cases of pulmonary neoplasia. in the healthy animal, % to % of the bal cells are pulmonary alveolar macrophages (see fig. - , a) . clearance, or a combination of both is the underlying pathogenetic mechanism in many pulmonary diseases ( fig. - ) . the anatomic configuration of the nasal cavity and bronchi plays a unique role in preventing or reducing the penetration of noxious material into the lungs, especially into the alveoli, which is the most vulnerable portion of the respiratory system. the narrow nasal meatuses and the coiled arrangement of the nasal conchae generate enormous turbulence of airflow, and as a result, physical forces are created that forcefully impact particles larger than µm onto the surface of the nasal mucosa ( fig. - ). although particles smaller than µm could escape trapping in the nasal cavity, these mediumsized particles meet a second barrier at the tracheal and bronchial in some instances, pathogenic organisms can also reach the pleura and lungs through penetrating injuries, such as gunshot wounds, migrating awns, or bites, or by direct extension from a ruptured esophagus or perforated diaphragm. the lungs, particularly the bronchioles and alveoli, are vulnerable to endogenous injury caused by the local generation of free radicals during inflammation or by toxic metabolites generated by club (clara) cells (see fig. - , b). inflammatory processes in the respiratory system, particularly those caused by infectious organisms, can spread to contiguous or distant tissues. for instance, rhinitis may spread into the sinuses causing rhinosinusitis. similarly, laryngeal inflammation may spread into the lungs when exudate in the larynx is aspirated. lung disease can have profound systemic effects when cytokines, produced locally during necrosis or inflammation, are released into circulation. as a result of the enormous vascular bed present in the lung, sepsis and septic shock often develop when proinflammatory molecules overwhelm the antiinflammatory response during the so-called "cytokine storm." it is axiomatic that a particle, microbe, or toxic gas must first gain entry to a vulnerable region of the respiratory system before it can induce an adaptive immune response or have a pathologic effect. the characteristics of size, shape, dispersal, and deposition of particles present in inspired air are studied in aerobiology. it is important to recognize the difference between deposition, clearance, and retention of inhaled particles. deposition is the process by which particles of various sizes and shapes are trapped within specific regions of the respiratory tract. clearance is the process by which deposited particles are destroyed, neutralized, or removed from the mucosal surfaces. the difference between what is deposited and what is cleared from the respiratory tract is referred to as retention. the main mechanisms involved in clearance are sneezing, coughing, mucociliary transport, and phagocytosis (table - ). abnormal retention of particles resulting from increased deposition, decreased same rate in all levels of a conducting system, a "bottleneck" effect would be created in major airways as the minor but more numerous airways enter the bronchi. for this reason, the mucociliary transport in proximal (rostral) airways is physiologically faster than that of the distal (caudal) ones. ciliary activity and mucus transport increase notably in response to stimuli such as in respiratory infections. the mucociliary blanket of the nasal cavity, trachea, and bronchi also plays an important role in preventing injury from toxic gases. if a soluble gas contacts the mucociliary blanket, it mixes with the mucus, thus reducing the concentration of gas reaching deep into the alveoli. in other words, mucus acts as a "scavenger system," whereby gases are solubilized and subsequently cleared from the respiratory tract via mucociliary transport. if ciliary transport is reduced (loss of cilia) or mucus production is excessive, coughing becomes an important mechanism for clearing the airways. in addition to the mechanical barrier and physical transport provided by the mucociliary escalator, other cells closely associated with ciliated epithelium contribute to the defense mechanism of the conducting and transitional systems. among the most notable are the microfold (m) cells, which are modified epithelial cells covering the bronchial-associated lymphoid tissue (balt), both of which are strategically situated at the corner of the bifurcation of bronchi and bronchioles, where inhaled particles often collide with the mucosa because of inertial forces. from here, inhaled particles and soluble antigens are phagocytosed and transported by macrophages, dendritic cells, and other professional antigen-presenting cells (apcs) into the balt, thus providing a unique opportunity for b and t lymphocytes to enter into close contact with inhaled pathogenic substances. pulmonary lymphocytes are not quiescent in the balt but are in continual traffic to other organs and contribute to both cellular (cytotoxic, helper, and suppressor t lymphocytes) and humoral immune responses. immunoglobulin a (iga), produced by mucosal plasma cells, and, to a lesser extent, immunoglobulin g (igg) and m (igm) play important roles in the local immunity of the conducting and transitional systems, especially with regard to preventing attachment of pathogens to the cilia. chronic airway diseases, especially those caused by infectious agents such as mycoplasmas or retroviruses, are often accompanied by severe hyperplasia of the balt. the mucociliary clearance terminates at the pharynx, where mucus, propelled caudally from the nasal cavity and cranially from the tracheobronchial tree, is eventually swallowed and thus eliminated from the conducting system of the respiratory tract. some respiratory pathogens, such as rhodococcus equi, can infect the intestines after having been removed and swallowed from the respiratory tract into the alimentary system. alveoli lack ciliated and mucus-producing cells; thus the defense mechanism against inhaled particles in the alveolar region cannot be provided by mucociliary clearance. instead, the main defense mechanisms of alveoli (exchange system) are phagocytosis provided by the pulmonary alveolar macrophages and antimicrobial molecules of the alveolar lining fluid ( fig. - ). pulmonary alveolar macrophages are highly phagocytic cells, which are not to be confused with pulmonary intravascular macrophages, and are derived largely from blood monocytes and, to a much lesser extent, from a slowly dividing population of interstitial macrophages. after a temporary adaptive stage within alveolar interstitium, blood monocytes reduce their glycolytic metabolism and increase their oxidative metabolism to function in an aerobic rather than an anaerobic environment. pulmonary alveolar macrophages contribute to the bifurcations. abrupt changes in the direction of air (inertia), which occurs at the branching of major airways, cause particles in the -to -µm size range to collide with the surface of bronchial mucosa (see fig. - ). because the velocity of inspired air at the level of the small bronchi and bronchioles has become rather slow, inertial and centrifugal forces no longer play a significant role in the trapping of inhaled particles. here, in the transitional (bronchiolar) and exchange (alveolar) regions, particles µm or smaller may come into contact with the mucosa by means of sedimentation because of gravitation or by diffusion as a result of brownian movement. infective aerosols containing bacteria and viruses are within the size range ( . to µm) that can gain access to the bronchiolar and alveolar regions. in addition to size, other factors, such as shape, length, electrical charge, and humidity, play an important role in mucosal deposition, retention, and pathogenicity of inhaled particles. for example, particles longer than µm may also reach the lower respiratory tract provided their mean aerodynamic diameter is less than µm. asbestos is a good example of a large but slender fiber that can bypass the filtrating mechanisms by traveling parallel to the airstream. once in the terminal bronchioles and alveoli, asbestos fibers cause asbestosis, a serious pulmonary disease in human beings. in summary, the anatomic features of the nasal cavity and airways provide an effective barrier, preventing the penetration of most large particles into the lungs. once larger particles are trapped in the mucosa of conducting airways and small particles are deposited on the surface of the nasal, tracheal, or bronchoalveolar mucosa, it is crucial that these exogenous materials be promptly removed to prevent or minimize injury to the respiratory system. for these purposes, the respiratory system is equipped with several defense mechanisms, all of which are provided by specialized cells operating in a remarkably well-coordinated manner. conducting system (nose, trachea, and bronchi) mucociliary clearance is the physical unidirectional movement and removal of deposited particles and gases dissolved in the mucus from the respiratory tract. mucociliary clearance, also referred to as the waste disposal system, is provided by the mucociliary blanket (mucociliary escalator) and is the main defense mechanism of the conducting system (nasal cavity, trachea, and bronchi) (see figs. - and - ). mucus acts primarily as a barrier and a vehicle, and it is a complex mixture of water, glycoproteins, immunoglobulins, lipids, and electrolytes. these substances are produced by goblet (mucous) cells, serous cells, submucosal glands, and fluid from transepithelial ion and water transport. once serous fluid and mucus are secreted onto the surface of the respiratory mucosa, a thin, double-layer film of mucus is formed on top of the cells. the outer layer of this film is in a viscous gel phase, whereas the inner layer, which is in a fluid or sol phase, is directly in contact with cilia (see fig. - and see e- fig. - ). the respiratory system of a healthy human produces approximately ml of mucus per day. each ciliated cell in the conducting system has approximately to motile and chemosensory cilia ( µm long), beating metachronously (forming a wave) at a ciliary beat frequency of approximately strokes per minute, and in a horse, for example, mucus moves longitudinally at a rate of up to mm per minute. rapid and powerful movement of cilia creates a series of waves that, in a continuous and synchronized manner, propel the mucus, exfoliated cells, and entrapped particles out of the respiratory tract to the pharynx. the mucus is finally swallowed or, when present in large amounts, is coughed up out of the conducting system. if mucus flow were to move at the activated alveolar macrophages. similarly, inhaled particles, such as dust, pollen, spores, carbon, or erythrocytes from intraalveolar hemorrhage, are all phagocytosed and eventually removed from alveoli by pulmonary alveolar macrophages. most alveolar macrophages leave the alveoli by migrating toward the bronchiolar (transitional) region until the mucociliary blanket is reached. once there, pulmonary macrophages are removed in the same way as any other particle: along the mucociliary flow to the pharynx and swallowed. in the cat, as many as million macrophages per hour move out from the alveoli into the conducting system and pharynx. destruction and removal of inhaled microbes and particles by alveolar macrophages is a well-orchestrated mechanism that engages many cells, receptors (i.e., toll-like receptors [tlrs]), and pulmonary secretions in the lung. the cell-to-cell interactions are complex and involve pulmonary alveolar macrophages, pneumonocytes, endothelial cells, lymphocytes, plasma cells, natural killer (nk) cells, and dendritic cells. antibodies are also important in the protection (acquired immune response) of the respiratory tract against inhaled pathogens. iga is the most abundant antibody in the nasal and tracheal secretions and prevents the attachment and absorption of antigens (immune exclusion). igg and, to a lesser extent, ige and igm promote the uptake and destruction of inhaled pathogens by phagocytic cells (immune elimination). igg is the most abundant antibody in the alveolar surface and acts primarily as an opsonizing antibody for alveolar macrophages and neutrophils. in addition to antibodies, there are several secretory molecules locally released into the alveoli that constitute the alveolar lining material and contribute to the pulmonary defense mechanisms. the most important of these antimicrobial products are transferrin, anionic peptides, and pulmonary surfactant (table - ). to facilitate phagocytosis and discriminate between "self" and "foreign" antigens, pulmonary alveolar macrophages are furnished with a wide variety of specific receptors on their cell surfaces. among the most important ones are fc receptors for antibodies; complement receptors (for c b, c a, and c a); tumor necrosis factor (tnf) receptor; and cd receptors, which facilitate phagocytosis and destruction of opsonized particles. toll-like receptors (tlrs) recognize microbial components, and apoptosis stimulating fragment (fas) receptors are involved in apoptosis and in the phagocytosis of apoptotic cells in the lung. "scavenger receptors," which are responsible for the recognition and uptake of foreign particulates, such as dust and fibers, are also present on pulmonary alveolar macrophages. lungs are also susceptible to hematogenously borne microbes, toxins, or emboli. the hepatic (kupffer cells) and splenic macrophages are the primary phagocytic cells responsible for removing circulating bacteria and other particles from the blood of dogs, some rodents, and human beings. in contrast, the cell responsible for the removal of circulating particles, bacteria, and endotoxin from the blood of ruminants, cats, pigs, and horses is mainly the pulmonary intravascular macrophage, a distinct population of phagocytes normally residing within the pulmonary capillaries (see fig. - ). in pigs, % of the pulmonary capillary surface is lined by pulmonary intravascular macrophages. in ruminants, % of intravenously injected tracer particles or bacteria are rapidly phagocytosed by these intravascular macrophages. studies have shown that an abnormally reduced number of kupffer cells in diseased liver results in a compensatory increase in pulmonary intravascular macrophages, even in animal species in which these phagocytic cells are normally absent from the lung. in some abnormal conditions, such as sepsis, pulmonary innate and adaptive immune response by rapidly attaching and phagocytosing bacteria and any other particles reaching the alveolar lumens. the number of free macrophages in the alveolar space is closely related to the number of inhaled particles reaching the lungs. this ability to increase, within hours, the number of available phagocytic cells is vital in protecting the distal lungs against foreign material, particularly when the inhaled particle load is high. unlike that of tissue macrophages, the life span of alveolar macrophages in the alveoli is notably short, only a few days, and thus they are continuously being replaced by newly migrated blood monocytes. alveolar phagocytosis plays a prominent role in the innate defense mechanism against inhaled bacteria without the need of an inflammatory reaction. bacteria reaching the alveoli are rapidly phagocytosed, and bactericidal enzymes present in lysosomes are discharged into the phagosome containing the bacteria (see b) . except for some facultative pathogens that are resistant to intracellular killing (e.g., mycobacterium tuberculosis, listeria monocytogenes, brucella abortus, rhodococcus equi, and some salmonella spp.), most bacteria reaching the lungs are rapidly destroyed by (ros) not only induce extensive pulmonary injury but also impair the defense and repair mechanisms in the lung. oxygen and free radical scavengers, such as catalase, superoxide dismutase, ubiquinone, and vitamins e and c, are largely responsible for protecting pulmonary cells against peroxidation. these scavengers are present in alveolar and bronchiolar epithelial cells and in the extracellular spaces of the pulmonary interstitium. in summary, the defense mechanisms are so effective in trapping, destroying, and removing bacteria that, under normal conditions, animals can be exposed to aerosols containing massive numbers of bacteria without any ill effects. if defense mechanisms are impaired, inhaled bacteria colonize and multiply in bronchi, bronchioles, and alveoli, and they produce infection, which can result in fatal pneumonia. similarly, when blood-borne pathogens, inhaled toxicants, or free radicals overwhelm the protective defense mechanisms, cells of the respiratory system are likely to be injured, often causing serious respiratory diseases. for many years, factors such as viral infections, toxic gases, stress, and pulmonary edema have been implicated in predisposing human beings and animals to secondary bacterial pneumonia. there are many pathways by which the defense mechanisms can be impaired; only those relevant to veterinary species are discussed. viral agents are notorious in predisposing human beings and animals to secondary bacterial pneumonias by what is known as viral-bacterial synergism. a good example of the synergistic effect of combined virus-bacterial infections is documented from epidemics of human beings with influenza virus in which the mortality rate has been significantly increased from secondary bacterial pneumonia. the most common viruses incriminated in predisposing animals to secondary bacterial pneumonia include influenza virus in pigs and horses; bovine herpesvirus (bohv- ), bovine parainfluenza virus (bpiv- ), and bovine respiratory syncytial virus (brsv) in cattle; canine distemper virus (cdv) in dogs; and felid herpesvirus (fehv- ) and feline calicivirus (fcv) in cats. the mechanism of the synergistic effect of viral-bacterial infections was previously believed to be the destruction of the mucociliary blanket and a concurrent reduction of mucociliary clearance, but in experimental studies, viral infections did not significantly reduce the physical removal of particles or bacteria out of the lungs. now, it is known that to days after a viral infection, the phagocytic function of pulmonary alveolar macrophages and, to a lesser extent, the mucociliary clearance are notably impaired (see fig. - ). other mechanisms by which viruses impair defense mechanisms are multiple and remain poorly understood (box - ). immunization against viral infections in many cases prevents or reduces the synergistic effect of viruses and thus the incidence of secondary bacterial pneumonia. certain gases also impair respiratory defense mechanisms, rendering animals more susceptible to secondary bacterial infections. for instance, hydrogen sulfide and ammonia, frequently encountered on farms, especially in buildings with poor ventilation, can impair pulmonary defense mechanisms and increase susceptibility to bacterial pneumonia. the effects of environmental pollutants on the defense mechanisms of human beings and animals living in crowded and polluted cities remain to be determined. excessive release of cytokines by pulmonary intravascular macrophages may result in acute lung injury. existing in an oxygen-rich environment and being the site of numerous metabolic reactions, the lungs also require an efficient defense mechanism against oxidant-induced cellular damage (oxidative stress). this form of damage is caused by inhaled oxidant gases (e.g., nitrogen dioxide, ozone, sulfur dioxide, or tobacco smoke), by xenobiotic toxic metabolites produced locally, by toxins reaching the lungs via the bloodstream (e.g., -methylindole and paraquat), or by free radicals (reactive oxygen species) released by phagocytic cells during inflammation. free radicals and reactive oxygen species anomalies localized congenital anomalies of the nasal cavity are rare in domestic animals and are often merely part of a more extensive craniofacial deformity (e.g., cyclops) or a component of generalized malformation (e.g., chondrodysplasia). congenital anomalies involving the nasal cavity and sinuses, such as choanal atresia (lack of communication between the nasal cavity and pharynx), some types of chondrodysplasia, and osteopetrosis, are incompatible with life. examples of nonfatal congenital anomalies include cystic nasal conchae, deviation of the nasal septum, cleft upper lip (harelip and cheiloschisis), hypoplastic turbinates, and cleft palate (palatoschisis) (see fig. - ). bronchoaspiration and aspiration pneumonia are common sequelae to cleft palate. nasal and paranasal sinus cysts are slowly growing and expansive lesions that mimic neoplasia and cause severe cranial deformation in horses. as in other organs or systems, it is extremely difficult to determine the actual cause (genetic vs. congenital) of anomalies based on pathologic evaluation. metabolic disturbances affecting the nasal cavity and sinuses are rare in domestic animals. immunodeficiency disorders, whether acquired or congenital, are often associated with increased susceptibility to viral, bacterial, and protozoal pneumonias. for example, human beings with acquired immunodeficiency syndrome (aids) are notably susceptible to pneumonia caused by proliferation of pneumocystis (carinii) jirovecii. a similar ubiquitous organism, which under normal circumstances is not pathogenic, is also found in the pneumonic lungs of immunosuppressed pigs, foals, dogs, and rodents. pigs infected with the porcine reproductive and respiratory syndrome (prrs) virus frequently develop pneumocystis carinii infection ( fig. - ) . arabian foals born with combined immunodeficiency disease easily succumb to infectious diseases, particularly adenoviral pneumonia. combined infections with two respiratory viruses, such as canine distemper virus (cdv) and canine adenovirus (cav- ), are sporadically reported in immunosuppressed puppies. also, large doses of chemotherapeutic agents, such as steroids and alkylating agents, cause immunosuppression in dogs, cats, and other animals, increasing susceptibility to secondary viral and bacterial infections. stress, uremia, endotoxemia, dehydration, starvation, hypoxia, acidosis, pulmonary edema, anesthesia, and ciliary dyskinesia are only some of the many conditions that have been implicated in impairing respiratory defense mechanisms and consequently predisposing animals to develop secondary bacterial pneumonia. the mechanisms by which each of these factors suppresses pulmonary defenses are diverse and sometimes not well understood. for example, hypoxia and pulmonary edema decrease phagocytic function of pulmonary alveolar macrophages and alter the production of surfactant (abnormal head tilt and abnormal gait), which in severe cases may lead to emaciation. based on the nature of exudate, rhinitis can be classified as serous, fibrinous, catarrhal, purulent, or granulomatous. these types of inflammatory reactions can progress from one to another in the course of the disease (i.e., serous to catarrhal to purulent), or in some instances exudates can be mixed, such as those seen in mucopurulent, fibrinohemorrhagic, or pyogranulomatous rhinitis. microscopic examination of impression smears or nasal biopsy, and bacterial or fungal cultures are generally required in establishing the cause of inflammation. common sequelae of rhinitis are hemorrhage, ulcers, and, in some cases, nasopharyngeal polyps (hyperplasia) arising from inflamed mucosa. rhinitis also can be classified according to the age of the lesions as acute, subacute, or chronic; to the severity of the insult as mild, moderate, or severe; and to the etiologic agent as viral, allergic, bacterial, mycotic, parasitic, traumatic, or toxic. serous rhinitis. serous rhinitis is the mildest form of inflammation and is characterized by hyperemia and increased production of a clear fluid locally manufactured by serous glands present in the nasal submucosa. serous rhinitis is of clinical interest only. it is caused by mild irritants or cold air, and it occurs during the early stages of viral infections, such as the common cold in human beings, upper respiratory tract infections in animals, or in mild allergic reactions. catarrhal rhinitis. catarrhal rhinitis is a slightly more severe process and has, in addition to serous secretions, a substantial increase in mucus production by hypersecretion of goblet cells and mucous glands. a mucous exudate is a thick, translucent, or slightly turbid viscous fluid, sometimes containing a few exfoliated cells, leukocytes, and cellular debris. in chronic cases, catarrhal rhinitis is characterized microscopically by notable hyperplasia of goblet cells. as the inflammation becomes more severe, the mucus is infiltrated with neutrophils, giving the exudate a cloudy appearance. this exudate is referred to as mucopurulent. purulent (suppurative) rhinitis. purulent (suppurative) rhinitis is characterized by a neutrophilic exudate, which occurs when the nasal mucosa suffers a more severe injury that generally is accompanied by mucosal necrosis and secondary bacterial infection. cytokines, leukotrienes, complement activation, and bacterial products cause exudation of leukocytes, especially neutrophils, which mix with nasal secretions, including mucus. grossly, the exudate in suppurative rhinitis is thick and opaque, but it can vary from white to green to brown, depending on the types of bacteria and type of leukocytes (neutrophils or eosinophils) present in the exudate . in severe cases, the nasal passages are completely blocked by the exudate. microscopically, neutrophils can be seen in the submucosa and mucosa and form plaques of exudate on the mucosal surface. neutrophils are commonly found marginated in vessels, in the lamina propria, and in between epithelial cells in their migration to the surface of the mucosa. fibrinous rhinitis. fibrinous rhinitis is a reaction that occurs when nasal injury causes a severe increase in vascular permeability, resulting in abundant exudation of plasma fibrinogen, which coagulates into fibrin. grossly, fibrin appears as a yellow, tan, or gray rubbery mat on nasal mucosa. fibrin accumulates on the surface and forms a distinct film of exudate sometimes referred to as pseudomembrane ( fig. - ). if this fibrinous exudate can be removed, leaving an intact underlying mucosa, it is termed a croupous or pseudodiphtheritic rhinitis. conversely, if the pseudomembrane is difficult to remove and leaves an ulcerated mucosa, it is referred to as diphtheritic or fibrinonecrotic rhinitis. the term diphtheritic was derived from human diphtheria, which causes a severe and destructive inflammatory process of the nasal, tonsillar, pharyngeal, and laryngeal mucosa. nasal amyloidosis. amyloidosis, the deposition of amyloid protein (fibrils with a β-pleated configuration) in various tissues, has been sporadically reported as a localized lesion in the nasal cavity of horses and human beings (see nasal amyloidosis, in disorders of horses). congestion and hyperemia. the nasal mucosa is well vascularized and is capable of rather dramatic variation in blood flow, whether passively as a result of interference with venous return (congestion) or actively because of vasodilation (hyperemia). congestion of the mucosal vessels is a nonspecific lesion commonly found at necropsy and presumably associated with the circulatory failure preceding death (e.g., heart failure, bloat in ruminants in which the increased intraabdominal pressure causes increased intrathoracic pressure impeding the venous return from the head and neck). hyperemia of the nasal mucosa is seen in early stages of inflammation, whether caused by irritation (e.g., ammonia and regurgitated feed), viral infections, secondary bacterial infections, toxemia, allergy, or trauma. hemorrhage. epistaxis is the clinical term used to denote blood flow from the nose (nosebleed) regardless of whether the blood originates from the nasal mucosa or from deep in the lungs, such as in horses with "exercise-induced pulmonary hemorrhage." unlike blood in the digestive tract, where the approximate anatomic location of the bleeding can be estimated by the color the blood imparts to fecal material, blood in the respiratory tract is always red. this fact is due to the rapid transport of blood out of the respiratory tract by the mucociliary blanket and during breathing. hemorrhages into the nasal cavity can be the result of local trauma, can originate from erosions of submucosal vessels by inflammation (e.g., guttural pouch mycosis), or can be caused by neoplasms. hemoptysis refers to the presence of blood in sputum or saliva (coughing or spitting blood) and is most commonly the result of pneumonia, lung abscesses, ulcerative bronchitis, pulmonary thromboembolisms or hemorrhage, and pulmonary neoplasia. inflammation of the nasal mucosa is called rhinitis, and inflammation of the sinuses is called sinusitis. these conditions usually occur together, although mild sinusitis can be undetected. clinically, rhinosinusitis is characterized by nasal discharge. rhinitis. the occurrence of infectious rhinitis presupposes an upset in the balance of the normal microbial flora of the nasal cavity. innocuous bacteria present normally protect the host through a process called competitive exclusion, whereby potential pathogens are kept at a harmless level. disruption of this protective mechanism can be caused by respiratory viruses, pathogenic bacteria, fungi, irritant gases, environmental changes, immunosuppression, local trauma, stress, or prolonged antibacterial therapy. inflammatory processes in the nasal cavity are not life-threatening and usually resolve completely. however, some adverse sequelae in cases of infectious rhinitis include bronchoaspiration of exudate leading to bronchopneumonia. chronic rhinitis often leads to destruction of the nasal conchae (turbinates), deviation of the septum, and, eventually, craniofacial deformation. also, nasal inflammation may extend into the sinuses causing sinusitis; into facial bones causing osteomyelitis; through the cribriform plate causing meningitis; into the eustachian tubes causing otitis media or guttural pouch empyema (eustachitis) in horses; and even into the inner ear causing otitis interna and vestibular syndrome the nasal septum has been removed to expose nasal conchae. the nasal mucosa is hyperemic and covered by yellow-white purulent exudate (arrows). inset, histological section showing submucosal congestion and edema and also large aggregates of neutrophils on the superficial mucosa (asterisk). h&e stain. (courtesy dr. a. lópez, atlantic veterinary college.) microscopically, the lesions include a perivascular edema with fibrin, a few neutrophils infiltrating the mucosa, and superficial plaques of exudate consisting of fibrin strands mixed with leukocytes and cellular debris covering a necrotic and ulcerated epithelium. fungal infections, such as aspergillosis, can cause a severe fibrinonecrotizing rhinitis. granulomatous rhinitis. granulomatous rhinitis is a reaction in the nasal mucosa and submucosa that is characterized by infiltration of numerous activated macrophages mixed with a few lymphocytes and plasma cells (figs. - and - ). in some cases, chronic inflammation leads to the formation of polypoid nodules that in severe cases are large enough to cause obstruction of the nasal passages ( fig. - ). granulomatous rhinitis is generally associated with chronic allergic inflammation or infection with specific organisms, such as fungi (see fig. - ), tuberculosis, systemic mycosis (see section on granulomatous pneumonia), and rhinosporidiosis ; also see fig. - ). in some cases, the cause of granulomatous rhinitis cannot be determined. sinusitis. sinusitis occurs sporadically in domestic animals and is frequently combined with rhinitis (rhinosinusitis), or it occurs as extend into the adjacent bone (osteomyelitis) or through the ethmoidal conchae into the meninges and brain (meningitis and encephalitis). nasal amyloidosis. amyloidosis, the deposition of amyloid protein (fibrils with a β-pleated configuration) in various tissues, has been sporadically reported as a localized lesion in the nasal cavity of horses. unlike amyloidoses in other organs of domestic animals where amyloid is generally of the reactive type (amyloid aa), equine nasal amyloidosis appears to be of the immunocytic type (amyloid al). affected horses with large amyloid masses have difficulty breathing because of nasal obstruction and may exhibit epistaxis and reduced athletic performance; on clinical examination, large, firm nodules resembling neoplasms (amyloidoma) can be observed in the alar folds, rostral nasal septum, and floor of nasal cavity. microscopic lesions are similar to those seen in other organs and consist of a deposition of hyaline amyloid material in nasal mucosa that is confirmed by a histochemical stain, such as congo red. progressive ethmoidal hematoma. progressive ethmoidal hematoma (peh) is important in older horses and is characterized clinically by chronic, progressive, often unilateral nasal bleeding. grossly or endoscopically, an ethmoidal hematoma appears as a single, soft, tumor-like, pedunculated, expansive, dark red mass arising from the mucosa of the ethmoidal conchae ( fig. - ) . microscopic examination reveals a capsule lined by epithelium and hemorrhagic stromal tissue infiltrated with abundant macrophages, most of which are siderophages. viral infections. viruses, such as equine viral rhinopneumonitis virus, influenza virus, adenovirus, and equine picornavirus, cause mild and generally transient respiratory infections in horses. the route of infection for these respiratory viruses is typically aerogenous. all of these infections are indistinguishable clinically; signs consist mainly of malaise, fever, coughing, conjunctivitis, and nasal a sequela to penetrating or septic wounds of the nasal, frontal, maxillary, or palatine bones; improper dehorning in young cattle, which exposes the frontal sinus; or maxillary tooth infection in horses and dogs (maxillary sinus). based on the type of exudate, sinusitis is classified as serous, catarrhal, fibrinous (rare), purulent, or granulomatous. paranasal sinuses have poor drainage; therefore exudate tends to accumulate, causing mucocele (accumulation of mucus) or empyema (accumulation of pus) ( fig. - ). chronic sinusitis may promised horses, particularly in arabian foals with inherited combined immunodeficiency disease. bacterial infections. strangles, glanders, and melioidosis of horses are all systemic bacterial diseases that cause purulent rhinitis and suppuration in various organs. these diseases are grouped as upper respiratory diseases because nasal discharge is often the most notable clinical sign. strangles. strangles is an infectious and highly contagious disease of equidae that is caused by streptococcus equi ssp. equi (streptococcus equi) . it is characterized by suppurative rhinitis and lymphadenitis (mandibular and retropharyngeal lymph nodes) with occasional hematogenous dissemination to internal organs. unlike streptococcus equi ssp. zooepidemicus (streptococcus zooepidemicus) and streptococcus dysgalactiae ssp. equisimilis (streptococcus equisimilis), streptococcus equi is not part of the normal nasal flora. infection occurs when susceptible horses come into contact with feed, exudate, or air droplets containing the bacterium. after penetrating through the nasopharyngeal mucosa, streptococcus equi drains to the regional lymph nodes-mandibular and retropharyngeal lymph nodes-via lymphatic vessels. the gross lesions in horses with strangles (mucopurulent rhinitis) correlate with clinical findings and consist of copious amounts of mucopurulent exudate in the nasal passages with notable hyperemia of the nasal mucosa. affected lymph nodes are enlarged and may contain abscesses filled with thick purulent exudate (purulent lymphadenitis). the term bastard strangles is used in cases in which hematogenous dissemination of streptococcus equi results in metastatic abscesses in such organs as the lungs, liver, spleen, kidneys, or brain or in the joints. this form of strangles is often fatal. common sequelae to strangles include bronchopneumonia caused by aspiration of nasopharyngeal exudate; laryngeal hemiplegia ("roaring"), resulting from compression of the recurrent laryngeal nerves by enlarged retropharyngeal lymph nodes; facial paralysis and horner syndrome caused by compression of sympathetic nerves that run dorsal to the medial retropharyngeal lymph node; and purpura hemorrhagica as a result of vasculitis caused by deposition of streptococcus equi antigen-antibody complexes in arterioles, venules, and capillaries of the skin and mucosal membranes. in severe cases, nasal infection extends directly into the paranasal sinuses or to the guttural pouches via the eustachian tubes, causing inflammation and accumulation of pus (guttural pouch empyema). rupture of abscesses in the mandibular and retropharyngeal lymph nodes leads to suppurative inflammation of adjacent subcutaneous tissue (cellulitis), and in severe cases the exudate escapes through cutaneous fistulas. strangles can affect horses of all ages, but it is most commonly seen in foals and young horses. it is clinically characterized by cough, nasal discharge, conjunctivitis, and painful swelling of regional lymph nodes. some horses become carriers and a source of infection to other horses. glanders. glanders is an infectious world organization for animal health (oie)-notifiable disease of equidae caused by burkholderia mallei (pseudomonas mallei) that can be transmitted to carnivores by consumption of infected horsemeat. human beings are also susceptible, and untreated infection is often fatal. this gramnegative bacterium has been listed as a potential agent for biologic warfare and bioterrorism. in the past, burkholderia mallei was found throughout the world, but today, glanders has been eradicated from most countries, except for some areas in north africa, asia, and eastern europe. there also have been sporadic outbreaks reported in brazil. the pathogenesis of glanders is not fully understood. results from experimental infections suggest that infection occurs discharge varying from serous to purulent. viral respiratory infections are common medical problems in adult horses. equine viral rhinopneumonitis. equine viral rhinopneumonitis (evr) is caused by two ubiquitous equine herpesviruses (ehv- and ehv- ) and may be manifested as a mild respiratory disease in weanling foals and young racehorses, as a neurologic disease (myeloencephalopathy), or as abortion in mares. the portal of entry for the respiratory form is typically aerogenous, and the disease is generally transient; thus the primary viral-induced lesions in the nasal mucosa and lungs are rarely seen at necropsy unless complicated by secondary bacterial rhinitis, pharyngitis, or bronchopneumonia. studies with polymerase chain reaction (pcr) techniques have demonstrated that, like other herpesviruses, ehv- and ehv- persist in the trigeminal ganglia for long periods of time (latency). reactivation because of stress or immunosuppression and subsequent shedding of the virus are the typical source of infection for susceptible animals on the farm. equine influenza. equine influenza is a common, highly contagious, and self-limiting upper respiratory infection of horses caused by aerogenous exposure to type a strains of influenza virus (h n [a/equi- ] and h n [a/equi- ]). equine influenza has high morbidity (outbreaks) but low mortality, and it is clinically characterized by fever, conjunctivitis, and serous nasal discharge. it occurs mainly in -to -year-old horses at the racetrack. as with human influenza, equine influenza is usually a mild disease, but occasionally it can cause severe bronchointerstitial pneumonia with pulmonary edema. in some horses, impaired defense mechanisms caused by the viral infection are complicated by a secondary bacterial bronchopneumonia caused by opportunistic organisms (streptococcus zooepidemicus, staphylococcus aureus, or bacteroides sp.) found in the normal flora of the upper respiratory tract. uncomplicated cases of equine influenza are rarely seen in the postmortem room. equine influenza virus (h n ) recently did an equine to canine "host-jump" causing extensive outbreaks of respiratory disease in dogs (see pneumonias of dogs). other equine respiratory viruses. equine picornavirus, adenovirus, and parainfluenza virus produce mild and transient upper respiratory infections (nasopharynx and trachea) in horses, unless complicated by secondary pathogens. in addition to reduced athletic performance, infected horses may have a temporary suppression of cell-mediated immunity leading to opportunistic infections such as pneumocystis carinii pneumonia. fatal adenoviral infections with severe pneumonia or enteritis occur commonly in immunocom- disorders of ruminants (cattle, sheep, and goats) infectious bovine rhinotracheitis. infectious bovine rhinotracheitis (ibr), or "rednose," occurs worldwide and is a disease of great importance to the cattle industry because of the synergism of the ibr virus with mannheimia haemolytica in producing pneumonia. the causative agent, bovine herpesvirus (bohv- ), has probably existed as a mild venereal disease in cattle in europe since at least the mid- s, but the respiratory form was not reported until intensive management feedlot systems were first introduced in north america around the s. typically, the disease is manifested as a transient, acute, febrile illness, which results in inspiratory dyspnea caused by obstruction of the airways by exudate only in very severe cases. other forms of bohv- infection include ulcerative rumenitis; enteritis; multifocal hepatitis in neonatal calves; nonsuppurative meningoencephalitis; infertility; and in experimental infections, mastitis, mammillitis, and ovarian necrosis. except for the encephalitic form, the type of disease caused by bohv- depends more on the site of entry than the viral strain. like other herpesviruses, bohv- also can remain latent in nerve ganglia, with recrudescence after stress or immunosuppression. this virus also causes bovine abortion, systemic infections of calves, and genital infections such as infectious pustular vulvovaginitis (ipv) and infectious balanoposthitis (ibp). the respiratory form of ibr is characterized by severe hyperemia and multifocal necrosis of nasal, pharyngeal, laryngeal, tracheal, and sometimes bronchial mucosa ( fig. - and see fig. - ). as in other respiratory viral infections, ibr lesions are microscopically characterized by necrosis and exfoliation of ciliated cells followed by repair. secondary bacterial infections of these areas of necrosis result in the formation of a thick layer of fibrinonecrotic material (diphtheritic) in the nasal, tracheal, and bronchial mucosa (see fig. - ). intranuclear inclusion bodies, commonly seen in herpesvirus infections, are rarely seen in field cases because inclusion bodies occur only during the early stages of the disease. the most important sequela to ibr is bronchopneumonia, which is caused either by direct aspiration of exudate from airways or as a result of an impairment in pulmonary defense mechanisms, thus predisposing the animal to secondary bacterial infection, most frequently mannheimia haemolytica (see pneumonic mannheimiosis discussion). postmortem diagnosis of ibr is confirmed by isolation of the virus or its identification by immunohistochemistry or pcr in affected tissues. other causes of rhinitis. nasal granulomas occur in cattle presumably as a result of repeated exposure to an unidentified inhaled antigen. nasal granulomas (atopic rhinitis) are reported mainly in cattle in australia, south africa, and the united kingdom, where affected cattle develop multiple, small, pink or red, polypoid nodules, starting in the nasal vestibule that in time extend into the caudal aspect of the nasal septum (see fig. - ). these nodules are composed of fibrovascular tissue mixed with lymphocytes (granulation tissue) superficially lined by hyperplastic epithelium with abundant mast cells and eosinophils in the lamina propria (nasal eosinophilia). the microscopic features suggest that hypersensitivity type i (immediate), type iii (immune complex), and type iv (delayed) may be involved in nasal granulomas of cattle. bovine (idiopathic) nasal granuloma must be differentiated from nasal mycetomas, nasal rhinosporidiosis, and nasal schistosomiasis, which also cause the formation of nodules in the nasal mucosa of cattle. an eosinophilic material consistent with the splendore-hoeppli phenomenon is occasionally observed in bovine mycotic granulomas. this phenomenon seen in some mycotic or bacterial infections is microscopically via the ingestion of contaminated feed and water and, very rarely, via inhalation of infectious droplets. the portals of entry are presumed to be the oropharynx or intestine, in which bacteria penetrate the mucosa and spread via lymph vessels to regional lymph nodes, then to the bloodstream, and thus hematogenously to the internal organs, particularly the lungs. lesions in the nasal cavity start as pyogranulomatous nodules in the submucosa; these lesions subsequently ulcerate, releasing copious amounts of burkholderia mallei-containing exudate into the nasal cavity (see fig. - , a) . finally, ulcerative lesions in conchal mucosa heal and are replaced by typical stellate (star-shaped), fibrous scars. in some cases, the lungs also contain numerous gray, hard, small ( to mm), miliary nodules (resembling millet seeds) randomly distributed in one or more pulmonary lobes because of the hematogenous route. microscopically, these nodules are typical chronic granulomas composed of a necrotic center, with or without calcification, surrounded by a layer of macrophages enclosed by a thick band of connective tissue infiltrated with macrophages, fewer giant cells, lymphocytes, and plasma cells. cutaneous lesions, often referred to as equine farcy, are the result of severe suppurative lymphangitis characterized by nodular thickening of extended segments of lymph vessels in the subcutaneous tissue of the legs and ventral abdomen (see fig. - , c). eventually, affected lymph vessels rupture and release large amounts of purulent exudate through sinuses to the surface of the skin. melioidosis (pseudoglanders). melioidosis (pseudoglanders) is an important, life-threatening disease of human beings, horses, cattle, sheep, goats, pigs, dogs, cats, and rodents caused by burkholderia pseudomallei (pseudomonas pseudomallei). this disease in horses is clinically and pathologically similar to glanders, hence the name pseudoglanders. in human beings, this infection can cause severe sepsis and septic shock and has also been considered to have potential for biologic welfare. melioidosis is currently present in southeast asia and, to a much lesser extent, in northern australia and some european countries where the causative organism is frequently found in rodents, feces, soil, and water. ingestion of contaminated feed and water appears to be the main route of infection; direct transmission between infected animals and insect bites has also been postulated as a possible mechanism of infection. after gaining entrance to the animal, burkholderia pseudomallei is disseminated by the bloodstream and causes suppuration and abscesses in most internal organs, such as nasal mucosa, joints, brain and spinal cord, lungs, liver, kidneys, spleen, and lymph nodes. the exudate is creamy or caseous and yellow to green. the pulmonary lesions in melioidosis are those of an embolic bacterial infection with the formation of pulmonary abscesses, which can become confluent. focal adhesive pleuritis develops where abscesses rupture through the pleura and heal. rhinosporidiosis. the protistan parasite, rhinosporidium seeberi, causes nasal infection in human beings, horses, mules, cattle, dogs, and cats. gross lesions vary from barely visible granulomas to large expansive polypoid nodules that may be mistaken as tumors. these granulomatous nodules are detected by direct observation when present in the nasal mucosa close to the nares or by rhinoscopy when located in the deep nasal cavity. the offending organism, rhinosporidium seeberi, is readily visible in histologic preparations and in impression smears, appearing as a large ( µm), oval sporangium containing thousands of endospores (see fig. - ). rhinosporidium seeberi was once considered a mycotic agent, but recent phylogenetic investigations suggest that it is an aquatic protistan parasite of the class mesomycetozoea. giant, basophilic, intranuclear inclusion bodies in the nasal epithelium, particularly in the nasal glands ( fig. - ). immunosuppressed piglets can develop a systemic cytomegalovirus infection characterized by necrosis of the liver, lungs, adrenal glands, and brain with intralesional inclusion bodies. inclusion body rhinitis is clinically a characterized by a deeply eosinophilic homogeneous material surrounded by bacteria or mycelia. it is thought to result from a localized antigen-antibody response in tissue. oestrus ovis. oestrus ovis (diptera: oestridae; nasal bot) is a brownish fly about the size of a honeybee that deposits its first-stage larvae in the nostrils of sheep in most areas of the world. microscopic larvae mature into large bots (maggots), which spend most of their larval stages in nasal passages and sinuses, causing irritation, inflammation, and obstruction of airways. mature larvae drop to the ground and pupate into flies. this type of parasitism in which living tissues are invaded by larvae of flies is known as myiasis ( fig. - ). although oestrus ovis is a nasal myiasis primarily of sheep, it sporadically affects goats, dogs, and sometimes human beings (shepherds). the presence of the larvae in nasal passages and sinuses causes chronic irritation and erosive mucopurulent rhinitis and sinusitis; bots of oestrus ovis can be found easily if the head is cut to expose the nasal passages and paranasal sinuses. rarely, larvae of oestrus ovis penetrate the cranial vault through the ethmoidal plate, causing direct or secondary bacterial meningitis. other causes of rhinitis. infectious rhinitis is only sporadically reported in goats, and most of these cases are caused by pasteurella multocida or mannheimia haemolytica. the lesions range from a mild serous to catarrhal or mucopurulent inflammation. foreign body rhinitis caused by plant material is sporadically seen cattle, sheep, and goats ( fig. - ). inclusion body rhinitis. inclusion body rhinitis is a disease of young pigs with high morbidity and low mortality caused by a porcine cytomegalovirus (suid herpesvirus- ) and characterized by a mild rhinitis. this virus commonly infects the nasal epithelium of piglets younger than weeks and causes a transient viremia. because this disease is seldom fatal, lesions are seen only incidentally or in euthanized animals. in uncomplicated cases, the gross lesion is hyperemia of the nasal mucosa, but with secondary bacterial infections, mucopurulent exudate can be abundant. microscopic lesions are typical and consist of a necrotizing, nonsuppurative rhinitis with toxigenic strains of pasteurella multocida. the only lesion associated with infection with bordetella bronchiseptica alone is a mild to moderate turbinate atrophy (nonprogressive atrophic rhinitis), but this bacterium actively promotes the colonization of the nasal cavity by pasteurella multocida. the toxigenic strains of pasteurella multocida produce potent cytotoxins that inhibit osteoblastic activity and promote osteoclastic reabsorption in nasal bones, particularly in the ventral nasal conchae, where abnormal bone remodeling results in progressive atrophy of conchae. the degree of conchal atrophy in pigs with atrophic rhinitis varies considerably, and in most pigs, the severity of the lesions does not correspond to the severity of the clinical signs. the best diagnostic method of evaluating this disease at necropsy is to make a transverse section of the snout between the first and second premolar teeth. in normal pigs, conchae are symmetric and fill most of the cavity, leaving only narrow airspaces (meatuses) between coiled conchae. the normal nasal septum is straight and divides the cavity into two mirror-image cavities. in contrast, the septum in pigs with atrophic rhinitis is generally deviated and the conchae appear smaller and asymmetric ( fig. - ). conchal atrophy causes dorsal and ventral meatuses to appear rather enlarged, and in the most advanced cases, the entire nasal conchae may be missing, leaving a large, empty space. it may seem logical to assume that after loss of conchae in an obligate nasal breather, such as the pig, the filtration defense mechanism of the nasal cavity would be impaired, thus enhancing the chances of aerogenous infections in the lung. however, the relationship between atrophic rhinitis, pneumonia, and growth rates in pigs is still controversial. osteoclastic hyperplasia and osteopenia of the conchae are the key microscopic lesions in atrophic rhinitis. depending on the stage of the disease, mucopurulent exudate may be found on the surface of the conchae. hyperplastic or metaplastic changes can occur in the nasal epithelium and glands, and infiltrates of lymphoplasmacytic cells can be present in the lamina propria. in summary, atrophic rhinitis is an important disease in pigs worldwide; morphologic characterized by a mild and transient rhinitis, causing sneezing, nasal discharge, and excessive lacrimation. atrophic rhinitis. a common worldwide disease of pigs, atrophic rhinitis (progressive atrophic rhinitis) is characterized by inflammation and atrophy of nasal conchae (turbinates). in severe cases, atrophy of the conchae may cause a striking facial deformity in growing pigs because of deviation of the nasal septum and nasal bones. the etiopathogenesis of atrophic rhinitis is complex and has been a matter of controversy for many years. pathogens historically associated with atrophic rhinitis include bordetella bronchiseptica, pasteurella multocida, haemophilus parasuis, and viral infections such as porcine cytomegalovirus (inclusion body rhinitis). in addition, predisposing factors have included genetic makeup, environment, and nutritional deficiencies. the cause of atrophic rhinitis is currently believed to be a combined infection by specific strains of bordetella bronchiseptica producing dermonecrotic toxin and linguatula serrata. linguatula serrata is a rare but highly specialized pentastomid parasite that shares some morphologic features with arthropods and annelids and causes infection when dogs consume uncooked ruminant meat containing infective larvae. it occurs primarily in carnivores, although sheep and goats may become aberrant hosts. human beings can also acquire the infection by ingesting raw ovine or caprine meat. the adult parasite is found throughout the nasal passages and sometimes can reach the sinuses and middle ear by moving through the exudate in the eustachian tubes. in common with other nasal parasites, linguatula serrata acts as an irritant, causing sneezing, catarrhal inflammation, and epistaxis. the eggs of this parasite leave the host in the exudate, which is coughed up or swallowed and eliminated in the feces. the nasal cavity and paranasal sinuses of dogs can occasionally be infested with other parasites, including mites (pneumonyssus caninum) and rhinosporidium seeberi (see figs. - and - ). allergic rhinitis. allergic rhinitis (hay fever; nasolacrimal urticaria), which is so common in human beings sensitized and reexposed to inhaled pollens or allergens, has been reported only sporadically in dogs and cats. hay fever in human beings and animals is a type i hypersensitivity reaction in which an ige-mediated degranulation of mast cells results in an acute rhinitis and conjunctivitis. microscopically, the nasal mucosa is edematous and infiltrated with numerous eosinophils, neutrophils, and some macrophages. clinically, allergic rhinitis is characterized by profuse serous nasal discharge and lacrimation. other causes of rhinitis. a nonspecific (idiopathic) chronic lymphoplasmacytic rhinitis is occasionally seen in dogs. immotile cilia syndrome (ciliary dyskinesia), a congenital disease, reduces mucociliary clearance and is an important factor in recurrent canine rhinosinusitis, bronchitis, bronchiectasis, and pneumonia. feline viral rhinotracheitis. feline viral rhinotracheitis (fvr) is a common, worldwide respiratory disease of cats caused by felid herpesvirus (fehv- ). the disease causes an impairment of pulmonary defense mechanisms predisposing cats to secondary bacterial pneumonia or to a coinfection with feline calicivirus. the virus also can remain latent in ganglia. the vast majority of cats that recover from fvr become carriers and shed fehv- , either spontaneously or following stress. susceptible animals, particularly kittens with low maternal immunity, become infected after exposure to a diseased or carrier cat. replication of fehv- in the nasal, conjunctival, pharyngeal, and, to a lesser extent, tracheal epithelium causes degeneration and exfoliation of cells. lesions caused by fehv- are fully reversible, but secondary infections with bacteria, such as pasteurella multocida, bordetella bronchiseptica, streptococcus spp., and mycoplasma felis, can cause a chronic, severe suppurative rhinitis and also conjunctivitis. intranuclear inclusion bodies are rarely seen in cats with fvr because inclusions are only present during the early stages of infection and have already disappeared by the time the cat is presented for diagnosis. respiratory sequelae to fvr can include chronic bacterial rhinitis and sinusitis with persistent purulent discharge; lysis of nasal bones, which can lead to conchal atrophy; permanent damage to the olfactory epithelium; and secondary bacterial pneumonia. in addition to rhinitis and interstitial pneumonia, fvr also causes ulcerative keratitis, hepatic necrosis, emaciation, abortion, and diagnosis is simple, but additional understanding of the pathogenesis will be necessary before effective preventive measures can be established. atrophic rhinitis is clinically characterized by sneezing, coughing, and nasal discharge. obstruction of the nasolacrimal duct is common and results in accumulation of dust and dried lacrimal secretions on the skin inferior to the medial canthus of the eye. viral infections. dogs have no specific viral infections affecting exclusively the nasal cavity or sinuses. acute rhinitis and sinusitis occurs as part of the canine infectious respiratory disease (cird) group caused by several distinct viruses, such as canine distemper virus, cav- and - , canine parainfluenza virus, reovirus, and canine herpesvirus. the viral lesions in the respiratory tract are generally transient, but the effect of the virus on other tissues and cells can be fatal, as in distemper encephalitis in dogs. bacterial infections. as in other species, secondary bacterial rhinitis, sinusitis, and pneumonia are possible sequelae of respiratory viral infections; bordetella bronchiseptica, escherichia coli, and pasteurella multocida are the most common isolates in dogs with bacterial rhinitis. mycotic infections. aspergillus spp. and penicillium spp. cause mycotic rhinitis and sinusitis in dogs (canine nasal aspergillosis) ( fig. - ). nasal biopsies reveal extensive necrosis of the nasal epithelium and thick plaques of fibrinopurulent exudate mixed with many fungal hyphae. cryptococcus neoformans and blastomyces dermatitides infections of the nasal cavity occur sporadically in dogs ( fig. - ). lesions are characterized by mucosal granulomas containing periodic acid-schiff (pas)-positive fungal organisms, and the infection is clinically characterized by mucopurulent nasal discharge. fvr; these two viral infections account for % of all cases of feline respiratory diseases. a febrile systemic hemorrhagic syndrome with high mortality (up to %) has been reported in cats infected with virulent strains of fcv. feline chlamydiosis. feline chlamydiosis is a persistent respiratory infection of cats caused by chlamydophila felis. infection results in a conjunctivitis (similar to the conjunctivitis seen in human trachoma caused by chlamydia trachomatis) and serous or mucopurulent rhinitis. in the past, chlamydophila felis was incriminated as the agent responsible for "feline pneumonitis," but its role in causing bronchointerstitial pneumonia in cats has been seriously challenged in recent years (see pneumonias of cats). mycotic infections. the most common mycotic infection in the feline nasal cavity is caused by cryptococcus neoformans and cryptococcus gatti, but not all animals exposed to these fungi necessarily develop cryptococcosis unless they are immunosuppressed. stillbirths. clinical signs of fvr infection are characterized by lethargy, oculonasal discharge, severe rhinitis, and conjunctivitis. feline calicivirus. feline rhinitis can be caused by different strains of feline calicivirus (fcv). it is an important infection of the respiratory tract of cats, and depending on the virulence of the strain, lesions vary from a mild oculonasal discharge to severe rhinitis, mucopurulent conjunctivitis, and ulcerative gingivitis and stomatitis. the lesions, in addition to rhinitis and conjunctivitis, include acute, diffuse interstitial pneumonia with necrotizing bronchiolitis (see pneumonias of cats) and in some cases prominent ulcers of the tongue and hard palate. primary viral lesions are generally transient, but secondary bacterial infections (bordetella bronchiseptica, pasteurella multocida, or escherichia coli) are a common complication. some kittens develop lameness after infection or vaccination with calicivirus because of an acute and self-limiting arthritis ("limping kitten syndrome"). carrier state and virus shedding from oronasal secretions and feces are natural sequelae after recovery from the acute phase of the disease. clinical and pathologic features of fcv disease are strikingly similar but not identical to those of hemorrhage, increased lacrimation as a result of obstruction of nasolacrimal ducts, and sneezing. in some instances, it is not possible to clinically or grossly differentiate neoplasms from hyperplastic nodules or granulomatous rhinitis. some neoplasms may infiltrate adjacent bone structures and produce notable facial deformities, loss of teeth, exophthalmus, and nervous signs. large neoplasms also project into the meatuses, narrow the lumen, and interfere with airflow, causing stertorous breathing (see . biopsies, as well as brush and imprint cytology, have proven effective in the antemortem diagnosis of nasal neoplasms, particularly in those of epithelial lineage. a unique group of nasal carcinomas (enzootic nasal tumors, enzootic intranasal tumors, and enzootic nasal carcinoma) of sheep and goats arise from the surface epithelium and glands of the ethmoidal conchae. these types of carcinomas are caused by betaretroviruses in sheep (entv- ) and goats (entv- ). the enzootic nasal tumor has been successfully transmitted to susceptible animals by the lesions vary from discrete nasal granulomas to large confluent masses of mucopurulent exudate filling the entire nasal cavity and paranasal sinuses. microscopic examination of the exudate reveals the typical thick-walled pas-positive organisms (see fig. - ). mycoplasma felis can also cause mucopurulent conjunctivitis and a mild upper respiratory infection, with clinical signs and lesions overlapping those seen with chlamydiosis, fvr, and fcr infections. respiratory infections and bronchopneumonia in cats may also be associated with the immunosuppressive effects of feline retroviruses such as feline leukemia virus (felv) and feline immunodeficiency virus (fiv). nasal aspergillosis and allergic rhinosinusitis are sporadically reported in cats (see disorders of the conducting system: species-specific diseases of the nasal cavity and paranasal sinuses: disorders of dogs: mycotic infections). neoplasms of the nasal cavity and paranasal sinuses may arise from any of the tissues forming these structures, including bone (osteoma or osteosarcoma), cartilage (chondroma or chondrosarcoma), connective tissue (fibroma or fibrosarcoma, myxoma or myxosarcoma), and blood vessels (hemangioma or hemangiosarcoma), and from all the different types of cells of glands and lining epithelium (adenoma, carcinoma, or adenocarcinoma). nasal tumors originating from stromal tissues, such as bone, cartilage, and connective tissue, are morphologically indistinguishable from those seen in other sites. in general, nasal neoplasms are rare in domestic animals, except for enzootic ethmoidal tumor (retroviral) in sheep and goats, which can occur in several animals in a herd (see the next section). in companion animals, nasal neoplasms are most common in dogs, particularly in medium to large breed dogs such as the collie, airedale terrier, basset hound, and german shepherd. the cat and the horse are less frequently affected. the main sites in order of frequency are the nasal passages and sinuses for dogs, the tip of the nose and nasal passages for cats, and the maxillary sinus and nasal passages for horses. the majority of neoplasms in the nasal cavity are malignant. benign nasal neoplasms (papilloma and adenoma) are rare and generally are either solitary or multiple, well-delineated nodules. in contrast, nasal carcinomas and nasal sarcomas are generally larger but vary in size and are often pale and multilobulated masses composed of fleshy to friable tissue (figs. - and - ). malignant neoplasms are locally invasive and tend to infiltrate sinuses, meninges, frontal brain, olfactory nerves, and vessels resulting in epistaxis. carcinomas vary from anaplastic (poorly differentiated) to well differentiated, in which cell and tissue morphology retains some glandular (adenocarcinoma) or squamous cell patterns. because nasal tumors in dogs and cats are usually large and invasive at the time of diagnosis, prognosis is usually poor and survival times are short. sarcomas originating in the nasal cavity and paranasal sinuses are less common than carcinomas. mesenchymal tumors can arise from bone (osteoma or osteosarcoma), cartilage (chondroma or chondrosarcoma), blood vessels (hemangioma or hemangiosarcoma), and connective tissue (fibroma or fibrosarcoma). overall, benign epithelial and mesenchymal tumors are less common than their malignant counterparts. secondary tumors in the nasal cavity are rare, with lymphoma being the most common secondary tumor in the nasal cavity of domestic animals ( fig. - ). nasal neoplasms become secondarily infected by bacteria, and clinical signs often overlap with those of infectious rhinitis and include catarrhal or mucopurulent nasal discharge, periodic anomalies congenital anomalies of the pharynx, guttural pouches, larynx, and trachea are rare in all species. depending on their location and severity, they may be inconsistent with postnatal life, pose little or no problem, interfere with quality of life, or manifest themselves in later life. if clinical signs of respiratory distress, such as stridor, coughing, dyspnea, or gagging, do occur, they are usually exacerbated by excitement, heat, stress, or exercise. brachycephalic airway syndrome. see disorders of the conducting system: species-specific diseases of the pharynx, guttural inoculation of cell-free tumor filtrates. enzootic nasal tumors are typically invasive but do not metastasize ( fig. - ). in some regions of the world, ethmoid tumors have been reported in horses and pigs, particularly in those farms where the endemic nasal tumors of ruminants are known to occur. nonneoplastic exophytic masses that resemble neoplasms are commonly found in horses, cats, and, to a lesser extent, other species. in horses, polyps tend to form in the ethmoidal region, whereas in cats, polyps are most frequently found in the nasopharynx and eustachian tubes. the pathogenesis of these benign growths is uncertain, although in many cases they follow chronic rhinitis or sinusitis. most recently, lymphatic obstruction secondary to inflammation has been postulated as the main culprit. grossly, polyps appear as firm, pedunculated nodules of various sizes protruding from the nasal mucosa into the nasal passages or nasopharynx ( fig. - ); the surface may be smooth, ulcerated, secondarily infected, and hemorrhagic. microscopically, polyps are characterized by a core of wellvascularized stromal tissue that contains inflammatory cells and are covered by pseudostratified or squamous epithelium (see fig. - ). nasal and paranasal sinus cysts are common idiopathic lesions in horses and are medically important because they clinically mimic table - for potential, suspected, or known genetic disorders. tracheal collapse and tracheal stenosis. tracheal collapse with reduction in tracheal patency occurs in toy, miniature, and brachycephalic breeds of dogs, in which the condition is also called tracheobronchial collapse or central airway collapse. the defect also occurs in horses, cattle, and goats. by radiographic, endoscopic, or gross examination, there is dorsoventral flattening of the trachea with concomitant widening of the dorsal tracheal membrane, which may then prolapse ventrally into the lumen ( fig. - ). most commonly, the defect extends the entire length of the trachea and only rarely affects the cervical portion alone. affected segments with a reduced lumen contain froth and even are covered by a diphtheritic membrane. in horses, the so-called scabbard trachea is characterized allergic reactions. grossly, the mucosa of the epiglottis and vocal cords is thickened and swollen, often protrudes dorsally onto the epiglottic orifice, and has a gelatinous appearance ( fig. - ). laryngeal and tracheal hemorrhage. hemorrhages in these sites occur as mucosal petechiae and are most commonly seen in coagulopathies; inflammation; septicemia and sepsis, particularly in pigs with classical swine fever (hog cholera); african swine fever or salmonellosis; and horses with equine infectious anemia. severe dyspnea and asphyxia before death can cause congestion, ecchymosis, and petechiae in the laryngeal and tracheal mucosa; this lesion must be differentiated from postmortem imbibition of hemoglobin in autolyzed carcasses (see chapter ). by lateral flattening so that the tracheal lumen is reduced to a narrow vertical slit. segmental tracheal collapse causing stenosis has been associated with congenital and acquired abnormalities. in severe cases, abnormal cartilaginous glycoproteins and loss of elasticity of tracheal rings causes the trachea to collapse. in some other cases, it is an acquired tracheal lesion that follows trauma, compression caused by extraluminal masses, peritracheal inflammation, and flawed tracheotomy or transtracheal aspirate techniques. other tracheal anomalies include tracheoesophageal fistula, which is most commonly found in human beings and sporadically in dogs and cattle. congenital fistulas can occur at any site of the cervical or thoracic segments of the trachea. acquired tracheoesophageal fistula can be a complication of improper intubation, tracheotomy, or esophageal foreign body. laryngeal hemiplegia. laryngeal hemiplegia (paralysis), sometimes called roaring in horses, is a common but obscure disease characterized by atrophy of the dorsal and lateral cricoarytenoid muscles (abductor and adductor of the arytenoid cartilage), particularly on the left side. muscular atrophy is most commonly caused by a primary denervation (recurrent laryngeal neuropathy) of unknown cause (idiopathic axonopathy) and, to a much lesser extent, secondary nerve damage (see the section on denervation atrophy in chapters and ). idiopathic laryngeal hemiplegia is an incurable axonal disease (axonopathy) of the cranial laryngeal nerve that affects mostly larger horses. secondary laryngeal hemiplegia is rare and occurs after nerve damage caused by other pathologic processes such as compression or inflammation of the left recurrent laryngeal nerve. the medial retropharyngeal lymph nodes are located immediately ventral to the floor of the guttural pouches. as a result of this close anatomic relationship, swelling or inflammation of the guttural pouches or retropharyngeal lymph nodes often results in secondary damage to the laryngeal nerve. common causes of secondary nerve damage (wallerian degeneration) include guttural pouch mycosis, retropharyngeal abscesses, inflammation because of iatrogenic injection into the nerves, neck injury, and metastatic neoplasms involving the retropharyngeal lymph nodes (e.g., lymphosarcoma). grossly, the affected laryngeal muscle in a horse with laryngeal hemiplegia is pale and smaller than normal (muscle atrophy) . microscopically, muscle fibers have lesions of denervation atrophy (see chapters and ). atrophy of laryngeal muscles also occurs in dogs as an inherited condition (siberian husky and bouvier des flanders), as a degenerative neuropathy in older dogs, secondary to laryngeal trauma in all species (e.g., choke chain damage), or secondary to hepatic encephalopathy in horses. the abnormal inspiratory sounds (roaring) during exercise in horses with laryngeal hemiplegia are caused by paralysis of the left dorsal and lateral cricoarytenoid muscles, which cause incomplete dilation of the larynx, obstruction of airflow, and vibration of vocal cords. laryngeal edema. laryngeal edema is a common feature of acute inflammation, but it is particularly important because swelling of the epiglottis and vocal cords can obstruct the laryngeal orifice, resulting in asphyxiation. laryngeal edema occurs in pigs with edema disease; in horses with purpura hemorrhagica; in cattle with acute interstitial pneumonia; in cats with systemic anaphylaxis; and in all species as a result of trauma, improper endotracheal tubing, inhalation of irritant gases (e.g., smoke), local inflammation, and animal species is classified as fibrinous, catarrhal, purulent, or granulomatous (figs. - and - ). chronic polypoid tracheitis occurs in dogs and cats, probably secondary to chronic infection. the most common causes of tracheitis are viral infections, such as those causing infectious bovine rhinotracheitis (see fig. - ), equine viral rhinopneumonitis, canine distemper, and feline rhinotracheitis. viral lesions are generally mild and transient but often become complicated with secondary bacterial infections. at the early stages, the mucosa is notably hyperemic and can show white foci of necrosis. in the most severe cases, the affected mucosa detaches from the underlying basement membrane, causing extensive tracheal ulceration. chemical tracheitis is also commonly seen after aspiration (see fig. - ). also, inhalation of fumes during barn fires can cause extensive injury and necrosis of the tracheal mucosa. in forensic cases, the presence of carbon pigment in the mucosal surface of trachea, bronchi, and bronchioles indicates that the burned animal was alive during the fire. parasitic infections of the larynx and trachea can cause obstruction with dramatic consequences, but burdens sufficient to cause such effects are not commonly seen in veterinary practice. besnoitiosis (besnoitia spp.) . besnoitiosis (besnoitia spp.) is caused by several species of this apicomplexan coccidian parasite, whose life cycle is still unknown. this parasite can cause pedunculated lesions on the skin, sclera, mucosa of the nasal cavity, and larynx of horses and donkeys, cattle, goats, and wild animals. besnoitiosis has been reported from africa, central and south america, north america, and europe. grossly, pale, round, exophytic nodules up to cm in diameter can be observed protruding from mucosal surfaces. microscopically, these nodules consist of finger-like projections covered by hyperplastic and sometimes ulcerated epithelium containing numerous thick-walled parasitic cysts with little inflammatory response. cattle. see disorders of cattle. inflammation of the pharynx, larynx, and trachea are important because of their potential to obstruct airflow and to lead to aspiration pneumonia. the pharynx is commonly affected by infectious diseases of the upper respiratory and upper digestive tracts, and the trachea can be involved by extension from both the lungs and larynx. pharyngeal obstruction and perforation. intraluminal foreign bodies in the pharynx, such as medicament boluses, apples, or potatoes, can move down and obstruct the larynx and trachea. also, pharyngeal obstruction can be caused by masses in the surrounding tissue, such as neoplasms of the thyroid gland, thymus, and parathyroid glands. a number of nonspecific insults can cause lesions and clinical signs. trauma may take the form of penetrating wounds in any species: perforation of the caudodorsal wall of the pharynx from the improper use of drenching or balling guns in sheep, cattle, and pigs; choking injury because of the use of collars in dogs and cats; and the shearing forces of bite wounds. the results of the trauma may be minimal (local edema and inflammation) or as serious as complete luminal obstruction by exudate. foreign bodies may be lodged anywhere in the pharyngeal region; the location and size determine the occurrence of dysphagia, regurgitation, dyspnea, or asphyxiation. pigs have a unique structure known as the pharyngeal diverticulum ( cm long in adult pigs), which is located in the pharyngeal wall rostral and dorsal to the esophageal entrance. it is important because barley awns may lodge in the diverticulum, causing an inflammatory swelling that affects swallowing. the diverticular wall may be perforated by awns or drenching syringes, which results in an exudate that can extend down the tissue planes between muscles of the neck and even into the mediastinum. the pharynx of the dog may also be damaged by trauma from chicken bones, sticks, and needles, resulting in the formation of a pharyngeal abscess. equine inflammation of the trachea (tracheitis). the types of injury and host inflammatory responses in the trachea are essentially the same as those described for the nasal mucosa. although tracheal mucosa is prone to aerogenous injury and necrosis, it has a remarkable capacity for repair. according to the exudate, tracheitis in all palate, are important causes of respiratory problems and reduced athletic performance in horses. an undersized epiglottis is prone to being entrapped below the arytenoepiglottic fold, causing an equine syndrome known as epiglottic entrapment. this syndrome also occurs in horses with lateral deviation and deformity of epiglottis, epiglottic cysts, or necrosis of the tip of the epiglottis. hypoplastic epiglottis also occurs in pigs. dorsal displacement of the soft palate, particularly during exercise, narrows the lumen of the nasopharynx and creates abnormal air turbulence in the conducting system of horses. epiglottic entrapment is clinically characterized by airway obstruction, exercise intolerance, respiratory noise, and cough. subepiglottic and pharyngeal cysts. anomalous lesions, such as subepiglottic and pharyngeal cysts, are occasionally seen in horses, particularly in standardbred and thoroughbred racehorses. these cysts vary in size ( to cm) and occur most commonly in the subepiglottic area and to a lesser extent in the dorsal pharynx, larynx, and soft palate. cysts are lined by squamous or pseudostratified epithelium and contain thick mucus. large cysts cause airway obstruction, reduced exercise tolerance, or dysphagia and predispose to bronchoaspiration of food. equine pharyngeal lymphoid hyperplasia. equine pharyngeal lymphoid hyperplasia, or pharyngitis with lymphoid follicular hyperplasia, is a common cause of partial upper airway obstruction in horses, particularly in -and -year-old racehorses. lymphoid hyperplasia is also seen in healthy horses as part of a response to mild chronic pharyngitis, which in many instances tends to regress with age in older animals. the cause is undetermined, but chronic bacterial infection combined with environmental factors may cause excessive antigenic stimulation and lymphoid hyperplasia. the gross lesions, visible endoscopically or at necropsy, consist of variably sized ( to mm) white foci located on the dorsolateral walls of the pharynx and extending into the openings of the guttural pouches and onto the soft palate. in severe cases, lesions may appear as pharyngeal polyps. microscopically, the lesions consist of large aggregates of lymphocytes and plasma cells in the pharyngeal mucosa. clinical signs consist of stertorous inspiration, expiration, or both. inflammation of guttural pouches. the guttural pouches of horses are large diverticula ( to ml) of the ventral portion of the auditory (eustachian) tubes. these diverticula are therefore exposed to the same pathogens as the pharynx and have drainage problems similar to the sinuses. although it is probable that various pathogens, including viruses, can infect them, the most common pathogens are fungi, which cause guttural pouch mycosis and guttural pouch empyema in the horse. eustachitis is the term used for inflammatory processes involving the eustachian (pharyngotympanic) tube. because of the close anatomic proximity of guttural pouches to the internal carotid arteries, cranial nerves (vii, ix, x, xi, and xii), and atlantooccipital joint, disease of these diverticula may involve these structures and cause a variety of clinical signs in horses. guttural pouch mycosis occurs primarily in stabled horses and is caused by aspergillus fumigatus and other aspergillus spp. infection is usually unilateral and presumably starts with the inhalation of spores from moldy hay. grossly, the mucosal surfaces of the dorsal and lateral walls of the guttural pouch mucosa are first covered by focal, rounded, raised plaques of diphtheritic (fibrinonecrotic) exudate, which with time can become confluent and grow into a large fibrinonecrotic mass ( fig. - ) . microscopically, the lesions are severe necrotic inflammation of the mucosa and submucosa with widespread vasculitis and intralesional fungal hyphae. necrosis of the mammomonogamus (syngamus) spp. mammomonogamus (syngamus) laryngeus is a nematode that is seen attached to the laryngeal mucosa of cattle in tropical asia and south america, and cats (gapeworm: mammomonogamus ierei) in the caribbean and southern united states. occasionally, human beings with a persistent cough or asthma-like symptoms have the parasite in the larynx or bronchi. oslerus (filaroides) osleri. see disorders of dogs. b a c empyema of guttural pouches is a sequela to suppurative inflammation of the nasal cavities, most commonly from streptococcus equi infection (strangles). in severe cases, the entire guttural pouch can be filled with purulent exudate ( fig. - ). the sequelae are similar to those of guttural pouch mycosis except that there is no erosion of the internal carotid artery. it is clinically characterized by nasal discharge, enlarged retropharyngeal lymph nodes, painful swelling of the parotid region, dysphagia, and respiratory distress. guttural pouch tympany develops sporadically in young horses when excessive air accumulates in the pouch from the one-way valve effect caused by inflammation or malformation of the eustachian tube. arabian and german warm-blooded horses are particularly susceptible to develop guttural pouch tympany. it is generally unilateral and characterized by nonpainful swelling of the parotid region. cattle. tracheal edema and hemorrhage syndrome of feeder cattle, also known as the honker syndrome or tracheal stenosis of feedlot cattle, is a poorly documented acute disease of unknown cause, most often seen during the summer months. severe edema and a few hemorrhages are present in the mucosa and submucosa of the dorsal surface of the trachea, extending caudally from the midcervical area as far as the tracheal bifurcation. on section, the tracheal mucosa is diffusely thickened and gelatinous. clinical signs include inspiratory wall of the guttural pouches can extend into the wall of the adjacent internal carotid artery causing hemorrhage into the lumen of the guttural pouch and recurrent epistaxis. invasion of the internal carotid artery causes arteritis, which can also lead to formation of an aneurysm and fatal bleeding into the guttural pouches. in other cases, the fungi may be angioinvasive, leading to the release of mycotic emboli into the internal carotid artery, generally resulting in multiple cerebral infarcts. dysphagia, another clinical sign seen in guttural pouch mycosis, is associated with damage to the pharyngeal branches of the vagus and glossopharyngeal nerves, which lie on the ventral aspect of the pouches. horner's syndrome results from damage to the cranial cervical ganglion and sympathetic fibers located in the caudodorsal aspect of the pouches. finally, equine laryngeal paralysis (hemiplegia) can result from damage to the laryngeal nerves as previously described in the section on laryngeal hemiplegia. pekingese, and others. the defects are a result of a mismatch of the ratio of soft tissue to cranial bone and the obstruction of airflow by excessive length of the palatine soft tissue. secondary changes, such as nasal and laryngeal edema caused by forceful inspiration, eventually lead to severe upper airway obstruction, respiratory distress, and exercise intolerance. tracheal hypoplasia. tracheal hypoplasia occurs most often in english bulldogs and boston terriers; the tracheal lumen is decreased in diameter throughout its length. canine infectious respiratory disease. canine infectious respiratory disease (cird), formerly called canine tracheobronchitis or kennel cough, is a highly contagious group of infectious diseases characterized clinically by an acute onset of coughing notably exacerbated by exercise. the term is nonspecific, much like the dyspnea that can progress to oral breathing, recumbency, and death by asphyxiation in less than hours. necrotic laryngitis. necrotic laryngitis (calf diphtheria, laryngeal necrobacillosis) is a common disease of feedlot cattle and cattle affected with other diseases, with nutritional deficiencies, or housed under unsanitary conditions. it also occurs sporadically in sheep and pigs. necrotic laryngitis, caused by fusobacterium necrophorum, is part of the syndrome termed necrotic stomatitis or laryngeal necrobacillosis, which can include lesions of the tongue, cheeks, palate, and pharynx. an opportunistic pathogen, fusobacterium necrophorum produces potent exotoxins and endotoxins after gaining entry either through lesions of viral infections, such as ibr and vesicular stomatitis in cattle, or after traumatic injury produced by feed or careless use of specula or balling guns. the gross lesions, regardless of location in the mouth or larynx (most common in the mucosa overlying the laryngeal cartilages), consist of well-demarcated, dry, yellow-gray, thick-crusted, and fibrinonecrotic exudate ( fig. - ) that in the early stages is bounded by a zone of active hyperemia. deep ulceration develops, and if the lesion does not result in death, healing is by granulation tissue formation. microscopically, the necrotic foci are first surrounded by congested borders, then by a band of leukocytes, and finally the ulcers heal by granulation tissue and collagen (fibrosis). the lesions can extend deep into the submucosal tissue. numerous bacteria are evident at the advancing edge. there are numerous and important sequelae to calf diphtheria; the most serious is death from severe toxemia or overwhelming fusobacteremia. sometimes, the exudate may be copious enough to cause laryngeal obstruction and asphyxiation or be aspirated and cause bronchopneumonia. the clinical signs of necrotic laryngitis are fever, anorexia, depression, halitosis, moist painful cough, dysphagia, and inspiratory dyspnea and ventilatory failure because of fatigue of the respiratory muscles (diaphragmatic and intercostal). laryngeal contact ulcers. ulcerative lesions in the larynx are commonly found in feedlot cattle. grossly, the laryngeal mucosa reveals circular ulcers (up to cm in diameter), which may be unilateral or bilateral and sometimes deep enough to expose the underlying arytenoid cartilages. the cause has not been established, but causal agents, such as viral, bacterial, and traumatic, have been proposed, along with increased frequency and rate of closure of the larynx (excessive swallowing and vocalization) when cattle are exposed to market and feedlot stresses such as dust, pathogens, and interruption of feeding. contact ulcers predispose a calf to diphtheria (fusobacterium necrophorum) and laryngeal papillomas. ulceration of the mucosa and necrosis of the laryngeal cartilages have also been described in calves, sheep, and horses under the term laryngeal chondritis. laryngeal abscesses involving the mucosa and underlying cartilage occur as a herd or flock problem in calves and sheep, presumably caused by a secondary infection with trueperella (arcanobacterium) pyogenes. anomalies brachycephalic airway syndrome. brachycephalic airway syndrome is a clinical term that refers to increased airflow resistance caused by stenotic nostrils and nasal meatuses and an excessively long soft palate. these abnormalities are present in brachycephalic canine breeds such as bulldogs, boxers, boston terriers, pugs, a "common cold" in human beings or bovine respiratory disease complex (brdc) in cattle. the infection occurs commonly as a result of mixing dogs from different origins such as occurs at commercial kennels, animal shelters, and veterinary clinics. between bouts of coughing, most animals appear normal, although some have rhinitis, pharyngitis, tonsillitis, or conjunctivitis; some with secondary pneumonia become quite ill. the pathogenesis of cird is complex, and many pathogens and environmental factors have been incriminated. bordetella bronchiseptica, canine adenovirus- (cav- ), and canine parainfluenza virus- (cpiv- ) are most commonly implicated. the severity of the disease is increased when more than one agent is involved or if there are extreme environmental conditions (e.g., poor ventilation). for example, dogs asymptomatically infected with bordetella bronchiseptica are more severely affected by superinfection with cav- than those not carrying the bacterium. other agents are sometimes isolated but of lesser significance and include canine adenovirus- (cav- : infectious canine hepatitis virus), reovirus type , canid herpesvirus- (cahv- ), canine respiratory coronavirus (crcov), and mycoplasma species. depending on the agents involved, gross and microscopic lesions are completely absent or they vary from catarrhal to mucopurulent tracheobronchitis, with enlargement of the tonsils and retropharyngeal and tracheobronchial lymph nodes. in dogs with bordetella bronchiseptica infection, the lesions are suppurative or mucopurulent rhinitis and tracheobronchitis, and suppurative bronchiolitis. in contrast, when lesions are purely viral, microscopic changes are focal necrosis of the tracheobronchial epithelium. sequelae can include spread either proximally or distally in the respiratory tract, the latter sometimes inducing chronic bronchitis and bronchopneumonia. oslerus (filaroides) osleri. oslerus (filaroides) osleri is a nematode parasite of dogs and other canidae that causes characteristic protruding nodules into the lumen at the tracheal bifurcation. they are readily seen on endoscopic examination or at necropsy. in severe cases, these nodules can extend cm cranially or caudally from the tracheal bifurcation and even into primary and secondary bronchi. the disease occurs worldwide, and oslerus osleri is considered the most common respiratory nematode of dogs. the gross lesions are variably sized, up to cm, submucosal nodules that extend up to cm into the tracheal lumen ( fig. - , a). microscopically, nodules contain adult parasites with a mild mononuclear cell reaction; with the death of the parasite, an intense foreign body reaction develops with neutrophils and giant cells b) . clinically, it can be asymptomatic, although it most often causes a chronic cough that can be exacerbated by exercise or excitement. severe infestations can result in dyspnea, exercise intolerance, cyanosis, emaciation, and even death in young dogs. neoplasms of the guttural pouches occur rarely in horses and are usually squamous cell carcinomas. laryngeal neoplasms are rare in dogs and extremely so in other species, although they have been reported in cats and horses. the most common laryngeal neoplasms in dogs are papillomas and squamous cell carcinomas. other less common tumors are laryngeal rhabdomyoma, previously referred to as laryngeal oncocytoma, and chondromas and osteochondromas. lymphoma involving the laryngeal tissue is sporadically seen in cats. when large enough to be obstructive, neoplasms may cause a change or loss of voice, cough, or respiratory distress with cyanosis, collapse, and syncope. other signs include dysphagia, anorexia, and exercise intolerance. the neoplasm is sometimes visible from the oral cavity and causes swelling of the neck. the prognosis is poor because most lesions recur after excision. tracheal neoplasms are even more uncommon than those of the larynx. the tracheal cartilage or mucosa can be the site of an osteochondroma, leiomyoma, osteosarcoma, mast cell tumor, and carcinoma. lymphoma in cats can extend from the mediastinum to involve the trachea. each lung is subdivided into various numbers of pulmonary lobes (see fig. - ). in the past, these were defined by anatomic fissures. however, in current anatomy, lobes are defined by the ramification of the bronchial tree. following this criterion, the left lung of all domestic species is composed of cranial and caudal lobes, whereas the right lung, depending on species, is composed of cranial, middle (absent in horse), caudal, and accessory lobes. each pulmonary lobe is further subdivided by connective tissue into pulmonary lobules, which in some species (cattle and pigs) are rather prominent and in others are much less conspicuous. from a practical standpoint, identification of the lungs among different species could be achieved by carefully observing the degree of lobation (external fissures) and the degree of lobulation (connective tissue between lobules). cattle and pigs have well-lobated and well-lobulated lungs; sheep and goats have well-lobated but poorly lobulated lungs; horses have both poorly lobated and poorly lobulated lungs and resemble human lungs; finally, dogs and cats have well-lobated but not well-lobulated lungs. the degree of lobulation determines the degree of air movement between the lobules. in pigs and cattle, movement of air between lobules is practically absent because of the thick connective tissue of the interlobular septa separating individual lobules. this movement of air between lobules and between adjacent alveoli (via the pores of kohn) constitutes what is referred to as collateral ventilation. this collateral ventilation is poor in cattle and pigs and good in dogs. the functional implications of collateral ventilation are discussed in the section on pulmonary emphysema. the lungs have an interconnecting network of interstitial stromal tissue supporting the blood and lymphatic vessels, nerves, bronchi, bronchioles, and alveoli. for purposes of simplicity, the pulmonary interstitium can be anatomically divided into three contiguous compartments: ( ) bronchovascular interstitium, where main bronchi and pulmonary vessels are situated; ( ) interlobular interstitium separating pulmonary lobules and supporting small blood and lymph vessels; and ( ) alveolar interstitium supporting the alveolar walls that contain pulmonary capillaries and alveolar epithelial cells (no lymphatic vessels here) (see discussion on the blood-air barrier in the section on alveoli). pulmonary changes, such as edema, emphysema, and inflammation, may affect one or more of these interstitial compartments. anomalies congenital anomalies of the lungs are rare in all species but are most commonly reported in cattle and sheep. compatibility with life largely depends on the type of structures involved and the proportion of functional tissue present at birth. accessory lungs are one of the most common anomalies and consist of distinctively lobulated masses of incompletely differentiated pulmonary tissue present in the thorax, abdominal cavity, or subcutaneous tissue virtually anywhere in the trunk. large accessory lungs can cause dystocia. ciliary dyskinesia (immotile cilia syndrome, kartagener's syndrome) is characterized by defective ciliary movement, which results in reduced mucociliary clearance because of a defect in the microtubules of all ciliated cells and, most important, in the ciliated respiratory epithelium and spermatozoa. primary ciliary dyskinesia often associated with situs inversus has been reported in dogs, which as a result usually have chronic recurrent rhinosinusitis, pneumonia, and infertility. pulmonary agenesis, pulmonary hypoplasia, abnormal lobulation, congenital emphysema, lung hamartoma, and congenital bronchiectasis are occasionally seen in domestic animals. congenital melanosis is a common incidental finding in pigs and ruminants and is usually seen at slaughter (fig. - ). it is characterized by black spots, often a few centimeters in diameter, in various organs, mainly the lungs, meninges, intima of the aorta, and caruncles of the uterus. melanosis has no clinical significance, and the texture of pigmented lungs remains unchanged. congenital emphysema is sporadically seen in dogs (e- fig. - ). pulmonary calcification ("calcinosis"). calcification of the lungs occurs in some hypercalcemic states, generally secondary to hypervitaminosis d or from ingestion of toxic (hypercalcemic) plants, such as solanum malacoxylon (manchester wasting disease), that contain vitamin d analogs. it is also a common sequela to uremia and hyperadrenocorticism in dogs and to pulmonary necrosis (dystrophic calcification) in most species. calcified lungs may fail to collapse when the thoracic cavity is opened and have a characteristic "gritty" texture ( fig. - ) . microscopically, lesions vary from calcification of the alveolar basement membranes (see by pathologists. recent investigations suggest that excessive lipid originates from the breakdown products of neoplastic cells. bronchial and bronchiolar obstructions such as those caused by lungworms can also cause alveolar lipidosis. the pathogenesis relates to the inability of alveolar macrophages that normally remove part of the surfactant lipids to exit the lung via the mucociliary escalator. exogenous lipid pneumonia. another form of lipid pneumonia occurs accidentally in cats or horses given mineral oil by their owners in an attempt to remove hairballs or treat colic (aspiration pneumonia). to achieve gaseous exchange, a balanced ratio of the volumes of air to capillary blood must be present in the lungs (ventilation/perfusion ratio), and the air and capillary blood must be in close proximity across the alveolar wall. a ventilation-perfusion mismatch occurs if pulmonary tissue is either collapsed (atelectasis) or overinflated (hyperinflation and emphysema). the term atelectasis means incomplete distention of alveoli and is used to describe lungs that have failed to expand with air at the time of birth (congenital or neonatal atelectasis) or lungs that have collapsed after inflation has taken place (acquired atelectasis or alveolar collapse) (figs. - and - ). during fetal life, lungs are not fully distended, contain no air, and are partially filled with a locally produced fluid known as fetal lung fluid. not surprisingly, lungs of aborted and stillborn fetuses sink when placed in water, whereas those from animals that have breathed float. at the time of birth, fetal lung fluid is rapidly reabsorbed and replaced by inspired air, leading to the normal distention of alveoli. congenital atelectasis occurs in newborns that fail to inflate their lungs after taking their first few breaths of air; it is caused by obstruction of airways, often as a result of aspiration of amniotic fluid and meconium (described in the section on meconium aspiration syndrome) (see fig. - ). congenital atelectasis also develops when alveoli cannot remain distended after initial aeration because of an alteration in quality and quantity of pulmonary surfactant produced by type ii pneumonocytes and club (clara) cells. this infant form of congenital atelectasis is referred to in human neonatology as infant respiratory distress syndrome (irds) or as hyaline membrane disease because of the clinical and microscopic features of the disease. it commonly occurs in babies who are premature or born to diabetic or alcoholic mothers and is occasionally found in animals, particularly foals and piglets. the pathetic, gasping attempts of affected foals and pigs to breathe have prompted the use of the name "barkers"; foals that survive may have brain damage from cerebral hypoxia (see chapter ) and are referred to as "wanderers" due to their aimless behavior and lack of a normal sense of fear. acquired atelectasis is much more common and occurs in two main forms: compressive and obstructive (see fig. - ). compressive atelectasis has two main causes: space-occupying masses in the pleural cavity, such as abscesses and tumors, or transferred pressures, such as that caused by bloat, hydrothorax, hemothorax, chylothorax, and empyema ( fig. - ). another form of compressive atelectasis occurs when the negative pressure in the thoracic cavity is lost because of pneumothorax. this form generally has massive atelectasis and thus is also referred to as lung collapse. obstructive (absorption) atelectasis occurs when there is a reduction in the diameter of the airways caused by mucosal edema and inflammation, or when the lumen of the airway is blocked by fig. - ) to heterotopic ossification of the lungs (e- fig. - ). in most cases, pulmonary calcification in itself has little clinical significance, although its cause (e.g., uremia or vitamin d toxicosis) may be very important. alveolar filling disorders are a heterogeneous group of lung diseases characterized by accumulation of various chemical compounds in the alveolar lumens. the most common are alveolar proteinosis, in which the alveoli are filled with finely granular eosinophilic material; pulmonary lipidosis, in which alveoli are filled with macrophages containing endogenous or exogenous lipid; and alveolar microlithiasis, in which the alveoli contain numerous concentric calcified "microliths" or "calcospherites." a similar but distinct concretion is known as corpora amylacea, which is an accumulation of laminated bodies composed of cellular debris, lipids, proteins, and possibly amyloid. for most alveolar filling disorders, there is little host response, and in many cases, it is an incidental finding. most of the alveolar filling disorders originate from inherited metabolic defects in which alveolar cells (epithelial or macrophages) cannot properly metabolize or remove lipids or proteins, whereas others result from an excessive synthesis of these substances in the lung. endogenous lipid (lipoid) pneumonia. endogenous lipid pneumonia is an obscure, subclinical pulmonary disease of cats and occasionally of dogs, which is unrelated to aspiration of foreign material. although the pathogenesis is not understood, it is presumed that lipids from pulmonary surfactant and from degenerated cells accumulate within alveolar macrophages. accumulation of surfactant lipids can occur in metabolic abnormalities of alveolar macrophages or in bronchial obstruction where surfactant-laden macrophages cannot exit the lungs via the mucociliary escalator. the gross lesions are multifocal, white, firm nodules scattered throughout the lungs (e- fig. - ) . microscopically, the alveoli are filled with foamy lipid-laden macrophages accompanied by interstitial infiltration of lymphocytes and plasma cells, fibrosis, alveolar epithelialization, and, in some cases, cholesterol clefts and lipid granulomas. lipid (lipoid) pneumonia occurs frequently in the vicinity of cancerous lung lesions in human beings, cats, and dogs. the reason for this association remains unknown and frequently unrecognized appearance of atelectasis is more common in species with poor collateral ventilation, such as cattle and pigs. the extent and location of obstructive atelectasis depends largely on the size of the affected airway (large vs. small) and on the degree of obstruction (partial vs. complete). atelectasis also occurs when large animals are kept recumbent for prolonged periods, such as during anesthesia (hypostatic atelectasis). the factors contributing to hypostatic atelectasis are a combination of blood-air imbalance, shallow breathing, airway obstruction because of mucus and fluid that has not been drained from bronchioles and alveoli, and from inadequate local production of surfactant. atelectasis can also be a sequel to paralysis of respiratory muscles and prolonged use of mechanical ventilation or general anesthesia in intensive care. in general, the lungs with atelectasis appear depressed below the surface of the normally inflated lung. the color is generally dark blue, and the texture is flabby or firm; they are firm if there is concurrent edema or other processes, such as can occur in ards or "shock" lungs (see the section on pulmonary edema). distribution and extent vary with the process, being patchy (multifocal) in congenital atelectasis, lobular in the obstructive type, and of various degrees in between in the compressive type. microscopically, the alveoli are collapsed or slitlike and the alveolar walls appear parallel and close together, giving prominence to the interstitial tissue even without any superimposed inflammation. pulmonary emphysema. pulmonary emphysema, often simply referred to as emphysema, is an extremely important primary disease in human beings, whereas in animals, it is always a secondary condition resulting from a variety of pulmonary lesions. in human medicine, emphysema is strictly defined as an abnormal permanent enlargement of airspaces distal to the terminal bronchiole, accompanied by destruction of alveolar walls (alveolar emphysema). this definition separates it from simple airspace enlargement or hyperinflation, in which there is no destruction of alveolar walls and which can occur congenitally (down syndrome) or be acquired with age (aging lung, sometimes misnamed "senile emphysema"). the pathogenesis of emphysema in human beings is still controversial, but current thinking overwhelmingly suggests that destruction of mucus plugs, exudate, aspirated foreign material, or lungworms (see fig. - ). when the obstruction is complete, trapped air in the lung eventually becomes reabsorbed. unlike the compression type, obstructive atelectasis often has a lobular pattern as a result of blockage of the airway supplying that lobule. this lobular lungs are extremely well-vascularized organs with a dual circulation provided by pulmonary and bronchial arteries. disturbances in pulmonary circulation have a notable effect on gaseous exchange, which may result in life-threatening hypoxemia and acidosis. in addition, circulatory disturbances in the lungs can have an impact on other organs, such as the heart and liver. for example, impeded blood flow in the lungs because of chronic pulmonary disease results in cor pulmonale, which is caused by unremitting pulmonary hypertension followed by cardiac dilation, right heart failure, chronic passive congestion of the liver (nutmeg liver), and generalized edema (anasarca). hyperemia is an active process that is part of acute inflammation, whereas congestion is the passive process resulting from decreased outflow of venous blood, as occurs in congestive heart failure ( fig. - ). in the early acute stages of pneumonia, the lungs appear notably red, and microscopically, blood vessels and alveolar capillaries are engorged with blood from alveolar walls is largely the result of an imbalance between proteases released by phagocytes and antiproteases produced in the lung as a defense mechanism (the protease-antiprotease theory). the destructive process in human beings is markedly accelerated by defects in the synthesis of antiproteases or any factor, such as cigarette smoking or pollution, that increases the recruitment of macrophages and leukocytes in the lungs. this theory originated when it was found that human beings with homozygous α -antitrypsin deficiency were remarkably susceptible to emphysema and that proteases (elastase) inoculated intratracheally into the lungs of laboratory animals produced lesions similar to those found in the disease. more than % of the problem relates to cigarette smoking, and airway obstruction is no longer considered to play a major role in the pathogenesis of emphysema in human beings. primary emphysema does not occur in animals, and thus no animal disease should be called simply emphysema. in animals, this lesion is always secondary to obstruction of outflow of air or is agonal at slaughter. secondary pulmonary emphysema occurs frequently in animals with bronchopneumonia, in which exudate plugging bronchi and bronchioles causes an airflow imbalance where the volume of air entering exceeds the volume leaving the lung. this airflow imbalance is often promoted by the so-called one-way valve effect caused by the exudate, which allows air into the lung during inspiration but prevents movement of air out of the lung during expiration. depending on the localization in the lung, emphysema can be classified as alveolar or interstitial. alveolar emphysema characterized by distention and rupture of the alveolar walls, forming variably sized air bubbles in pulmonary parenchyma, occurs in all species. interstitial emphysema occurs mainly in cattle, presumably because of their wide interlobular septa, and lack of collateral ventilation in these species does not permit air to move freely into adjacent pulmonary lobules. as a result, accumulated air penetrates the alveolar and bronchiolar walls and forces its way into the interlobular connective tissue, causing notable distention of the interlobular septa. it is also suspected that forced respiratory movements predispose to interstitial emphysema when air at high pressure breaks into the loose connective tissue of the interlobular septa ( fig. - ). sometimes these bubbles of trapped air in alveolar or interstitial emphysema become confluent, forming large (several centimeters in diameter) pockets of air that are referred to as bullae (singular: bulla) (see e- fig. - ); the lesion is then called bullous emphysema. this lesion is not a specific type of emphysema and does not indicate a different disease process but, rather, is a larger accumulation of air at one focus. in the most severe cases, air moves from the interlobular septa into the connective tissue surrounding the main stem bronchi and major vessels (bronchovascular bundles), and from here it leaks into the mediastinum, causing pneumomediastinum first, and eventually exits via the thoracic inlet into the cervical and thoracic subcutaneous tissue causing subcutaneous emphysema. note that mild and even moderate alveolar emphysema is difficult to judge at necropsy and by light microscopy unless special techniques are used to prevent collapse of the lung when the thorax is opened. these techniques include plugging of the trachea or intratracheal perfusion of fixative ( % neutral-buffered formalin) before the thorax is opened to prevent collapse of the lungs. important diseases that cause secondary pulmonary emphysema in animals include small airway obstruction (e.g., heaves) in horses and pulmonary edema and emphysema (fog fever) in cattle (see fig. - ) and exudates in bronchopneumonia. congenital emphysema occurring secondary to bronchial cartilage hypoplasia with subsequent bronchial collapse is occasionally reported in dogs. severe and persistent cases of heart failure, the lungs fail to collapse because of edema and pulmonary fibrosis. terminal pulmonary congestion (acute) is frequently seen in animals euthanized with barbiturates and should not be mistaken for an antemortem lesion. hypostatic congestion is another form of pulmonary congestion that results from the effects of gravity and poor circulation on a highly vascularized tissue, such as the lung. this type of gravitational congestion is characterized by the increase of blood in the lower side of the lung, particularly the lower lung of animals in lateral recumbency, and is most notable in horses and cattle. the affected portions of the lung appear dark red and can have a firmer texture. in animals and human beings who have been prostrated for extended periods of time, hypostatic congestion may be followed by hypostatic edema, and hypostatic pneumonia as edema interferes locally with the bacterial defense mechanisms. pulmonary hemorrhage. pulmonary hemorrhages can occur as a result of trauma, coagulopathies, and disseminated intravascular coagulation (dic), vasculitis, sepsis, and pulmonary thromboembolism from jugular thrombosis or from embolism of exudate from a hepatic abscess that has eroded the wall and ruptured into the caudal vena cava (cattle). a gross finding often confused with intravital pulmonary hemorrhage is the result of severing both the trachea and the carotid arteries simultaneously at slaughter. blood is aspirated from the transected trachea into the lungs, forming a random pattern of irregular red foci ( to mm) in one or more lobes. these red foci are readily visible on both the pleural and the cut surfaces of the lung, and free blood is visible in the lumens of bronchi and bronchioles. rupture of a major pulmonary vessel with resulting massive hemorrhage occurs occasionally in cattle when a growing abscess in a lung invades and disrupts the wall of a major pulmonary artery or vein ( fig. - ). in most cases, animals die rapidly, often with spectacular hemoptysis, and on postmortem examination, bronchi are filled with blood (see fig. - ). pulmonary edema. in normal lungs, fluid from the vascular space slowly but continuously passes into the interstitial tissue, where it is rapidly drained by the pulmonary and pleural lymphatic vessels. clearance of alveolar fluid across the alveolar epithelium is also a major mechanism of fluid removal from the lung. edema develops when the rate of fluid transudation from pulmonary vessels into the interstitium or alveoli exceeds that of lymphatic and alveolar removal ( fig. - ). pulmonary edema can be physiologically classified as cardiogenic (hydrostatic; hemodynamic) and noncardiogenic (permeability) types. hydrostatic (cardiogenic) pulmonary edema develops when there is an elevated rate of fluid transudation because of increased hydrostatic pressure in the vascular compartment or decreased osmotic pressure in the blood. once the lymph drainage has been overwhelmed, fluid accumulates in the perivascular spaces, causing distention of the bronchovascular bundles and alveolar interstitium, and eventually leaks into the alveolar spaces. causes of hemodynamic pulmonary edema include congestive heart failure (increased hydrostatic pressure); iatrogenic fluid overload; and disorders in which blood osmotic pressure is reduced, such as with hypoalbuminemia seen in some hepatic diseases, nephrotic syndrome, and protein-losing enteropathy. hemodynamic pulmonary edema also occurs when lymph drainage is impaired, generally secondary to neoplastic invasion of lymphatic vessels. permeability edema (inflammatory) occurs when there is excessive opening of endothelial gaps or damage to the cells that constitute the blood-air barrier (endothelial cells or type i pneumonocytes). hyperemia. pulmonary congestion is most frequently caused by heart failure, which results in stagnation of blood in pulmonary vessels, leading to edema and egression of erythrocytes into the alveolar spaces. as with any other foreign particle, erythrocytes in alveolar spaces are rapidly phagocytosed (erythrophagocytosis) by pulmonary alveolar macrophages. when extravasation of erythrocytes is severe, large numbers of macrophages with brown cytoplasm may accumulate in the bronchoalveolar spaces. the brown cytoplasm is the result of accumulation of considerable amounts of hemosiderin; these macrophages filled with iron pigment (siderophages) are generally referred to as heart failure cells ( fig. - ). the lungs of animals with chronic heart failure usually have a patchy red appearance with foci of brown discoloration because of accumulated hemosiderin. in this type of edema is an integral and early part of the inflammatory response, primarily because of the effect of inflammatory mediators, such as leukotrienes, platelet-activating factor (paf), cytokines, and vasoactive amines released by neutrophils, macrophages, mast cells, lymphocytes, endothelial cells, and type ii pneumonocytes. these inflammatory mediators increase the permeability of the blood-air barrier. in other cases, permeability edema results from direct damage to the endothelium or type i pneumonocytes, allowing plasma fluids to move freely from the vascular space into the alveolar lumen ( fig. - and see fig. - ). because type i pneumonocytes are highly vulnerable to some pneumotropic viruses (influenza and brsv), toxicants (nitrogen dioxide [no ], sulfur dioxide [so ], hydrogen sulfide [h s], and -methylindole), and particularly to free radicals, it is not surprising that permeability edema commonly accompanies many viral or toxic pulmonary diseases. a permeability edema also occurs when endothelial cells in the lung are injured by bacterial toxins, sepsis, ards, dic, anaphylactic shock, milk allergy, paraquat toxicity, adverse drug reactions, and smoke inhalation (e- fig. - ) . the concentration of protein in edematous fluid is greater in permeability edema (exudate) than in hemodynamic edema (transudate); this difference has been used clinically in human medicine to differentiate one type of pulmonary edema from another. microscopically, because of the higher concentration of protein, edema fluid in lungs with inflammation or damage to the blood-air barrier tends to stain more intensely eosinophilic than that of the hydrostatic edema from heart failure. grossly, the edematous lungs-independent of the cause-are wet and heavy. the color varies, depending on the degree of congestion or hemorrhage, and fluid may be present in the pleural cavity. if edema is severe, the bronchi and trachea contain considerable amounts of foamy fluid, which originates from the mixing of edema fluid and air ( fig. - ). on cut surfaces, the lung parenchyma oozes fluid like a wet sponge. in cattle and pigs that have distinct lobules, the lobular pattern becomes rather accentuated because of edematous distention of lymphatic vessels in the interlobular septa and the edematous interlobular septum itself ( fig. - ). severe pulmonary edema may be impossible to differentiate from peracute pneumonia; (h&e)-stained sections (see fig. - ), particularly if a fixative such as zenker's solution, which precipitates protein, is used. acute respiratory distress syndrome. acute (adult) respiratory distress syndrome (ards; shock lung) is an important condition in human beings and animals characterized by pulmonary hypertension, intravascular aggregation of neutrophils in the lungs, acute lung injury, diffuse alveolar damage, permeability edema, and formation of hyaline membranes ( fig. - ) . these membranes are a mixture of plasma proteins, fibrin, surfactant, and cellular debris from necrotic pneumonocytes (see fig. - , b) . the pathogenesis of ards is complex and multifactorial but in general terms can be defined as diffuse alveolar damage that results from lesions in distant organs, from generalized systemic diseases, or from direct injury to the lung. sepsis, major trauma, aspiration of gastric contents, extensive burns, and pancreatitis are some of the disease entities known to trigger ards. all these conditions provoke "hyperreactive macrophages" to directly or indirectly generate overwhelming amounts of cytokines causing what is known as a "cytokine storm." the main cytokines that trigger ards are tnf-α, interleukin (il)- , il- , and il- , which prime neutrophils previously recruited in the lung capillaries and alveoli to release cytotoxic enzymes and free radicals. these substances cause severe and diffuse endothelial and alveolar damage that culminates in a fulminating pulmonary edema (see fig. - ). ards occurs in domestic animals and explains why pulmonary edema is one of the most common lesions found in many animals dying of sepsis, toxemia, aspiration of gastric contents, and pancreatitis, for example. a familial form of ards has been reported in dalmatians. the pulmonary lesions in this syndrome are further discussed in the sections on interstitial pneumonia and aspiration pneumonia in dogs. neurogenic pulmonary edema is another distinctive but poorly understood form of life-threatening lung edema in human beings that follows cns injury and increased intracranial pressure (i.e., head injury, brain edema, brain tumors, or cerebral hemorrhage). this type of pulmonary edema can be experimentally reproduced in laboratory animals by injecting fibrin into the fourth ventricle. it involves both hemodynamic and permeability pathways presumably from massive sympathetic stimulation and overwhelming release of catecholamines. neurogenic pulmonary edema has sporadically been reported in animals with brain injury or severe seizures or after severe stress and excitement. pulmonary embolism. with its vast vascular bed and position in the circulation, the lung acts as a safety net to catch emboli before they reach the brain and other tissues. however, this positioning is often to its own detriment. the most common pulmonary emboli in domestic animals are thromboemboli, septic (bacterial) emboli, fat emboli, and tumor cell emboli. pulmonary thromboembolism (pte) refers to both local thrombus formation and translocation of a thrombus present elsewhere in the venous circulation ( fig. - ). fragments released inevitably reach the lungs and become trapped in the pulmonary vasculature ( fig. - and see fig. - ). small sterile thromboemboli are generally of little clinical or pathologic significance because they can be rapidly degraded and disposed of by the fibrinolytic system. larger thromboemboli may cause small airway constriction, reduced surfactant production, pulmonary edema, and atelectasis resulting in hypoxemia, hyperventilation, and dyspnea. parasites (e.g., dirofilaria immitis and angiostrongylus vasorum), endocrinopathies (e.g., hyperadrenocorticism and hypothyroidism), glomerulopathies, and hypercoagulable states can be responsible for pulmonary arterial thrombosis and pulmonary thromboembolism in dogs (e- fig. - ) . pieces of this fact is not surprising because pulmonary edema occurs in the very early stages of inflammation (see e- fig. - ) . careful observation of the lungs at the time of necropsy is critical because diagnosis of pulmonary edema cannot be reliably performed microscopically. this is due in part to the loss of the edema fluid from the lungs during fixation with % neutral-buffered formalin and in part to the fact that the fluid itself stains very poorly or not at all with eosin because of its low protein content (hemodynamic edema). a protein-rich (permeability) edema is easier to visualize microscopically because it is deeply eosinophilic in hematoxylin and eosin fig. - ) . , normal alveolar capillary externally covered by type i and type ii pneumonocytes and internally by vascular endothelium (see fig. - for more detail). , at the early stages of sepsis, proinflammatory cytokines (interleukin [il- ] and tumor necrosis factor [tnf]) cause circulating neutrophils to adhere to the endothelial surface. following a "cytokine storm," the marginated neutrophils further activated by inflammatory mediators suddenly release their cytoplasmic granules (proteolytic enzymes and elastases myeloperoxidase) into the surrounding milieu (arrows). , enzymes released by these neutrophils cause injury to type i pneumonocytes (arrows) and endothelial cells (arrowheads), disrupting the blood-air barrier and causing permeability edema (curved arrows), alveolar hemorrhage (double-headed arrow), and exocytosis of neutrophils into the alveolar space (double-headed arrow). , extravasated plasma proteins admixed with surfactant and cell debris form thick hyaline membranes along the alveolar wall. , in the unlikely event that the animal survives, the healing process starts with alveolar macrophages removing cellular debris, reabsorption of edema, and hyperplasia of type ii pneumonocytes (double-headed curved arrow) that subsequently differentiate into type i pneumonocytes (see fig. b a recognized in the bovine lung after strong pneumatic stunning at slaughter (captive bolt) ( fig. - , a) . although obviously not important as an antemortem pulmonary lesion, brain emboli are intriguing as a potential risk for public health control of bovine spongiform encephalopathy (bse). fragments of hair can also embolize to the lung following intravenous injections (see fig. - , b). hepatic emboli formed by circulating pieces of fragmented liver occasionally become trapped in the pulmonary vasculature after severe abdominal trauma and hepatic rupture (see fig. - , c) . megakaryocytes trapped in alveolar capillaries are a common but incidental microscopic finding in the lungs of all species, particularly dogs (see fig. - , d) . tumor emboli (e.g., osteosarcoma and hemangiosarcoma in dogs and uterine carcinoma in cattle) can be numerous and striking and the ultimate cause of death in malignant neoplasia. in experimental studies, cytokines released during pulmonary inflammation are chemotactic for tumor cells and promote pulmonary metastasis. pulmonary infarcts. because of a dual arterial supply to the lung, pulmonary infarction is rare and generally asymptomatic. however, pulmonary infarcts can be readily caused when pulmonary thrombosis and embolism are superimposed on an already compromised pulmonary circulation such as occurs in congestive heart c d thrombi breaking free from a jugular, femoral, or uterine vein can cause pulmonary thromboembolism. pulmonary thromboembolisms occur in heavy horses after prolonged anesthesia (deep vein thrombosis), recumbent cows ("downer cow syndrome"), or in any animal undergoing long-term intravenous catheterization in which thrombi build up in the catheter and then break off (see fig. - ). septic emboli, pieces of thrombi contaminated with bacteria or fungi and broken free from infected mural or valvular thrombi in the heart and vessels, eventually become entrapped in the pulmonary circulation. pulmonary emboli originate most commonly from bacterial endocarditis (right side) and jugular thrombophlebitis in all species, hepatic abscesses that have eroded and discharged their contents into the caudal vena cava in cattle, and septic arthritis and omphalitis in farm animals (see fig. - ). when present in large numbers, septic emboli may cause unexpected death because of massive pulmonary edema; survivors generally develop pulmonary arteritis and thrombosis and embolic (suppurative) pneumonia, which may lead to pulmonary abscesses. bone marrow and bone emboli can form after bone fractures or surgical interventions of bone. these are not as significant a problem in domestic animals as they are in human beings. brain emboli (i.e., pieces of brain tissue) in the pulmonary vasculature reported in severe cases of head injury in human beings have recently been pulmonary macrophages (alveolar, intravascular, and interstitial), which have an immense biologic armamentarium, are the single most important effector cell and source of cytokines for all stages of pulmonary inflammation. these all-purpose phagocytic cells modulate the recruitment and trafficking of blood-borne leukocytes in the lung through the secretion of chemokines (see e- table - ). before reviewing how inflammatory cells are recruited in the lungs, three significant features in pulmonary injury must be remembered: ( ) leukocytes can exit the vascular system through the alveolar capillaries, unlike in other tissues, where postcapillary venules are the sites of leukocytic diapedesis (extravasation); ( ) the intact lung contains within alveolar capillaries a large pool of resident leukocytes (marginated pool); and ( ) additional neutrophils are sequestered within alveolar capillaries within minutes of a local or systemic inflammatory response. these three pulmonary idiosyncrasies, along with the enormous length of the capillary network in the lung, explain why recruitment and migration of leukocytes into alveolar spaces develops so rapidly. experimental studies with aerosols of endotoxin or gram-negative bacteria have shown that within minutes of exposure, there is a significant increase in capillary leukocytes, and by hours the alveolar lumen is filled with neutrophils. not surprisingly, the bal fluid collected from patients with acute pneumonia contains large amounts of inflammatory mediators such as tnf-α, il- , and il- . also, the capillary endothelium of patients with acute pneumonia has increased "expression" of adhesion molecules, which facilitate the migration of leukocytes from capillaries into the alveolar interstitium and from there into the alveolar lumen. in allergic pulmonary diseases, eotaxin and il- are primarily responsible for recruitment and trafficking of eosinophils in the lung. movement of plasma proteins into the pulmonary interstitium and alveolar lumen is a common but poorly understood phenomenon in pulmonary inflammation. leakage of fibrinogen and plasma proteins into the alveolar space occurs when there is structural damage to the blood-air barrier. this leakage is also promoted by some types of cytokines that enhance procoagulant activity, whereas others reduce fibrinolytic activity. excessive exudation of fibrin into the alveoli is particularly common in ruminants and pigs. the fibrinolytic system plays a major role in the resolution of pulmonary inflammatory diseases. in some cases, excessive plasma proteins leaked into alveoli mix with necrotic type i pneumonocytes and pulmonary surfactant, forming microscopic eosinophilic bands (membranes) along the lining of alveolar septa. these membranes, known as hyaline membranes, are found in specific types of pulmonary diseases, particularly in ards, and in cattle with acute interstitial pneumonias such as bovine pulmonary edema and emphysema and extrinsic allergic alveolitis (see pneumonias of cattle). in the past few years, nitric oxide has been identified as a major regulatory molecule of inflammation in a variety of tissues, including the lung. produced locally by macrophages, pulmonary endothelium, and pneumonocytes, nitric oxide regulates the vascular and bronchial tone, modulates the production of cytokines, controls the recruitment and trafficking of neutrophils in the lung, and switches on/off genes involved in inflammation and immunity. experimental work has also shown that pulmonary surfactant upregulates the production of nitric oxide in the lung, supporting the current view that pneumonocytes are also pivotal in amplifying and downregulating the inflammatory and immune responses in the lung (see e- table - ). as the inflammatory process becomes chronic, the types of cells making up cellular infiltrates in the lung change from mainly neutrophils to largely mononuclear cells. this shift in cellular composition is accompanied by an increase in specific cytokines, such as failure. it also occurs in dogs with torsion of a lung lobe (fig. - ) . the gross features of infarcts vary considerably, depending on the stage, and they can be red to black, swollen, firm, and cone or wedge shaped, particularly at the lung margins. in the early acute stage, microscopic lesions are severely hemorrhagic, and this is followed by necrosis. in or days, a border of inflammatory cells develops, and a few days later, a large number of siderophages are present in the necrotic lung. if sterile, pulmonary infarcts heal as fibrotic scars; if septic, an abscess may form surrounded by a thick fibrous capsule. in the past three decades, an information explosion has increased the overall understanding of pulmonary inflammation, with so many proinflammatory and antiinflammatory mediators described to date that it would be impossible to review them all here (see chapters and ). pulmonary inflammation is a highly regulated process that involves a complex interaction between cells imported from the blood (platelets, neutrophils, eosinophils, mast cells, and lymphocytes) and pulmonary cells (type i and ii pneumonocytes; endothelial and club [clara] cells; alveolar and intravascular macrophages; and stromal interstitial cells, such as mast cells, interstitial macrophages, fibroblasts, and myofibroblasts). blood-borne leukocytes, platelets, and plasma proteins are brought into the areas of inflammation by an elaborate network of chemical signals emitted by pulmonary cells and resident leukocytes. long-distance communication between pulmonary cells and blood cells is largely done by soluble cytokines; once in the lung, imported leukocytes communicate with pulmonary and vascular cells through adhesion and other inflammatory molecules. the best known inflammatory mediators are the complement system (c a, c b, and c a), coagulation factors (factors v and vii), arachidonic acid metabolites (leukotrienes and prostaglandins), cytokines (interleukins, monokines, and chemokines), adhesion molecules (icam and vcam), neuropeptides (substance p, tachykinins, and neurokinins), enzymes and enzyme inhibitors (elastase and antitrypsin), oxygen metabolites (o •, oh•, and h o ), antioxidants (glutathione), and nitric oxide (e -table - ). acting in concert, these and many other molecules send positive or negative signals to initiate, maintain, and, it is hoped, resolve the inflammatory process without causing injury to the lung. chapter respiratory system, mediastinum, and pleurae e- with several episodes of hemorrhage are characterized by large areas of dark brown discoloration, largely in the caudal lung lobes. microscopically, lesions are alveolar hemorrhages, abundant alveolar macrophages containing hemosiderin (siderophages), mild alveolar fibrosis, and occlusive remodeling of pulmonary veins. recurrent airway obstruction. recurrent airway obstruction (rao) of horses, also referred to as copd, heaves, chronic bronchiolitis-emphysema complex, chronic small airway disease, alveolar emphysema, and "broken wind," is a common clinically asthma-like syndrome of horses and ponies. rao is characterized by recurrent respiratory distress, chronic cough, poor athletic performance, airway neutrophilia, bronchoconstriction, mucus hypersecretion, and airway obstruction. the pathogenesis is still obscure, but genetic predisposition, t h (allergic) immune response, and the exceptional sensitivity of airways to environmental allergens (hyperreactive airway disease) have been postulated as the basic underlying mechanisms. what makes small airways hyperreactive to allergens is still a matter of controversy. epidemiologic and experimental studies suggest that it could be the result of preceding bronchiolar damage caused by viral infections; ingestion of pneumotoxicants ( -methylindole); or prolonged exposure to organic dust, endotoxin, and environmental allergens (molds). it has been postulated that sustained inhalation of dust particles, whether antigenic or not, upregulates the production of cytokines (tnf-α, il- , and monokine-inducible protein ) and neuropeptides (neurokinin a [nka], neurokinin b [nkb], and substance p), attracting neutrophils into the bronchioloalveolar region and promoting leukocyte-induced bronchiolar injury. summer pasture-associated obstructive pulmonary disease (spaopd) is a seasonal airway disease also reported in horses with similar clinical and pathologic findings. more recently, the term inflammatory airway disease (iad) has been introduced in equine medicine to describe rao-like syndrome in young horses to years old. the lungs of horses with heaves are grossly unremarkable, except for extreme cases in which alveolar emphysema may be present. microscopically, the lesions are often remarkable and include goblet cell metaplasia in bronchioles; plugging of bronchioles with mucus mixed with few eosinophils and neutrophils (see fig. - ); peribronchiolar infiltration with lymphocytes, plasma cells, and variable numbers of eosinophils; and hypertrophy of smooth muscle in bronchi and bronchioles. in severe cases, accumulation of mucus leads to the complete obstruction of bronchioles and alveoli and resultant alveolar emphysema characterized by enlarged "alveoli" from the destruction of alveolar walls. feline asthma syndrome. feline asthma syndrome, also known as feline allergic bronchitis, is a clinical syndrome in cats of any age characterized by recurrent episodes of bronchoconstriction, cough, or dyspnea. the pathogenesis is not well understood but is presumed to originate, as in human asthma, as a type i hypersensitivity (igemast cell reaction) to inhaled allergens. dust, cigarette smoke, plant and household materials, and parasitic proteins have been incriminated as possible allergens. this self-limited allergic disease responds well to steroid therapy; thus it is rarely implicated as a primary cause of death except when suppressed defense mechanisms allow a secondary bacterial pneumonia. bronchial biopsies from affected cats at the early stages reveal mild to moderate inflammation characterized by mucosal edema and infiltration of leukocytes, particularly eosinophils. increased numbers of circulating eosinophils (blood eosinophilia) are present in some but not all cats with feline asthma. il- , interferon-γ (ifn-γ), and interferon-inducible protein (ip- ), which are chemotactic for lymphocytes and macrophages. under appropriate conditions, these cytokines activate t lymphocytes, regulate granulomatous inflammation, and induce the formation of multinucleated giant cells such as in mycobacterial infections. inflammatory mediators locally released from inflamed lungs also have a biologic effect in other tissue. for example, pulmonary hypertension and right-sided heart failure (cor pulmonale) often follows chronic alveolar inflammation, not only as a result of increased pulmonary blood pressure but also from the effect of inflammatory mediators on the contractibility of smooth muscle of the pulmonary and systemic vasculature. cytokines, particularly tnf-α, that are released during inflammation are associated, both as cause and as effect, with the systemic inflammatory response syndrome (sirs), sepsis, severe sepsis with multiple organ dysfunction, and septic shock (cardiopulmonary collapse). as it occurs in any other sentinel system where many biologic promoters and inhibitors are involved (coagulation, the complement and immune systems), the inflammatory cascade could go into an "out-of-control" state, causing severe damage to the lungs. acute lung injury (ali), extrinsic allergic alveolitis, ards, pulmonary fibrosis, and asthma are archetypical diseases that ensue from an uncontrolled production and release of cytokines (cytokine storm). as long as acute alveolar injury is transient and there is no interference with the normal host response, the entire process of injury, degeneration, necrosis, inflammation, and repair can occur in less than days. on the other hand, when acute alveolar injury becomes persistent or when the capacity of the host for repair is impaired, lesions can progress to an irreversible stage in which restoration of alveolar structure is no longer possible. in diseases, such as extrinsic allergic alveolitis, the constant release of proteolytic enzymes and free radicals by phagocytic cells perpetuates alveolar damage in a vicious circle. in other cases, such as in paraquat toxicity, the magnitude of alveolar injury can be so severe that type ii pneumonocytes, basement membranes, and alveolar interstitium are so disrupted that the capacity for alveolar repair is lost. fibronectins and transforming growth factors (tgfs) released from macrophages and other mononuclear cells at the site of chronic inflammation regulate the recruitment, attachment, and proliferation of fibroblasts. in turn, these cells synthesize and release considerable amounts of ecm (collagen, elastic fibers, or proteoglycans), eventually leading to fibrosis and total obliteration of normal alveolar architecture. in summary, in diseases in which there is chronic and irreversible alveolar damage, lesions invariably progress to a stage of terminal alveolar and interstitial fibrosis. for pneumonia, see section species-specific pneumonia of domestic animals. exercise-induced pulmonary hemorrhage. exercise-induced pulmonary hemorrhage (eiph) is a specific form of pulmonary hemorrhage in racehorses that occurs after exercise and clinically is characterized by epistaxis. because only a small percentage of horses with bronchoscopic evidence of hemorrhage have clinical epistaxis, it is likely that eiph goes undetected in many cases. the pathogenesis is still controversial, but current literature suggests laryngeal paralysis, bronchiolitis, and extremely high pulmonary vascular and alveolar pressures during exercise, alveolar hypoxia, and preexisting pulmonary injury as possible causes. eiph is seldom fatal; postmortem lesions in the lungs of horses that have been affected disease may be known by different names. in pigs, for instance, enzootic pneumonia and mycoplasma pneumonia refer to the same disease caused by mycoplasma hyopneumoniae. the word pneumonitis has been used by some as a synonym for pneumonia; however, others have restricted this term to chronic proliferative inflammation generally involving the alveolar interstitium and with little or no evidence of exudate. in this chapter, the word pneumonia is used for any inflammatory lesion in the lungs, regardless of whether it is exudative or proliferative, alveolar, or interstitial. on the basis of texture, distribution, appearance, and exudation, pneumonias can be grossly diagnosed into four morphologically distinct types: bronchopneumonia, interstitial pneumonia, embolic pneumonia, and granulomatous pneumonia. by using this classification, it is possible at the time of a necropsy to predict with some degree of certainty the likely cause (virus, bacteria, fungi, or parasites), routes of entry (aerogenous vs. hematogenous), and possible sequelae. these four morphologic types allow the clinician or pathologists to predict the most likely etiology and therefore facilitate the decision as to what samples need to be taken and which tests should be requested to the diagnostic laboratory (i.e., histopathology, bacteriology, virology, or toxicology). however, overlapping of these four types of pneumonias is possible, and sometimes two morphologic types may be present in the same lung. the criteria used to classify pneumonias grossly into bronchopneumonia, interstitial pneumonia, embolic pneumonia, and granulomatous pneumonia are based on morphologic changes, including distribution, texture, color, and general appearance of the affected lungs (table - ). distribution of the inflammatory lesions in the lungs can be ( ) cranioventral, as in most bronchopneumonias; ( ) multifocal, as in embolic pneumonias; ( ) diffuse, as in interstitial pneumonias; or ( ) locally extensive, as in granulomatous in the most advanced cases, chronic bronchoconstriction and excess mucus production may result in smooth muscle hyperplasia and obstruction of the bronchi and bronchioles and infiltration of the airway mucosa by eosinophils. a syndrome known as canine asthma has been reported in dogs but is not as well characterized as the feline counterpart. few subjects in veterinary pathology have caused so much debate as the classification of pneumonias. historically, pneumonias in animals have been classified or named based on the following: . presumed cause, with names such as viral pneumonia, pasteurella pneumonia, distemper pneumonia, verminous pneumonia, chemical pneumonia, and hypersensitivity pneumonitis . type of exudation, with names such as suppurative pneumonia, fibrinous pneumonia, and pyogranulomatous pneumonia . morphologic features, with names such as gangrenous pneumonia, proliferative pneumonia, and embolic pneumonia . distribution of lesions, with names such as focal pneumonia, cranioventral pneumonia, diffuse pneumonia, and lobar pneumonia . epidemiologic attributes, with names such as enzootic pneumonia, contagious bovine pleuropneumonia, and "shipping fever" . geographic regions, with names such as montana progressive pneumonia . miscellaneous attributes, with names such as atypical pneumonia, cuffing pneumonia, progressive pneumonia, aspiration pneumonia, pneumonitis, farmer's lung, and extrinsic allergic alveolitis until a universal and systematic nomenclature for animal pneumonias is established, veterinarians should be acquainted with this heterogeneous list of names and should be well aware that one the parts of the face with the tip of your finger has been advocated by some pathologists. the texture of a normal lung is comparable to the texture of the center of the cheek. firm consolidation is comparable to the texture of the tip of the nose, and hard consolidation is comparable to the texture of the forehead. the term consolidation is frequently used to describe a firm or hard lung filled with exudate. pneumonias ( fig. - ) . texture of pneumonic lungs can be firmer or harder (bronchopneumonias), more elastic (rubbery) than normal lungs (interstitial pneumonias), or have a nodular feeling (granulomatous pneumonias). describing in words the palpable difference between the texture of a normal lung compared with the firm or hard texture of a consolidated lung can be a difficult undertaking. an analogy illustrating this difference based on touching changes in the gross appearance of pneumonic lungs include abnormal color, the presence of nodules or exudate, fibrinous or fibrous adhesions, and the presence of rib imprints on serosal surfaces (see fig. - ). on cut surfaces, pneumonic lungs may have exudate, hemorrhage, edema, necrosis, abscesses, bronchiectasis, granulomas or pyogranulomas, and fibrosis, depending on the stage. palpation and careful observation of the lungs are essential in the diagnosis of pneumonia. (for details, see the section on examination of the respiratory tract.) bronchopneumonia refers to a particular morphologic type of pneumonia in which injury and the inflammatory process take place primarily in the bronchial, bronchiolar, and alveolar lumens. bronchopneumonia is undoubtedly the most common type of pneumonia seen in domestic animals and is with few exceptions characterized grossly by cranioventral consolidation of the lungs (fig. - and see fig. - ). the reason why bronchopneumonias in animals are almost always restricted to the cranioventral portions of the lungs is not well understood. possible factors contributing to this topographic selectivity within the lungs include ( ) gravitational sedimentation of the exudate, ( ) greater deposition of infectious organisms, ( ) inadequate defense mechanisms, ( ) reduced vascular perfusion, ( ) shortness and abrupt branching of airways, and ( ) regional differences in ventilation. the term cranioventral in veterinary anatomy is the equivalent of "anterosuperior" in human anatomy. the latter is defined as "in front (ventral) and above (cranial)." thus, applied to the lung of animals, "cranioventral" means the ventral portion of the cranial lobe. however, by common usage in veterinary pathology, the term cranioventral used to describe the location of lesions in pneumonias has come to mean "cranial and ventral." thus it includes pneumonias affecting not only the ventral portion of the cranial lobe (true cranioventral) but also those cases in which the pneumonia has involved the ventral portions of adjacent lung lobes-initially the middle and then caudal on the right and the caudal lobe on the left side. bronchopneumonias are generally caused by bacteria and mycoplasmas, by bronchoaspiration of feed or gastric contents, or by improper tubing. as a rule, the pathogens causing bronchopneumonias arrive in the lungs via inspired air (aerogenous), either from infected aerosols or from the nasal flora. before establishing infection, pathogens must overwhelm or evade the pulmonary defense mechanisms. the initial injury in bronchopneumonias is centered on the mucosa of bronchioles; from there, the inflammatory process can spread downward to distal portions of the alveoli and upward to the bronchi. typically, for bronchopneumonias, the inflammatory exudates collect in the bronchial, bronchiolar, and alveolar lumina leaving the alveolar interstitium relatively unchanged, except for hyperemia and possibly edema. through the pores of kohn, the exudate can spread to adjacent alveoli until most or all of the alveoli in an individual lobule are involved. if the inflammatory process cannot control the inciting cause of injury, the lesions spread rapidly from lobule to lobule through alveolar pores and destroyed alveolar walls until an entire lobe or large portion of a lung is involved. the lesion tends to spread centrifugally, with the older lesions in the center, and exudate can be coughed up and then aspirated into other lobules, where the inflammatory process starts again. at the early stages of bronchopneumonia, the pulmonary vessels are engorged with blood (active hyperemia), and the bronchi, bronchioles, and alveoli contain some fluid (permeability edema). in cases in which pulmonary injury is mild to moderate, cytokines locally released in the lung cause rapid recruitment of neutrophils and alveolar macrophages into bronchioles and alveoli ( fig. - and see fig. - ). when pulmonary injury is much more severe, proinflammatory cytokines induce more pronounced vascular changes by further opening endothelial gaps, thus increasing vascular permeability resulting in leakage of plasma fibrinogen (fibrinous exudates) and sometimes hemorrhage in the alveoli. alterations in permeability can be further exacerbated by structural damage to pulmonary capillaries and vessels directly caused by microbial toxins. filling of alveoli, bronchioles, and small bronchi with inflammatory exudate progressively obliterates airspaces, and as a consequence of this process, portions of severely affected (consolidated) lungs sink to the bottom of the container when placed in fixative. the replacement of air by exudate also changes the texture of the lungs, and depending on the severity of bronchopneumonia, the texture varies from firmer to harder than normal. the term consolidation is used at gross examination when the texture of pneumonic lung becomes firmer or harder than normal as a result of loss of airspaces because of exudation and atelectasis. (for details, see the discussion of lung texture in the section on classification of pneumonias in domestic animals). inflammatory consolidation of the lungs has been referred to in the past as hepatization because the affected lung had the appearance and texture of liver. the process was referred to as red hepatization in acute cases in which ( ) congestion, ( ) red hepatization (liver texture), ( ) gray hepatization, and ( ) resolution. because of the use of effective antibiotics and prevention, pneumococcal pneumonia and its four classic stages are rarely seen; thus this terminology has been largely abandoned. currently, the term bronchopneumonia is widely used for both suppurative and fibrinous consolidation of the lungs because both forms of inflammation have essentially the same pathogenesis in which the pathogens reach the lung by the aerogenous route, injury occurs initially in the bronchial and bronchiolar regions, and the inflammatory process extends centrifugally deep into the alveoli. it must be emphasized that it is the severity of pulmonary injury that largely determines whether bronchopneumonia becomes suppurative or fibrinous. in some instances, however, it is difficult to discriminate between suppurative and fibrinous bronchopneumonia because both types can coexist (fibrinosuppurative bronchopneumonia), and one type can progress to the other. suppurative bronchopneumonia. suppurative bronchopneumonia is characterized by cranioventral consolidation of lungs (see figs. - and - ), with typically purulent or mucopurulent exudate present in the airways. this exudate can be best demonstrated by expressing intrapulmonary bronchi, thus forcing exudate out of the bronchi (see fig. - ). the inflammatory process in suppurative bronchopneumonia is generally confined to individual lobules, and as a result of this distribution, the lobular pattern of the lung becomes notably emphasized. this pattern is particularly obvious in cattle and pigs because these species have prominent lobulation of the lungs. the gross appearance often resembles an irregular checkerboard because of an admixture of normal and abnormal (consolidated) lobules (see fig. - ). because of this typical lobular distribution, suppurative bronchopneumonias are also referred to as lobular pneumonias. different inflammatory phases occur in suppurative bronchopneumonia where the color and appearance of consolidated lungs varies considerably, depending on the virulence of offending organisms and chronicity of the lesion. the typical phases of suppurative bronchopneumonia can be summarized as follows: . during the first hours when bacteria are rapidly multiplying, the lungs become hyperemic and edematous. . soon after, neutrophils start filling the airways, and by hours the parenchyma starts to consolidate and becomes firm in texture. . three to days later, hyperemic changes are less obvious, but the bronchial, bronchiolar, and alveolar spaces continue to fill with neutrophils and macrophages, and the affected lung sinks when placed in formalin. at this stage, the affected lung has a gray-pink color, and on cut surface, purulent exudate can be expressed from bronchi. . in favorable conditions where the infection is under control of the host defense mechanisms, the inflammatory processes begin to regress, a phase known as resolution. complete resolution in favorable conditions could take to weeks. . in animals in which the lung infection cannot be rapidly contained, inflammatory lesions can progress into a chronic phase. approximately to days after infection, the lungs become pale gray and take a "fish flesh" appearance. this appearance is the result of purulent and catarrhal inflammation, obstructive atelectasis, mononuclear cell infiltration, peribronchial and peribronchiolar lymphoid hyperplasia, and early alveolar fibrosis. complete resolution is unusual in chronic bronchopneumonia, and lung scars, such as pleural and pulmonary fibrosis; bronchiectasis as a consequence of chronic destructive bronchitis (see bronchiectasis [dysfunction/responses to injury and patterns of injury]); atelectasis; pleural adhesions; and lung abscesses may remain unresolved there was notable active hyperemia with little exudation of neutrophils; conversely, the process was referred to as gray hepatization in those chronic cases in which hyperemia was no longer present, but there was abundant exudation of neutrophils and macrophages. this terminology, although used for and applicable to human pneumonias, is rarely used in veterinary medicine primarily because the evolution of pneumonic processes in animals does not necessarily follow the red-to-gray hepatization pattern. bronchopneumonia can be subdivided into suppurative bronchopneumonia if the exudates are predominantly composed of neutrophils and fibrinous bronchopneumonia if fibrin is the predominant component of the exudates (see table - ). it is important to note that some veterinarians use the term fibrinous pneumonia or lobar pneumonia as a synonym for fibrinous bronchopneumonia, and bronchopneumonia or lobular pneumonia as a synonym for suppurative bronchopneumonia. human pneumonias for many years have been classified based on their etiology and morphology, which explains why pneumococcal pneumonia (streptococcus pneumoniae) has been synonymous with lobar pneumonia. in the old literature, four distinct stages of pneumococcal pneumonia were described as lumen of bronchiole capillary pulmonary defense mechanisms to allow them to colonize the lungs and establish an infection. suppurative bronchopneumonia can also result from aspiration of bland material (e.g., milk). pulmonary gangrene may ensue when the bronchopneumonic lung is invaded by saprophytic bacteria (aspiration pneumonia). fibrinous bronchopneumonia. fibrinous bronchopneumonia is similar to suppurative bronchopneumonia except that the predominant exudate is fibrinous rather than neutrophilic. with only a few exceptions, fibrinous bronchopneumonias also have a cranioventral distribution (fig. - and see fig. - ) . however, exudation is not restricted to the boundaries of individual pulmonary lobules, as is the case in suppurative bronchopneumonias. instead, the inflammatory process in fibrinous pneumonias involves numerous contiguous lobules and the exudate moves quickly through pulmonary tissue until the entire pulmonary lobe is rapidly affected. because of the involvement of the entire lobe and pleural surface, fibrinous bronchopneumonias are also referred to as lobar pneumonias or pleuropneumonias. in general terms, fibrinous bronchopneumonias are the result of more severe pulmonary injury and thus cause death earlier in the sequence of the inflammatory process than suppurative bronchopneumonias. even in cases in which fibrinous bronchopneumonia involves % or less of the total area, clinical signs and death can occur as a result of severe toxemia and sepsis. the gross appearance of fibrinous bronchopneumonia depends on the age and severity of the lesion and on whether the pleural surface or the cut surface of the lung is viewed. externally, early stages of fibrinous bronchopneumonias are characterized by severe congestion and hemorrhage, giving the affected lungs a characteristically intense red discoloration. a few hours later, fibrin starts to permeate and accumulate on the pleural surface, giving the pleura a ground glass appearance and eventually forming plaques of fibrinous exudate over a red, dark lung (see fig. - ). at this stage, a yellow fluid starts to accumulate in the thoracic cavity. the color of fibrin deposited over the pleural surface is also variable. it can be bright yellow when the exudate is formed primarily by fibrin, tan when fibrin is mixed with blood, and gray when a large number of leukocytes and fibroblasts are part of the fibrinous plaque in more chronic cases. because of the tendency of fibrin to deposit on the pleural surface, some pathologists use the term pleuropneumonia as a synonym for fibrinous bronchopneumonia. on the cut surface, early stages of fibrinous bronchopneumonia appear as simple red consolidation. in more advanced cases ( hours), fibrinous bronchopneumonia is generally accompanied by notable dilation and thrombosis of lymph vessels and edema of interlobular septa (see fig. - , b) . this distention of the interlobular septa gives affected lungs a typical marbled appearance. distinct focal areas of coagulative necrosis in the pulmonary parenchyma are also common in fibrinous bronchopneumonia such as in shipping fever pneumonia and contagious bovine pleuropneumonia. in animals that survive the early stage of fibrinous bronchopneumonia, pulmonary necrosis often develops into pulmonary "sequestra," which are isolated pieces of necrotic lung encapsulated by connective tissue. pulmonary sequestra result from extensive necrosis of lung tissue either from severe ischemia (infarct) caused by thrombosis of a major pulmonary vessel such as in contagious bovine pleuropneumonia or from the effect of necrotizing toxins released by pathogenic bacteria such as mannheimia haemolytica. sequestra in veterinary pathology should not be confused with "bronchopulmonary sequestration," a term used in human pathology to describe a congenital malformation in which whole lobes or parts of the lung develop without normal connections to the airway or vascular systems. for a long time. "enzootic pneumonias" of ruminants and pigs are typical examples of chronic suppurative bronchopneumonias. microscopically, acute suppurative bronchopneumonias are characterized by hyperemia, abundant neutrophils, macrophages, and cellular debris within the lumen of bronchi, bronchioles, and alveoli (see fig. - ). recruitment of leukocytes is promoted by cytokines, complement, and other chemotactic factors that are released in response to alveolar injury or by the chemotactic effect of bacterial toxins, particularly endotoxin. in most severe cases, purulent or mucopurulent exudates completely obliterate the entire lumen of bronchi, bronchioles, and alveoli. if suppurative bronchopneumonia is merely the response to a transient pulmonary injury or a mild infection, lesions resolve uneventfully. within to days, cellular exudate can be removed from the lungs via the mucociliary escalator, and complete resolution may take place within weeks. in other cases, if injury or infection is persistent, suppurative bronchopneumonia can become chronic with goblet cell hyperplasia, an important component of the inflammatory process. depending on the proportion of pus and mucus, the exudate in chronic suppurative bronchopneumonia varies from mucopurulent to mucoid. a mucoid exudate is found in the more chronic stages when the consolidated lung has a "fish flesh" appearance. hyperplasia of balt is another change commonly seen in chronic suppurative bronchopneumonias; it appears grossly as conspicuous white nodules (cuffs) around bronchial walls (cuffing pneumonia). this hyperplastic change merely indicates a normal reaction of lymphoid tissue to infection. further sequelae of chronic suppurative bronchopneumonia include bronchiectasis (see figs. - and - ), pulmonary abscesses, pleural adhesions (from pleuritis) ( fig. - ) , and atelectasis and emphysema from completely or partially obstructed bronchi or bronchioles (e.g., bronchiectasis). clinically, suppurative bronchopneumonias can be acute and fulminating but are often chronic, depending on the etiologic agent, stressors affecting the host, and immune status. the most common pathogens causing suppurative bronchopneumonia in domestic animals include pasteurella multocida, bordetella bronchiseptica, trueperella (arcanobacterium) pyogenes, streptococcus spp., escherichia coli, and several species of mycoplasmas. most of these organisms are secondary pathogens requiring a preceding impairment of the fulminating hemorrhagic bronchopneumonia can be caused by highly pathogenic bacteria such as bacillus anthracis. although the lesions in anthrax are primarily related to a severe septicemia and sepsis, anthrax should always be suspected in animals with sudden death and exhibiting severe acute fibrinohemorrhagic pneumonia, splenomegaly, and multisystemic hemorrhages. animals are considered good sentinels for anthrax in cases of bioterrorism. interstitial pneumonia refers to that type of pneumonia in which injury and the inflammatory process take place primarily in any microscopically, in the initial stage of fibrinous bronchopneumonia, there is massive exudation of plasma proteins into the bronchioles and alveoli, and as a result, most of the airspaces become obliterated by fluid and fibrin. leakage of fibrin and fluid into alveolar lumina is due to extensive disruption of the integrity and increased permeability of the blood-air barrier. fibrinous exudates can move from alveolus to alveolus through the pores of kohn. because fibrin is chemotactic for neutrophils, these types of leukocytes are always present a few hours after the onset of fibrinous inflammation. as inflammation progresses ( to days), fluid exudate is gradually replaced by fibrinocellular exudates composed of fibrin, neutrophils, macrophages, and necrotic debris ( fig. - ). in chronic cases (after days), there is notable fibrosis of the interlobular septa and pleura. in contrast to suppurative bronchopneumonia, fibrinous bronchopneumonia rarely resolves completely, thus leaving noticeable scars in the form of pulmonary fibrosis and pleural adhesions. the most common sequelae found in animals surviving an acute episode of fibrinous bronchopneumonia include alveolar fibrosis and bronchiolitis obliterans, in which organized exudate becomes attached to the bronchiolar lumen (see fig. - ) . these changes are collectively referred to as bronchiolitis obliterans organizing pneumonia (boop), a common microscopic finding in animals with unresolved bronchopneumonia. other important sequelae include pulmonary gangrene, when saprophytic bacteria colonize necrotic lung; pulmonary sequestra; pulmonary fibrosis; abscesses; and chronic pleuritis with pleural adhesions. in some cases, pleuritis can be so extensive that fibrous adhesions extend onto the pericardial sac. pathogens causing fibrinous bronchopneumonias in domestic animals include mannheimia (pasteurella) haemolytica (pneumonic mannheimiosis), histophilus somni (formerly haemophilus somnus), actinobacillus pleuropneumoniae (porcine pleuropneumonia), mycoplasma bovis, and mycoplasma mycoides ssp. mycoides small colony type (contagious bovine pleuropneumonia). fibrinous broncho- a b n alveolar epithelium. inhaled antigens, such as fungal spores, combine with circulating antibodies and form deposits of antigen-antibody complexes (type iii hypersensitivity) in the alveolar wall, which initiate a cascade of inflammatory responses and injury (allergic alveolitis). hematogenous injury to the vascular endothelium occurs in septicemias, sepsis, dic, larva migrans (ascaris suum), toxins absorbed in the alimentary tract (endotoxin) or toxic metabolites locally generated in the lungs ( -methylindole and paraquat), release of free radicals in alveolar capillaries (ards), and infections with endotheliotropic viruses (canine adenovirus and classical swine fever [hog cholera]). interstitial pneumonias in domestic animals and human beings are subdivided based on morphologic features into acute and chronic. it should be kept in mind, however, that not all acute interstitial pneumonias are fatal and that they do not necessarily progress to the chronic form. acute interstitial pneumonias. acute interstitial pneumonias begin with injury to either type i pneumonocytes or alveolar capillary endothelium, which provokes a disruption of the blood-air barrier and a subsequent exudation of plasma proteins into the alveolar space (see fig. - ) . this leakage of proteinaceous fluid into the alveolar lumen constitutes the exudative phase of acute interstitial pneumonia. in some cases of diffuse alveolar damage, exuded plasma proteins mix with lipids and other components of pulmonary surfactant and form elongated membranes that become partially attached to the alveolar basement membrane and bronchiolar walls. these membranes are referred to as hyaline membranes because of their hyaline appearance (eosinophilic, homogeneous, and amorphous) microscopically (see figs. - and - ). in addition to intraalveolar exudation of fluid, inflammatory edema and neutrophils accumulate in the alveolar interstitium and cause thickening of the alveolar walls. this acute exudative phase is generally followed a few days later by the proliferative phase of acute interstitial pneumonia, which is characterized by hyperplasia of type ii pneumonocytes to replace the lost type i pneumonocytes (see fig. - ). type ii pneumonocytes are in fact progenitor cells that differentiate and replace necrotic type i pneumonocytes (see fig. - ) . as a consequence, the alveolar walls become increasingly thickened. this process is in part the reason why lungs become rubbery on palpation, what prevents their normal collapse after the thorax is opened, and why the cut surface of the lung has a "meaty" appearance (see fig. - ). of the three layers of the alveolar walls (endothelium, basement membrane, and alveolar epithelium) and the contiguous bronchiolar interstitium (see fig. - ) . this morphologic type of pneumonia is the most difficult to diagnose at necropsy and requires microscopic confirmation because it is easily mistaken in the lung showing congestion, edema, hyperinflation, or emphysema. in contrast to bronchopneumonias, in which distribution of lesions is generally cranioventral, in interstitial pneumonias, lesions are more diffusely distributed and generally involve all pulmonary lobes, or in some cases, they appear to be more pronounced in the dorsocaudal aspects of the lungs (see fig. - ). three important gross features of interstitial pneumonia are ( ) the failure of lungs to collapse when the thoracic cavity is opened, ( ) the occasional presence of rib impressions on the pleural surface of the lung indicating poor deflation, and ( ) the lack of visible exudates in airways unless complicated with secondary bacterial pneumonia. the color of affected lungs varies from diffusely red in acute cases to diffusely pale gray to a mottled red, pale appearance in chronic ones. pale lungs are caused by severe obliteration of alveolar capillaries (reduced blood-tissue ratio), especially evident when there is fibrosis of the alveolar walls. the texture of lungs with uncomplicated interstitial pneumonia is typically elastic or rubbery, but definitive diagnosis based on texture alone is difficult and requires histopathologic examination. on a cut surface, the lungs may appear and feel more "meaty" (having the texture of raw meat) and have no evidence of exudate in the bronchi or pleura (fig. - ). in acute interstitial pneumonias, particularly in cattle, there is frequently pulmonary edema (exudative phase) and interstitial emphysema secondary to partial obstruction of bronchioles by edema fluid and strenuous air gasping before death. because edema tends to gravitate into the cranioventral portions of the lungs, and emphysema is often more obvious in the dorsocaudal aspects, acute interstitial pneumonias in cattle occasionally have a gross cranioventral-like pattern that may resemble bronchopneumonia, although the texture is different. lungs are notably heavy because of the edema and the infiltrative and proliferative changes. the pathogenesis of interstitial pneumonia is complex and can result from aerogenous injury to the alveolar epithelium (type i and ii pneumonocytes) or from hematogenous injury to the alveolar capillary endothelium or alveolar basement membrane. aerogenous inhalation of toxic gases (i.e., ozone and no ) or toxic fumes (smoke inhalation) and infection with pneumotropic viruses (influenza, herpesviruses, or canine distemper virus) can damage the figure - interstitial pneumonia, lung, feeder pig. a, the lung is heavy, pale, and rubbery in texture. it also has prominent costal (rib) imprints (arrows), a result of hypercellularity of the interstitium and the failure of the lungs to collapse when the thorax was opened. b, transverse section. the pulmonary parenchyma has a "meaty" appearance and some edema, but no exudate is present in airways or on the pleural surface. this type of lung change in pigs is highly suggestive of a viral pneumonia. (courtesy dr. a. lópez, atlantic veterinary college.) pneumonia are centered in the alveolar wall and its interstitium, a mixture of desquamated epithelial cells, macrophages, and mononuclear cells are usually present in the lumens of bronchioles and alveoli. ovine progressive pneumonia, hypersensitivity pneumonitis in cattle and dogs, and silicosis in horses are good veterinary examples of chronic interstitial pneumonia. pneumoconioses (silicosis and asbestosis), paraquat toxicity, pneumotoxic antineoplastic drugs (bleomycin), and extrinsic allergic alveolitis (farmer's lung) are well-known examples of diseases that lead to chronic interstitial pneumonias in human beings. massive pulmonary migration of ascaris larvae in pigs also causes interstitial pneumonia ( fig. - ). there is an insidious and poorly understood group of chronic idiopathic interstitial diseases, both in human beings and in animals, that eventually progress to terminal interstitial fibrosis. these were originally thought to be the result of repeated cycles of alveolar injury, inflammation, and fibroblastic/myoblastic response to an unknown agent. however, aggressive antiinflammatory therapy generally fails to prevent or reduce the severity of fibrosis. now, it is acute interstitial pneumonias are often mild and transient, especially those caused by some respiratory viruses, such as those responsible for equine and porcine influenza. these mild forms of pneumonia are rarely seen in the postmortem room because they are not fatal and do not leave significant sequelae (see the section on defense mechanisms/barrier systems). in severe cases of acute interstitial pneumonias, animals may die of respiratory failure, usually as a result of diffuse alveolar damage, a profuse exudative phase (leakage of proteinaceous fluid) leading to a fatal pulmonary edema. examples of this type of fatal acute interstitial pneumonia are bovine pulmonary edema and emphysema, and ards in all species. chronic interstitial pneumonia. when the source of alveolar injury persists, the exudative and proliferative lesions of acute interstitial pneumonia can progress into a morphologic stage referred to as chronic interstitial pneumonia. the hallmark of chronic interstitial pneumonia is fibrosis of the alveolar walls (with or without intraalveolar fibrosis) and the presence of lymphocytes, macrophages, fibroblasts, and myofibroblasts in the alveolar interstitium (figs. - and - ). in other cases, these chronic changes are accompanied by hyperplasia and persistence of type ii pneumonocytes, squamous metaplasia of the alveolar epithelium, microscopic granulomas, and hyperplasia of smooth muscle in bronchioles and pulmonary arterioles. it should be emphasized that although the lesions in interstitial the term bronchointerstitial pneumonia is used in veterinary pathology to describe cases in which microscopic lesions share some histologic features of both bronchopneumonia and interstitial pneumonia (e- fig. - ). this combined type of pneumonia is in fact frequently seen in many viral infections in which viruses replicate and cause necrosis in bronchial, bronchiolar, and alveolar cells. damage to the bronchial and bronchiolar epithelium causes an influx of neutrophils similar to that in bronchopneumonias, and damage to alveolar walls causes proliferation of type ii pneumonocytes, similar to that which takes place in the proliferative phase of acute interstitial pneumonias. it is important to emphasize that bronchointerstitial pneumonia is a microscopic not a gross diagnosis. examples include uncomplicated cases of respiratory syncytial virus infections in cattle and lambs, canine distemper, and influenza in pigs and horses. embolic pneumonia refers to a particular type of pneumonia in which gross and microscopic lesions are multifocally distributed in all pulmonary lobes. by definition, lung injury is hematogenous, and the inflammatory response is typically centered in pulmonary arterioles and alveolar capillaries. lungs act as a biologic filter for circulating particulate matter. sterile thromboemboli, unless extremely large, are rapidly dissolved and removed from the pulmonary vasculature by fibrinolysis, causing little, if any, ill effects. experimental studies have confirmed that most types of bacteria when injected intravenously (bacteremia) are phagocytosed by pulmonary intravascular macrophages, or they bypass the lungs and are finally trapped by macrophages in the liver, spleen, joints, or other organs. to cause pulmonary infection, circulating bacteria must first attach to the pulmonary endothelium with specific binding proteins or simply attach to intravascular fibrin and then evade phagocytosis by intravascular macrophages or leukocytes. septic thrombi facilitate entrapment of bacteria in the pulmonary vessels and provide a favorable environment to escape phagocytosis. once trapped in the pulmonary vasculature, usually in small arterioles or alveolar capillaries, offending bacteria disrupt endothelium and basement membranes, spread from the vessels to the interstitium and then to the surrounding lung, finally forming a new nidus of infection. embolic pneumonia is characterized by multifocal lesions randomly distributed in all pulmonary lobes (see fig. - and e-figs. - and - ). early lesions in embolic pneumonia are characterized grossly by the presence of very small ( to mm), white foci surrounded by discrete, red, hemorrhagic halos ( fig. - ). unless emboli arrive in massive numbers, causing fatal pulmonary edema, embolic pneumonia is seldom fatal; therefore these acute lesions are rarely seen at postmortem examination. in most instances, if unresolved, acute lesions rapidly progress to pulmonary abscesses. these are randomly distributed in all pulmonary lobes and are not restricted to the cranioventral aspects of the lungs, as is the case of abscesses developing from suppurative bronchopneumonia. the early microscopic lesions in embolic pneumonias are always focal or multifocal ( fig. - ) ; thus they differ from those of endotoxemia or septicemia, in which endothelial damage and interstitial reactions (interstitial pneumonia) are diffusely distributed in the lungs. when embolic pneumonia or its sequela (abscesses) is diagnosed at necropsy, an attempt should be made to locate the source of septic emboli. the most common sources are hepatic abscesses that have ruptured into the caudal vena cava in cattle, omphalophlebitis in farm animals, chronic bacterial skin or hoof infections, and a contaminated catheter in all species (see fig. - ) . valvular or mural endocarditis in the right heart is a common source of septic emboli and embolic pneumonia in all species. most frequently, bacterial proposed that a genetic mutation alters the cell-cell communication between epithelial and mesenchymal cells in the lung. this aberrant cellular communication leads to an overexpression of inflammatory and repair molecules (i.e., il- , il- , tgf-β , and caveolin), leading to increased apoptosis and interstitial deposition of extracellular matrix (ecm). the chronic interstitial (restrictive) diseases in human medicine include "idiopathic pulmonary fibrosis," "nonspecific interstitial pneumonia," "unusual interstitial pneumonia," and "cryptogenic organizing pneumonia," also referred to as idiopathic bronchiolitis obliterans organizing pneumonia (idiopathic boop). feline idiopathic pulmonary fibrosis is an example of this type of progressive interstitial disease in veterinary medicine. it has been reported that in rare cases, chronic alveolar remodeling and interstitial fibrosis can progress to lung cancer. the lung has numerous circular areas of hemorrhage distributed randomly throughout all lung lobes (embolic pattern [see fig. - ]). these foci arise from injury to the microvasculature in alveolar septa and the visceral pleura secondary to lodgment of bacterial or fungal emboli (septic emboli) from valvular or mural endocarditis in the right heart or from other bacterial or fungal diseases where the bacterium or fungus gains access to the circulatory system as occurs in many bacterial and fungal enteritides or pneumonias caused by salmonella spp., e. coli, or aspergillus spp. the pathogenesis of granulomatous pneumonia shares some similarities with that of interstitial and embolic pneumonias. not surprisingly, some pathologists group granulomatous pneumonias within one of these types of pneumonias (e.g., granulomatous interstitial pneumonia). what makes granulomatous pneumonia a distinctive type is not so much the portal of entry or site of initial injury in the lungs but, rather, the unique type of inflammatory response that results in the formation of granulomas, which can be easily recognized at gross and microscopic examination. as a rule, agents causing granulomatous pneumonias are resistant to intracellular killing by phagocytic cells and to the acute inflammatory response, allowing prolonged persistence of these agents in tissues. the most common causes of granulomatous pneumonia in animals include systemic fungal diseases, such as cryptococcosis (cryptococcus neoformans and cryptococcus gatti), coccidioidomycosis (coccidioides immitis), histoplasmosis (histoplasma capsulatum), and blastomycosis (blastomyces dermatitidis) (see fig. - ). in most of these fungal diseases, the port of entry is aerogenous, and from the lungs the fungi disseminate systemically to other organs, particularly the lymph nodes, liver, and spleen. filamentous fungi such as aspergillus spp. or mucor spp. can also reach the lung by the hematogenous route. granulomatous pneumonia is also seen in some bacterial diseases, such as tuberculosis (mycobacterium bovis) in all species and rhodococcus equi in horses. sporadically, aberrant parasites such as fasciola hepatica in cattle and aspiration of foreign bodies can also cause granulomatous pneumonia (e- fig. - granulomatous pneumonia is characterized by the presence of variable numbers of caseous or noncaseous granulomas randomly distributed in the lungs (see fig. - ). on palpation, lungs have a typical nodular character given by well-circumscribed, variably sized nodules that generally have a firm texture, especially if calcification has occurred ( fig. - ) . during postmortem examination, granulomas in the lungs occasionally can be mistaken for neoplasms. microscopically, pulmonary granulomas are composed of a center of necrotic tissue, surrounded by a rim of macrophages (epithelioid cells) and giant cells and an outer delineated layer of connective tissue commonly infiltrated by lymphocytes and plasma cells ( fig. - ). unlike other types of pneumonias, the causative agent in granulomatous pneumonia can, in many cases, be identified isolates from septic pulmonary emboli in domestic animals are trueperella (arcanobacterium) pyogenes (cattle), fusobacterium necrophorum (cattle, pigs, and human beings), erysipelothrix rhusiopathiae (pigs, cattle, dogs, and human beings), streptococcus suis (pigs), staphylococcus aureus (dogs and human beings), and streptococcus equi (horses). granulomatous pneumonia refers to a particular type of pneumonia in which aerogenous or hematogenous injury is caused by organisms or particles that cannot normally be eliminated by phagocytosis and that evoke a local inflammatory reaction with numerous alveolar and interstitial macrophages, lymphocytes, a few neutrophils, and sometimes giant cells. the term granulomatous is used here to describe an anatomic pattern of pneumonia typically characterized by the presence of granulomas. g g but yet unproven that viral infections may also predispose horses to airway hyperresponsiveness and recurrent airway obstruction (rao). equine influenza. equine influenza is an important and highly contagious flulike respiratory disease of horses characterized by high morbidity and low mortality and explosive outbreaks in susceptible populations. it is an oie-notifiable disease. two antigenically unrelated subtypes of equine influenza virus have been identified (h n [a/equi- ] and h n [a/equi- ]). the course of the disease is generally mild and transient, and its importance is primarily because of its economic impact on horse racing. the types of injury and host response in the conducting system are described in the section on disorders of the nasal cavity and paranasal sinuses of horses. uncomplicated lesions in the lungs are mild and self-limiting bronchointerstitial pneumonia. in fatal cases, the lungs are hyperinflated with coalescing areas of dark red discoloration. microscopically, there is a bronchointerstitial pneumonia characterized by necrotizing bronchiolitis that is followed by hyperplastic bronchiolitis, hyperplasia of type ii pneumonocytes, hyaline membranes in alveoli, and sporadic multinucleated giant cells. the microscopic changes are ards in severe and fatal cases. the influenza virus antigen can be readily demonstrated in ciliated cells and alveolar macrophages. clinical signs are characterized by fever, cough, abnormal lung sounds (crackles and wheezes), anorexia, and depression. secondary bacterial infections (streptococcus equi, streptococcus zooepidemicus, staphylococcus aureus, and escherichia coli) commonly complicate equine influenza. equine viral rhinopneumonitis. equine viral rhinopneumonitis (evr), or equine herpesvirus infection, is a respiratory disease of young horses that is particularly important in weanlings between and months of age and to a much lesser extent in young foals and adult horses. the causative agents are ubiquitous equine herpesviruses (ehv- and ehv- ) that in addition to respiratory disease can cause abortion in pregnant mares and neurologic disease (equine herpes myeloencephalopathy) (see the section on disorders of the nasal cavity and paranasal sinuses of horses). the respiratory form of evr is a mild and a transient bronchointerstitial pneumonia seen only by pathologists when complications with secondary bacterial infections cause a fatal bronchopneumonia microscopically in sections by pas reaction or grocott-gomori's methenamine silver (gms) stain for fungi or by an acid-fast stain for mycobacteria. viral infections of the respiratory tract, particularly equine viral rhinopneumonitis and equine influenza, are important diseases of horses throughout the world. the effects of these and other respiratory viruses on the horse can be manifested in three distinct ways. first, as pure viral infections, their severity may range from mild to severe, making them a frequent interfering factor in training and athletic performance. second, superimposed infections by opportunistic bacteria, such as streptococcus spp., escherichia coli, klebsiella pneumoniae, rhodococcus equi, and various anaerobes, can cause fibrinous or suppurative bronchopneumonias. third, it is possible equine henipavirus (hendra virus). fatal cases of a novel respiratory disease in horses and human beings suddenly appeared in approximately in hendra, a suburb of brisbane, australia. this outbreak was attributed to a newly recognized zoonotic virus that was tentatively named equine morbillivirus. now called hendra virus (hev), this emerging viral pathogen is currently classified as a member of the genus henipavirus (includes hendra virus and nipah virus), in the family paramyxoviridae. fruit bats (flying foxes) act as natural reservoirs and are involved in the transmission by poorly understood mechanisms. the lungs of affected horses are severely edematous with gelatinous distention of pleura and subpleural lymph vessels. microscopically, the lungs have diffuse alveolar edema associated with vasculitis, thrombosis, and the presence of multinucleated syncytial cells in the endothelium of small pulmonary blood vessels and alveolar capillaries. the lymphatic vessels are notably distended with fluid. the characteristic inclusion bodies seen in other paramyxovirus infections are not seen in horses; however, the virus can be easily detected by immunohistochemistry in pulmonary endothelial cells and alveolar epithelial cells (pneumonocytes). clinical signs are nonspecific and include fever, anorexia, respiratory distress, and nasal discharge. equine multinodular pulmonary fibrosis. equine multinodular pulmonary fibrosis is a lung disease characterized by well-demarcated fibrotic nodular lesions in the lung (e- fig. - ). until recently, the pathogenesis was unclear, but recent studies proposed equine herpesvirus (ehv- ) as the putative etiology. grossly, the lungs show multifocal to coalescing, firm tan nodules scattered in all pulmonary lobes, which resemble pulmonary neoplasia. microscopically, alveolar walls are thickened due to collagen deposition, infiltration of lymphocytes and macrophages, and cuboidal cells lining the alveolar walls. the alveolar lumens contain neutrophils and macrophages, some of which may contain a large eosinophilic intranuclear inclusion body. typical clinical signs include weight loss, low-grade fever, and progressive exercise intolerance. this condition has a poor prognosis. rhodococcus equi. rhodococcus equi is an important cause of morbidity and mortality in foals throughout the world. this facultative intracellular gram-positive bacterium causes two major forms of disease: the first involves the intestine, causing ulcerative enterocolitis, and the second severe and often fatal bronchopneumonia. although half of foals with pneumonia have ulcerative enterocolitis, it is rare to find animals with intestinal lesions alone. occasionally, infection disseminates to lymph nodes, joints, bones, the genital tract, and other organs. because rhodococcus equi is present in soil and feces of herbivores (particularly foals), it is common for the disease to become enzootic on farms ("hot spots") where the organism has been shed earlier by infected foals. serologic evidence of infection in horses is widespread, yet clinical disease is sporadic and largely restricted to young foals or to adult horses with severe immunosuppression. virulence factors encoded by plasmids (virulenceassociated protein a [vapa gene]) are responsible for the survival and replication of rhodococcus equi in macrophages, thus determining the evolution of the disease. this bacterium has also been sporadically incriminated with infections in cattle, goats, pigs, dogs, and cats, and quite often in immunocompromised human beings, for example, those infected with the aids virus, after organ transplantation, or undergoing chemotherapy. it is still debatable whether natural infection starts as a bronchopneumonia (aerogenous route) from which rhodococcus equi reaches the intestine via swallowed sputum or whether infection starts as an enteritis (oral route) with a subsequent bacteremia into the lungs. (streptococcus equi, streptococcus zooepidemicus, or staphylococcus aureus) . uncomplicated lesions in evr are seen only in aborted fetuses or in foals that die within the first few days of life. they consist of focal areas of necrosis ( . to mm) in various organs, including liver, adrenal glands, and lungs. in some cases, intranuclear inclusion bodies are microscopically observed in these organs. outbreaks of interstitial pneumonia in donkeys have been attributed to multiple strains of asinine herpesviruses (ahv- and - ). clinically, horses and donkeys affected with the respiratory form of evr exhibit fever, anorexia, conjunctivitis, cough, and nasal discharge. equine viral arteritis. equine viral arteritis (eva), a pansystemic disease of horses, donkeys, and mules caused by an arterivirus (equine arteritis virus [eav]), occurs sporadically throughout the world, sometimes as an outbreak. this virus infects and causes severe injury to macrophages and endothelial cells. gross lesions are hemorrhage and edema in many sites, including lungs, intestine, scrotum, and periorbital tissues and voluminous hydrothorax and hydroperitoneum. the basic lesion is fibrinoid necrosis and inflammation of the vessel walls (vasculitis), particularly the small muscular arteries (lymphocytic arteritis), which is responsible for the edema and hemorrhage that explain most of the clinical features. pulmonary lesions are those of interstitial pneumonia with hyperplasia of type ii pneumonocytes and vasculitis with abundant edema in the bronchoalveolar spaces and distended pulmonary lymphatic vessels. viral antigen can be detected by immunoperoxidase techniques in the walls and endothelial cells of affected pulmonary vessels and in alveolar macrophages. clinical signs are respiratory distress, fever, abortion, diarrhea, colic, and edema of the limbs and ventral abdomen. respiratory signs are frequent and consist of serous or mucopurulent rhinitis and conjunctivitis with palpebral edema. like most viral respiratory infections, eva can predispose horses to opportunistic bacterial pneumonias. african horse sickness. african horse sickness (ahs) is an arthropod-borne, oie-notifiable disease of horses, mules, donkeys, and zebras that is caused by an orbivirus (family reoviridae) and characterized by respiratory distress or cardiovascular failure. ahs has a high mortality rate-up to % in the native population of horses in africa, the middle east, india, pakistan, and, most recently, spain and portugal. although the ahs virus is transmitted primarily by insects (culicoides) to horses, other animals, such as dogs, can be infected by eating infected equine flesh. the pathogenesis of african horse sickness remains unclear, but this equine orbivirus has an obvious tropism for pulmonary and cardiac endothelial cells and, to a lesser extent, mononuclear cells. based on clinical signs (not pathogenesis), african horse sickness is arbitrarily divided into four different forms: pulmonary, cardiac, mixed, and mild. the pulmonary form is characterized by severe respiratory distress and rapid death because of massive pulmonary edema, presumably from viral injury to the pulmonary endothelial cells. grossly, large amounts of froth are present in the airways, lungs fail to collapse, subpleural lymph vessels are distended, and the ventral parts of the lungs are notably edematous (see fig. - ) . in the cardiac form, recurrent fever is detected, and heart failure results in subcutaneous and interfascial edema, most notably in the neck and supraorbital region. the mixed form is a combination of the respiratory and cardiac forms. finally, the mild form, rarely seen in postmortem rooms, is characterized by fever and clinical signs resembling those of equine influenza; it is in most cases transient and followed by a complete recovery. this mild form is most frequently seen in donkeys, mules, and zebras and in horses with some degree of immunity. detection of viral antigen for diagnostic purposes can be done by immunohistochemistry in paraffin-embedded tissues. chapter respiratory system, mediastinum, and pleurae clinically, rhodococcus equi infection can be acute, with rapid death caused by severe bronchopneumonia, or chronic, with depression, cough, weight loss, and respiratory distress. in either form, there may be diarrhea, arthritis, osteomyelitis, or subcutaneous abscess formation. parascaris equorum. parascaris equorum is a large nematode (roundworm) of the small intestine of horses; the larval stages migrate through the lungs as ascarid larvae do in pigs. it is still unclear whether migration of parascaris equorum larvae can cause significant pulmonary lesions under natural conditions. experimentally, migration of larvae results in coughing, anorexia, weight loss, and small necrotic foci and petechial hemorrhages in the liver, hepatic and tracheobronchial lymph nodes, and lungs. microscopically, eosinophils are prominent in the interstitium and airway mucosa during the parasitic migration and in focal granulomas caused by dead larvae in the lung. dictyocaulus arnfieldi. dictyocaulus arnfieldi is not a very pathogenic nematode, but it should be considered if there are signs of coughing in horses that are pastured together with donkeys. donkeys are considered the natural hosts and can tolerate large numbers of parasites without ill effects. dictyocaulus arnfieldi does not usually become patent in horses, so examination of fecal samples is not useful; bal is only occasionally diagnostic because eosinophils (but not parasites) are typically found in the lavage fluid. mature parasites (up to cm in length) cause obstructive bronchitis, edema, and atelectasis, particularly along the dorsocaudal lung. the microscopic lesion is an eosinophilic bronchitis similar to the less acute infestations seen in cattle and sheep with their dictyocaulus species. the results of experimental studies suggest that natural infection likely starts from inhalation of infected dust or aerosols. once in the lung, rhodococcus equi is rapidly phagocytosed by alveolar macrophages, but because of defective phagosome-lysosome fusion and premature lysosomal degranulation, bacteria survive and multiply intracellularly, eventually leading to the destruction of the macrophage. interestingly, rhodococcus equi appears to be easily killed by neutrophils but not macrophages. released cytokines and lysosomal enzymes and bacterial toxins are responsible for extensive caseous necrosis of the lungs and the recruitment of large numbers of neutrophils, macrophages, and giant cells containing intracellular gram-positive organisms in their cytoplasm. depending on the stage of infection and the immune status and age of affected horses, pulmonary lesions induced by rhodococcus equi can vary from pyogranulomatous to granulomatous pneumonia. in young foals, the infection starts as a suppurative cranioventral bronchopneumonia, which progresses within a few days into small variable-size pulmonary abscesses. these abscesses rapidly transform into pyogranulomatous nodules, some of which become confluent and form large masses of caseous exudate ( fig. - ). microscopically, the early lesion starts with neutrophilic infiltration, followed by an intense influx of alveolar macrophages into the bronchoalveolar spaces. this type of histiocytic inflammation persists for a long period of time because rhodococcus equi is a facultative intracellular organism that survives the bactericidal effects of equine alveolar macrophages. in the most chronic cases, the pulmonary lesions culminate with the formation of large caseonecrotic masses with extensive fibrosis of the surrounding pulmonary parenchyma. pcr analysis of tracheobronchial aspirates has successfully been used as an alternative to bacteriologic culture in the diagnosis of rhodococcus equi infection in live foals. b a rhinotracheitis (ibr)/bovine herpes virus (bohv- ), bovine parainfluenza virus (bpiv- ), and bovine respiratory syncytial virus (brsv); and noninfectious interstitial pneumonias, such as bovine pulmonary edema and emphysema, reinfection syndrome, and many others. bovine enzootic pneumonia. enzootic pneumonia, sometimes simply referred to as calf pneumonia, is a multifactorial disease caused by a variety of etiologic agents that produces an assortment of lung lesions in young, intensively housed calves. the hostmicrobial-environmental triad is central in the pathogenesis of this disease. morbidity is often high (up to %), but fatalities are uncommon (> %) unless management is poor or unless new, virulent pathogens are introduced by additions to the herd. enzootic pneumonia is also called viral pneumonia because it often begins with an acute respiratory infection with bpiv- , brsv, or possibly with one or more of several other viruses (adenovirus, bohv- , reovirus, bovine coronavirus [bcov] , and bovine rhinitis virus). mycoplasmas, notably mycoplasma dispar, mycoplasma bovis, ureaplasma, and possibly chlamydophila, may also be primary agents. following infection with any of these agents, opportunistic bacteria, such as pasteurella multocida, trueperella (arcanobacterium) pyogenes, histophilus somni, mannheimia haemolytica, and escherichia coli, can cause a secondary suppurative bronchopneumonia, the most serious stage of enzootic pneumonia. the pathogenesis of the primary invasion and how it predisposes the host to invasion by the opportunists are poorly understood, but it is likely that there is impairment of pulmonary defense mechanisms. environmental factors, including air quality (poor ventilation), high relative humidity, and animal crowding, have been strongly incriminated. the immune status of the calf also plays an important role in the development and severity of enzootic pneumonia. calves with bovine leukocyte adhesion deficiency (blad), which prevents the migration of neutrophils from the capillaries, are highly susceptible to bronchopneumonia. lesions are variable and depend largely on the agents involved and on the duration of the inflammatory process. in the acute phases, lesions caused by viruses are those of bronchointerstitial pneumonia, which are generally mild and transient, and therefore are seen only sporadically at necropsy. microscopically, the lesions are necrotizing bronchiolitis, necrosis of type i pneumonocytes with hyperplasia of type ii pneumonocytes, and mild interstitial and alveolar edema. in the case of bpiv- and brsv infection, intracytoplasmic inclusion bodies and the formation of large multinucleated syncytia, resulting from the fusion of infected bronchiolar and alveolar epithelial cells, can also be observed in the lungs (fig. - ) . airway hyperreactivity has been described in calves after brsv infection; however, the significance of this syndrome in relation to enzootic pneumonia of calves is still under investigation. the mycoplasmas also can cause bronchiolitis, bronchiolar and alveolar necrosis, and an interstitial reaction, but in contrast to viral-induced pneumonias, mycoplasmal lesions tend to progress to a chronic stage characterized by striking peribronchiolar lymphoid hyperplasia (cuffing pneumonia). when complicated by secondary bacterial infections (e.g., pasteurella multocida and trueperella pyogenes), viral or mycoplasmal lesions change from a pure bronchointerstitial to a suppurative bronchopneumonia (fig. - ) . in late stages of bronchopneumonia, the lungs contain a creamy-mucoid exudate in the airways and later often have pulmonary abscesses and bronchiectasis (see fig. - ) . note that the same viruses and mycoplasmas involved in the enzootic pneumonia complex can also predispose cattle to other diseases, such as pneumonic mannheimiosis (mannheimia aspiration pneumonia. aspiration pneumonia is often a devastating sequela to improper gastric tubing of horses, particularly exogenous lipid pneumonia from mineral oil delivered into the trachea in treatment of colic. gross and microscopic lesions are described in detail in the section on aspiration pneumonias of cattle. opportunistic infections. chlamydophila (chlamydia) spp., obligatory intracellular zoonotic pathogens, can cause systemic infection in many mammalian and avian species; in horses, they can also cause keratoconjunctivitis, rhinitis, pneumonia, abortion, polyarthritis, enteritis, hepatitis, and encephalitis. serologic studies suggest that infection without apparent disease is common in horses. horses experimentally infected with chlamydophila psittaci develop mild and transient bronchointerstitial pneumonia. there are unconfirmed reports suggesting a possible association between these organisms and recurrent airway obstruction in horses. detection of chlamydial organisms in affected tissue is not easy and requires special laboratory techniques such as pcr, immunohistochemistry, and fluorescent antibody tests. horses are only sporadically affected with mycobacteriosis (mycobacterium avium complex, mycobacterium tuberculosis, and mycobacterium bovis). the intestinal tract and associated lymph nodes are generally affected, suggesting an oral route of infection with subsequent hematogenous dissemination to the lungs. the tubercles (granulomas) differ from those in ruminants and pigs, being smooth, gray, solid, sarcoma-like nodules without grossly visible caseous necrosis or calcification (e- fig. - ) . microscopically, the tubercles are composed of macrophages, epithelioid cells, and multinucleated giant cells. fibrosis increases with time, accounting in part for the sarcomatous appearance. adenovirus infections occur commonly in arabian foals with combined immunodeficiency (cid), a hereditary lack of b and t lymphocytes. in cases of adenoviral infection, large basophilic or amphophilic inclusions are present in the nuclei of tracheal, bronchial, bronchiolar, alveolar, renal, and intestinal epithelial cells. as it occurs in other species, infection with a unique fungal pathogen known as pneumocystis carinii typically occurs in immunosuppressed or immunoincompetent individuals such as arabian foals with cid (see fig. - ). diagnosis of pneumocystis carinii requires microscopic examination of lungs and special stains. idiopathic interstitial pneumonia. interstitial and bronchointerstitial pneumonias of undetermined cause that can progress to severe pulmonary fibrosis have been reported in foals and young horses. the gross and microscopic lesions are reminiscent of those of bovine pulmonary edema and emphysema or ards. the lungs are notably congested and edematous and microscopically are characterized by necrosis of the bronchiolar epithelium, alveolar edema, hyperplasia of type ii pneumonocytes, and hyaline membranes. the cause of this form of equine interstitial pneumonia is not known, but toxic and particularly viral causes have been proposed. bovine respiratory disease complex (brdc) and acute undifferentiated respiratory disease are general terms often used by clinicians to describe acute and severe bovine respiratory illness of clinically undetermined cause. these terms do not imply any particular type of pneumonia and therefore should not be used in pathology reports. clinically, the brd complex includes bovine enzootic pneumonia (multifactorial etiology); pneumonic mannheimiosis (mannheimia haemolytica); respiratory histophilosis (histophilus somni), previously known as respiratory hemophilosis (haemophilus somnus); mycoplasma bovis; respiratory viral infections, such as infectious bovine .e chapter respiratory system, mediastinum, and pleurae pneumonic mannheimiosis (shipping fever) is the most important respiratory disease of cattle in north america, particularly in feedlot animals that have been through the stressful marketing and assembly processes. mannheimia haemolytica biotype a, serotype is the etiologic agent most commonly responsible for the severe pulmonary lesions. a few investigators still consider that pasteurella multocida and other serotypes of mannheimia haemolytica are also causes of this disease. even after many years of intense investigation, from the gross lesions to the molecular aspects of the disease, the pathogenesis of pneumonic mannheimiosis remains incompletely understood. experiments have established that mannheimia haemolytica a alone is usually incapable of causing disease because it is rapidly cleared by pulmonary defense mechanisms. these findings may explain why mannheimia haemolytica, despite being present in the nasal cavity of healthy animals, only sporadically causes disease. for mannheimia haemolytica to be established as a pulmonary infection, it is first required that stressors impair the defense mechanisms and allow the bacteria to colonize the lung (see section on impairment of defense haemolytica). clinically, enzootic pneumonia is usually mild, but fatal cases are occasionally seen even in farms with optimal health management. pneumonic mannheimiosis (shipping fever). shipping fever (transit fever) is a vague clinical term used to denote acute respiratory diseases that occur in cattle several days or weeks after shipment. the disease is characterized by a severe fibrinous bronchopneumonia, reflecting the fact that death generally occurs early or at an acute stage. because mannheimia haemolytica (formerly pasteurella haemolytica) is most frequently isolated from affected lungs, the names pneumonic mannheimiosis and pneumonic pasteurellosis have been used synonymously. it is known that pneumonic mannheimiosis can occur in animals that have not been shipped and that organisms other than mannheimia haemolytica can cause similar lesions. therefore the term shipping fever should be relinquished in favor of more specific names, such as pneumonic mannheimiosis or respiratory histophilosis. irregular areas of coagulative necrosis are typically bordered by a rim of elongated cells often referred to as oat-shaped cells or oat cells that are degenerating neutrophils mixed with a few alveolar macrophages (see fig. - ). in the early stages of necrosis, there is no evidence of vascular thrombosis, suggesting that necrosis is primarily caused by the cytotoxin of mannheimia haemolytica and is not the result of an ischemic change. the interlobular septa become distended with protein-rich edematous fluid, and the lymphatic vessels contain fibrin thrombi. the trachea and bronchi can have considerable amounts of blood and exudate, which are transported by the mucociliary escalator or coughed up from deep within the lungs, but the walls of the trachea and major bronchi may or may not be involved. because of the necrotizing process, sequelae to pneumonic mannheimiosis can be serious and can include abscesses, encapsulated sequestra (isolated pieces of necrotic lung), chronic pleuritis, fibrous pleural adhesions, and bronchiectasis. clinically, pneumonic mannheimiosis is characterized by a severe toxemia that can kill animals even when considerable parts of the lungs remain functionally and structurally normal. cattle usually become depressed, febrile ( ° to ° f [ ° to ° c]), and anorexic and have a productive cough, encrusted nose, mucopurulent nasal exudate, shallow respiration, or an expiratory grunt. hemorrhagic septicemia. pneumonic mannheimiosis should not be confused with hemorrhagic septicemia (septicemic pasteurellosis) of cattle and water buffalo (bubalus bubalis) caused by inhalation or ingestion of serotypes :b and :e of pasteurella multocida. this oie-notifiable disease does not occur in north america and currently is reported only from some countries in asia, africa, and recently in germany. in contrast to pneumonic mannheimiosis, in which lesions are always confined to the lower respiratory tract, the bacteria of hemorrhagic septicemia always disseminates hematogenously to other organs. at necropsy, typically, generalized petechiae are present on the serosal surfaces of the intestine, heart, and lungs and in skeletal muscles. superficial and visceral lymph nodes are swollen and hemorrhagic. variable lesions include edematous and hemorrhagic lungs with or without consolidation; hemorrhagic enteritis; blood-tinged fluid in the thorax and abdomen; and subcutaneous edema of the head, neck, and ventral abdomen. bacteria can be cultured from blood, and animals have high fever and die rapidly ( % case fatality). respiratory histophilosis (haemophilosis). respiratory histophilosis is part of the histophilus somni (haemophilus somnus) disease complex, which has at least eight different clinicopathologic forms, each one involving different organs. this complex includes septicemia, encephalitis (known as thrombotic meningoencephalitis [tme]), pneumonia (respiratory histophilosis), pleuritis, myocarditis, arthritis, ophthalmitis, conjunctivitis, otitis, and abortion. the portals of entry for the different forms of histophilosis have not been properly established. the respiratory form of bovine histophilosis is the result of the capacity of the bacterium to induce both suppurative and fibrinous bronchopneumonia (e- fig. - ). the latter is in some cases indistinguishable from that of pneumonic mannheimiosis. the pathogenesis of respiratory histophilosis is still poorly understood, and the disease cannot be reproduced consistently by administration of histophilus somni alone. like mannheimia haemolytica, it requires predisposing factors such as stress or a preceding viral infection. histophilus somni is often isolated from the lungs of calves with enzootic pneumonia. the capacity of histophilus somni to cause septicemia and localized infections in the lungs, brain, eyes, ear, heart, mammary gland, male and female genital organs, or placenta is perhaps attributable to specific virulence factors, such as immunoglobulin-binding proteins (igbps) and lipooligosaccharide (los). also, histophilus mechanisms). these stressors include weaning, transport, fatigue, crowding, mixing of cattle from various sources, inclement weather, temporary starvation, and viral infections. horizontal transmission of viruses and mannheimia haemolytica occurs during crowding and transportation of cattle. viruses that most commonly predispose cattle to pneumonic mannheimiosis include bohv- , bpiv- , and brsv. once established in the lungs, mannheimia haemolytica causes lesions by means of different virulence factors, which include endotoxin, lipopolysaccharide, adhesins, and outer membrane proteins; however, the most important is probably the production of a leukotoxin (exotoxin), which binds and kills bovine macrophages and neutrophils. the fact that this toxin exclusively affects ruminant leukocytes probably explains why mannheimia haemolytica is a respiratory pathogen in cattle and sheep but not in other species. during mannheimia haemolytica infection, alveolar macrophages, neutrophils, and mast cells release maximum amounts of proinflammatory cytokines, particularly tnf-α, il- , il- , adhesion molecules, histamine, and leukotrienes. by locally releasing enzymes and free radicals, leukocytes further contribute to the injury and necrosis of bronchiolar and alveolar cells. the gross lesions of acute and subacute pneumonic mannheimiosis are the prototypic fibrinous bronchopneumonia, with prominent fibrinous pleuritis ( fig. - and see fig. - ) and pleural effusion. lesions are always cranioventral and usually ventral to a horizontal line through the tracheal bifurcation. the interlobular septa are distended by yellow, gelatinous edema and fibrin. the "marbling" of lobules is the result of intermixing areas of coagulation necrosis, interlobular interstitial edema, and congestion ( fig. - ) . microscopically, lung lesions are evident hours after experimental infection in which neutrophils fill the bronchial, bronchiolar, and alveolar spaces. within to hours, the cytotoxic effect of mannheimia haemolytica is manifested by necrosis of individual alveolar cells and fibrin begins to exude into the alveoli from increased permeability of the air-blood barrier. these changes are exacerbated by endothelial swelling, altered platelet function, increased procoagulant activity, and diminished profibrinolytic activity in the lungs. by hours, alveolar macrophages start to appear in the bronchoalveolar space. at this time, large and the pulmonary defense mechanisms. lung lesions are typically those of a chronic bronchopneumonia with numerous well-delineated caseonecrotic nodules (fig. - and e-fig. - ) . microscopically, lesions are quite characteristic and consist of distinct areas of pulmonary necrosis centered on bronchi or bronchioles. the lesion is formed by a core of fine eosinophilic granular debris surrounded by a rim of neutrophils, macrophages, and fibroblasts (see fig. - ) . although the origin of the caseonecrotic lesions is under investigation, recent studies incriminate reactive oxygen species (ros) and reactive nitrogen species (rns) as the major contributors for cell injury in the lung. the diagnosis is confirmed by isolation or somni has the ability to undergo structural and antigenic variation, evade phagocytosis by promoting leukocytic apoptosis, inhibit intracellular killing, reduce transferrin concentrations, and induce endothelial apoptosis in the lungs of affected calves. mixed pulmonary infections of histophilus somni, mannheimia haemolytica, pasteurella multocida, trueperella pyogenes, and mycoplasmas are fairly common in calves. mycoplasma bovis pneumonia. mycoplasma bovis is the most common mycoplasma sp. isolated from pneumonic lungs of cattle in europe and north america. pulmonary infection is exacerbated by stress or any other adverse factor (e.g., viral infection) that depresses n control programs for infectious disease. it was eradicated from north america in and from australia in the s, but it is still enzootic in large areas of africa, asia, and eastern europe. the etiologic agent, mycoplasma mycoides ssp. mycoides small colony type, was the first mycoplasma isolated and is one of the most pathogenic of those that infect domestic animals. natural infection occurs in cattle and asian buffalo. the portal of entry is aerogenous, and infections occur when a susceptible animal inhales infected droplets. the pathogenic mechanisms are still inadequately understood but are suspected to involve toxin and galactan production, unregulated production of tnf-α, ciliary dysfunction, immunosuppression, and immune-mediated vasculitis. vasculitis and thrombosis of pulmonary arteries, arterioles, veins, and lymphatic vessels lead to lobular infarction. the name of the disease is a good indication of the gross lesions. it is a severe, fibrinous bronchopneumonia (pleuropneumonia) similar to that of pneumonic mannheimiosis (see figs. - and - ) but having a more pronounced "marbling" of the lobules because of extensive interlobular edema and lymphatic thrombosis. typically, % to % of lesions are in the caudal lobes (not cranioventrally), and pulmonary sequestra (necrotic lung encapsulated by connective tissue) are more frequent and larger than pneumonic mannheimiosis. unilateral lesions are common in this disease. microscopically, the appearance again is like that of pneumonic mannheimiosis, except that vasculitis and thrombosis of pulmonary arteries, arterioles, and capillaries are much more obvious and are clearly the major cause of the infarction and thrombosis of lymphatic vessels in interlobular septa. mycoplasma mycoides ssp. mycoides small colony type remains viable in the sequestra for many years, and under stress (e.g., starvation), the fibrous capsule may break down releasing mycoplasma into the airways, thus becoming a source of infection for other animals. clinical signs are those of severe sepsis, including fever, depression, and anorexia followed by severe respiratory signs such as opened-mouth breathing, dyspnea and coughing, and crepitation and pleural friction on thoracic auscultation. vaccination is highly effective in preventing the disease. bovine tuberculosis. tuberculosis is an ancient, communicable, worldwide, chronic disease of human beings and domestic animals. it continues to be a major problem in human beings in underdeveloped countries, and it is on the rise in some industrialized nations, largely because of the immunosuppressive effects of aids, immigration, and movement of infected animals across borders. the world health organization (who) estimates that more than million people die of tuberculosis and million new cases appear each year, mostly in developing countries. mycobacterium tuberculosis is transmitted between human beings, but where unpasteurized milk is consumed, mycobacterium bovis from the milk of cattle with mammary tuberculosis is also an important cause of human tuberculosis. mycobacterium bovis infections have also been reported in a number of domestic and wild mammalian species; in some countries, wildlife reservoirs exist and may act as a source of infection for cattle. bovine tuberculosis is primarily caused by mycobacterium bovis, but infection with mycobacterium tuberculosis, the pathogen of human tuberculosis, and mycobacterium caprae (formerly mycobacterium bovis ssp. caprae/mycobacterium tuberculosis ssp. caprae) can occur sporadically. tuberculosis can be acquired by several routes, but infection of the lungs by inhalation of mycobacterium bovis is the most common in adult cattle, whereas ingestion of infected milk is more predominant in young animals. organisms belonging to the mycobacterium avium complex can also infect cattle, but for infection caused by these organisms, the term atypical mycobacteriosis (not tuberculosis) is currently preferred. immunohistochemical labeling of tissue sections for mycoplasma antigens. mycoplasma bovis is also incriminated in arthritis, otitis, mastitis, abortion, and keratoconjunctivitis. contagious bovine pleuropneumonia. contagious bovine pleuropneumonia is an oie-notifiable disease of historic interest in veterinary medicine because it was the object of early national a b c than % of bovine cases, a chronic, moist cough can progress to dyspnea. enlarged tracheobronchial lymph nodes can contribute to the dyspnea by impinging on airways, and the enlargement of caudal mediastinal nodes can compress the caudal thoracic esophagus and cause bloating. interstitial pneumonias. atypical interstitial pneumonia (aip) is a vague clinical term well entrenched in veterinary literature but one that has led to enormous confusion among veterinarians. it was first used to describe acute or chronic forms of bovine pneumonia that did not fit in any of the "classic" forms because of the lack of exudate and lack of productive cough. microscopically, the criteria for diagnosis of aip in cattle were based on the absence of obvious exudate and the presence of edema, interstitial emphysema (see the section on pulmonary emphysema), hyaline membranes, hyperplasia of type ii pneumonocytes, and alveolar fibrosis with interstitial cellular infiltrates. at that time, any pulmonary disease or pulmonary syndrome that had a few of the previously mentioned lesions was traditionally diagnosed as aip, and grouping all these different syndromes together was inconsequential because their etiopathogenesis were then unknown. field and laboratory investigations have demonstrated that most of the bovine syndromes previously grouped under aip have rather different causes and pathogeneses ( fig. - ) . furthermore, what was "atypical" in the past has become so common that it is fairly routine nowadays to find "typical cases" of aip. for all these reasons, investigators, largely from britain, proposed that all these syndromes previously clustered into aip should be named according to their specific cause or pathogenesis. the most common bovine syndromes characterized by edema, emphysema, hyaline membranes, and hyperplasia of type ii pneumonocytes include bovine pulmonary edema and emphysema (fog fever), "extrinsic allergic alveolitis" (hypersensitivity pneumonitis), "reinfection syndromes" (hypersensitivity to dictyocaulus sp. or brsv), milk allergy, ingestion of moldy potatoes, paraquat toxicity, toxic silo gases, mycotoxins, and others. acute bovine pulmonary edema and emphysema (fog fever). acute bovine pulmonary edema and emphysema (abpee), known in britain as fog fever (no association with atmospheric conditions), occurs in cattle usually grazing "fog" pastures (i.e., aftermath or foggage, regrowth after a hay or silage has been cut). epidemiologically, abpee usually occurs in adult beef cattle in the fall when there is a change in pasture from a short, dry grass to a lush, green grass. it is generally accepted that l-tryptophan present in the pasture is metabolized in the rumen to -methylindole, which in turn is absorbed into the bloodstream and carried to the lungs. mixed function oxidases present in the nonciliated bronchiolar epithelial (club) cells metabolize -methylindole into a highly pneumotoxic compound that causes extensive and selective necrosis of bronchiolar cells and type i pneumonocytes (fig. - and see fig. - ) and increases alveolar permeability, leading to edema, thickening of the alveolar interstitium, and alveolar and interstitial emphysema. -methylindole also interferes with the lipid metabolism of type ii pneumonocytes. the gross lesions are those of a diffuse interstitial pneumonia with severe alveolar and interstitial edema and interlobular emphysema (see fig. - , a) . the lungs are expanded, pale, and rubbery in texture, and the lesions are most notable in the caudal lobes. microscopically, the lesions are alveolar and interstitial edema and emphysema, formation of characteristic hyaline membranes within alveoli (see fig. - , b) , and in those animals that survive for several days, hyperplasia of type ii pneumonocytes and alveolar interstitial fibrosis. respiratory infection usually starts when inhaled bacilli reach the alveoli and are phagocytosed by pulmonary alveolar macrophages. if these cells are successful in destroying the bacteria, infection is averted. however, mycobacterium bovis, being a facultative pathogen of the monocytic-macrophage system, may multiply intracellularly, kill the macrophage, and initiate infection. from this first nidus of infection, bacilli spread aerogenously via airways within the lungs and eventually via the lymph vessels to tracheobronchial and mediastinal lymph nodes. the initial focus of infection at the portal of entry (lungs) plus the involvement of regional lymph nodes is termed the primary (ghon) complex of tuberculosis. if the infection is not contained within this primary complex, bacilli disseminate via the lymph vessels to distant organs and other lymph nodes by the migration of infected macrophages. hematogenous dissemination occurs sporadically when a granuloma containing mycobacteria erodes the wall of a blood vessel, causes vasculitis, and allows the granuloma to discharge mycobacteria into the alveolar circulation. if dissemination is sudden and massive, mycobacteria are widely disseminated and numerous small foci of infection develop in many tissues and organs and the process is referred to as miliary tuberculosis (like millet seeds). the host becomes hypersensitive to the mycobacterium, which enhances the cell-mediated immune defenses in early or mild infections but can result in host-tissue destruction in the form of caseous necrosis. the evolution and dissemination of the pulmonary infection are closely regulated by cytokines and tnf-α production by alveolar macrophages. unlike abscesses that tend to grow rather fast, granulomas evolve slowly at the site of infection. the lesion starts with few macrophages and neutrophils ingesting the offending organism, but because mycobacterium organisms are resistant to phagocytosis, infected macrophages eventually die, releasing viable bacteria, lipids, and cell debris. cell debris accumulates in the center of the lesion, whereas viable bacteria and bacterial lipids attract additional macrophages and a few lymphocytes at the periphery of the lesion. some of these newly recruited macrophages are activated by local lymphocytes and become large phagocytic cells with abundant cytoplasm resembling epithelial cells, thus the term epithelioid macrophages. multinucleated giant cells (also macrophages) appear at the edges of the lesion, and finally the entire focus of inflammatory process becomes surrounded by fibroblasts and connective tissue (see fig. - ). it may take weeks or months for a granuloma to be grossly visible. bovine tuberculosis, the prototype for granulomatous pneumonia, is characterized by the presence of a few or many caseated granulomas (see fig. - ). the early gross changes are small foci (tubercles) most frequently seen in the dorsocaudal, subpleural areas. with progression, the lesions enlarge and become confluent with the formation of large areas of caseous necrosis. calcification of the granulomas is a typical finding in bovine tuberculosis. single nodules or clusters occur on the pleura and peritoneum, and this presentation has been termed pearl disease. microscopically, the tubercle is composed of mononuclear cells of various types. in young tubercles, which are noncaseous, epithelioid and langhans' giant cells are at the center, surrounded by lymphocytes, plasma cells, and macrophages. later, caseous necrosis develops at the center, secondary to the effects of cell-mediated hypersensitivity and enclosed by fibrosis at the periphery. acid-fast organisms may be numerous but more often are difficult to find in histologic section or smears. clinically, the signs of tuberculosis relate to the dysfunction of a particular organ system or to general debilitation, reduced milk production, and emaciation. in the pulmonary form, which is more grossly, the postmortem lesions vary from subtle, gray, subpleural foci (granulomatous inflammation) to severe lesions, in which the lungs are firm and heavy and have a "meaty appearance" because of interstitial pneumonia (e- fig. - ) with type ii pneumonocyte hyperplasia, lymphocytic infiltration, and interstitial fibrosis. characteristically, discrete noncaseous granulomas formed in response to the deposition of antigen-antibody complexes are scattered throughout the lungs. chronic cases of extrinsic allergic alveolitis can eventually progress to diffuse fibrosing alveolitis. clinically, it can be acute or chronic; the latter has a cyclical pattern of exacerbation during winter months. weight loss, coughing, and poor exercise tolerance are clinical features. full recovery can occur if the disease is recognized and treated early. reinfection syndrome. hypersensitivity to reinfection with larvae of dictyocaulus viviparus is another allergic syndrome manifested in the lungs that causes signs and lesions indistinguishable from abpee, with the exception of eosinophils and possibly larvae in the alveolar exudate. the hypersensitivity reaction in the lung causes diffuse alveolar damage and edema, necrosis of type i pneumonocytes, and hyperplasia of type ii pneumonocytes. in the later stages of the disease, there is formation of small granulomas with interstitial infiltrates of mononuclear cells. it has been suggested but not confirmed that emphysema with diffuse proliferative alveolitis and formation of hyaline membranes can also occur sporadically in the late stages of brsv infection in cattle. presumably, this disease shares many similarities with "atypical" infections occasionally seen in children with respiratory syncytial virus (rsv human strain), in which a hypersensitivity to the virus or virus-induced augmentation of the immune response results in hypersensitivity pneumonitis (see fig. - ). brsv infection is also known to enhance hypersensitivity to environmental allergens in cattle. other forms of bovine interstitial pneumonia. inhalation of manure ("pit") gases, such as nitrogen dioxide (no ), hydrogen interstitial cell infiltrates, fibrosis, emphysema acute proliferation phase hyperplasia of type ii pneumonocytes clinically, severe respiratory distress develops within days of the abrupt pasture change, and cattle develop expiratory dyspnea, oral breathing, and evidence of emphysema within the lungs and even subcutaneously along the back. experimentally, reducing ruminal conversion of l-tryptophan to -methylindole prevents the development of abpee. a number of other agents cause virtually the same clinical and pathologic syndrome as is seen in abpee. the pathogenesis is assumed to be similar, although presumably other toxic factors are specific for each syndrome. one of these pneumotoxic factors is -ipomeanol, which is found in moldy sweet potatoes contaminated with the fungus fusarium solani. mixed function oxidases in the lungs activate -ipomeanol into a potent pneumotoxicant capable of producing irreversible oxidative injury to type i pneumonocytes and bronchiolar epithelial cells, presumably through lipoperoxidation of cell membranes. similarly, purple mint (perilla frutescens), stinkwood (zieria arborescens), and rapeseed and kale (brassica species) also cause pulmonary edema, emphysema, and interstitial pneumonia. extrinsic allergic alveolitis. extrinsic allergic alveolitis (hypersensitivity pneumonitis), one of the most common allergic diseases in cattle, is seen mainly in housed adult dairy cows in the winter. this disease shares many similarities with its human counterpart known as farmer's lung, which results from a type iii hypersensitivity reaction to inhaled organic antigens, most commonly microbial spores, mainly of the thermophilic actinomycete, saccharopolyspora rectivirgula (micropolyspora faeni), commonly found in moldy hay. this is followed by an antibody response to inhaled spores and local deposition of antigen-antibody complexes (arthus reaction) in the lungs (see fig. - ). because it affects only a few animals of the herd or the sporadic person working in a farm, it is presumed that intrinsic host factors, such as dysregulation of dendritic cells, t lymphocytes, igg, interleukins, ifn-γ, and surfactant, are involved in the pathogenesis of the disease. chapter respiratory system, mediastinum, and pleurae e- figure - interstitial pneumonia, adult cow. note meaty appearance of the pulmonary parenchyma and mild edematous distention of the interlobular septa. inset, thick hyaline membranes (arrows) lining hypercellular alveolar walls. hypersensitivity pneumonia was suspected. (courtesy dr. a. lópez, atlantic veterinary college.) gases, inhalation of no (silo gas) also causes bronchiolitis, edema, and interstitial pneumonia and, in survivors, bronchiolitis obliterans ("silo filler's disease"). smoke inhalation resulting from barn or house fires is sporadically seen by veterinarians and pathologists. in addition to skin burns, animals involved in fire accidents suffer extensive thermal injury produced by the heat on the nasal and laryngeal mucosa, and severe chemical irritation caused by inhalation of combustion gases and particles in the lung. animals that survive or are rescued from fires frequently develop nasal, laryngeal, and tracheal edema, and pulmonary hemorrhage and alveolar edema, which are caused by chemical injury to the blood-air barrier or by ards caused by the excessive production of free radicals during the pulmonary inflammatory response (see e- fig. - ) . microscopic examination of the lungs often reveals carbon particles (soot) on mucosal surfaces of the conducting system. verminous pneumonia (dictyocaulus viviparus). pulmonary lesions in parasitic pneumonias vary from interstitial pneumonia caused by migrating larvae to chronic bronchitis from intrabronchial adult parasites, to granulomatous pneumonia, which is caused by dead larvae, aberrant parasites, or eggs of parasites. in many cases, an "eosinophilic syndrome" in the lungs is characterized by infiltrates of eosinophils in the pulmonary interstitium and bronchoalveolar spaces and by blood eosinophilia. atelectasis and emphysema secondary to the obstruction of airways by parasites and mucous secretions are also common findings in parasitic pneumonias. the severity of these lesions relates to the numbers and size of the parasites and the nature of the host reaction, which sometimes includes hypersensitivity reactions (see section on reinfection syndrome). a common general term for all of these diseases is verminous pneumonia, and the adult nematodes are often visible grossly in the airways ( fig. - ) . dictyocaulus viviparus is an important pulmonary nematode (lungworm) responsible for a disease in cattle referred to as verminous pneumonia or verminous bronchitis. adult parasites live in the bronchi of cattle, mainly in the caudal lobes, and cause severe bronchial irritation, bronchitis, and pulmonary edema, which in turn are responsible for lobular atelectasis and interstitial emphysema. atelectasis is confined to the lobules of the lungs ventilated by the obstructed bronchi (dorsocaudal). interstitial emphysema (interlobular) is caused by forced expiratory movements against a partially obstructed single bronchus. in addition to the inflammation of bronchial mucosa, bronchoaspiration of larvae and eggs also causes an influx of leukocytes into the bronchoalveolar space (alveolitis). verminous pneumonia is most commonly seen in calves during their first summer grazing pastures that are repeatedly used from year to year, particularly in regions of europe that have a moist cool climate. the parasite can overwinter in pastures, even in climates as cold as canada's, and older animals may be carriers for a considerable length of time. at necropsy, lesions appear as dark or gray, depressed, wedgeshaped areas of atelectasis involving few or many lobules usually along the dorsocaudal aspect of the lungs. on cut surface, edematous foam and mucus mixed with white, slender (up to -mm long) nematodes are visible in the bronchi (see fig. - ). in the most severe cases, massive numbers of nematodes fill the bronchial tree. microscopically, the bronchial lumens are filled with parasites admixed with mucus because of goblet cell hyperplasia, and there is squamous metaplasia of the bronchial and bronchiolar epithelium because of chronic irritation. there are also inflammatory infiltrates in the bronchial mucosa; alveolar edema; hyperplasia of balt sulfide (h s), and ammonia (nh ), from silos or sewage can be a serious hazard to animals and human beings. at toxic concentrations, these gases cause necrosis of bronchiolar cells and type i pneumonocytes and fulminating pulmonary edema that causes asphyxiation and rapid death (see fig. - ) . like other oxidant secretory granules released by club cells contain several proteins, such as surfactantlike protein, antiinflammatory protein (cc ), and bronchiolar lining proteins. b, ros produced by club cells are also absorbed into capillaries within the lamina propria and are transferred by the circulatory system to pulmonary capillaries where they disrupt the air-blood barrier, causing degeneration and necrosis of type i pneumonocytes. this process leads to leakage of plasma fluid (alveolar edema [pink color]) and extravasation of erythrocytes (alveolar hemorrhage) and neutrophils (inflammation). ingested pneumotoxicants can be metabolized by the liver, leading to release of ros into the circulatory system that then disrupts the air-blood barrier in a similar manner. fig. - ). microscopically, there are focal intraalveolar hemorrhages caused by larvae migrating through the alveolar walls. some larvae admixed with edematous fluid and cellular exudate (including eosinophils) may be visible in bronchioles and alveoli. the alveolar walls are thickened because of edema and a few inflammatory cells. clinical signs include cough and expiratory dyspnea to the point of oral breathing. hydatid cysts, the intermediate stage of echinococcus granulosus, can be found in the lungs and liver and other viscera of sheep and to a lesser extent in cattle, pigs, goats, horses, and human beings. the adult stage is a tapeworm that parasitizes the intestine of canidae. hydatidosis is still an important zoonosis in some countries, and perpetuation of the parasite life cycle results from animals being fed uncooked offal from infected sheep and consumption of uninspected meat. hydatid cysts are generally to cm in diameter, and numerous cysts can be found in the viscera of affected animals ( fig. - ). each parasitic cyst is filled with clear fluid; numerous daughter cysts attach to the wall, each containing several "brood capsules" with protoscolices inside. hydatid cysts have little clinical significance in animals but are economically important because of carcass condemnation. aspiration pneumonias. the inhalation of regurgitated ruminal contents or iatrogenic deposition of medicines or milk into the trachea can cause severe and often fatal aspiration pneumonia. bland substances, such as mineral oil, may incite only a mild suppurative or histiocytic bronchopneumonia, whereas some "home remedies" or ruminal contents are highly irritating and cause a fibrinous, necrotizing bronchopneumonia. the right cranial lung lobe tends to be more severely affected because the right cranial bronchus is the most cranial branch and enters the ventrolateral aspect of the trachea. however, the distribution may vary when animals aspirate while in lateral recumbency. in some severe cases, pulmonary necrosis can be complicated by infection with saprophytic organisms present in ruminal contents, causing fatal gangrenous pneumonia. aspiration pneumonia should always be considered in animals whose swallowing has been compromised-for example, those with cleft palate or hypocalcemia (milk fever). on the other hand, neurological diseases such as encephalitis (e.g., rabies) or encephalopathy (e.g., lead poisoning) should be investigated in animals in which the cause of aspiration pneumonia could not be caused by persistent immunologic stimuli; hypertrophy and hyperplasia of bronchiolar smooth muscle because of increased contraction and decreased muscle relaxation; and a few eosinophilic granulomas around the eggs and dead larvae. these granulomas, grossly, are gray, noncaseated nodules ( to mm in diameter) and may be confused with those seen at the early stages of tuberculosis. the clinical signs (coughing) vary with the severity of infection, and severe cases can be confused clinically with interstitial pneumonias. expiratory dyspnea and death can occur with heavy parasitic infestations when there is massive obstruction of airways. a different form of bovine pneumonia, an acute allergic reaction known as reinfection syndrome, occurs when previously sensitized adult cattle are exposed to large numbers of larvae (dictyocaulus viviparus). lesions in this syndrome are those of a hypersensitivity pneumonia as previously described. other lung parasites. ascaris suum is the common intestinal roundworm of pigs; larvae cannot complete their life cycle in calves, but the larvae can migrate through the lungs and cause severe pneumonia and death of calves within weeks of infection. infection is usually acquired from the soil on which infested pigs were previously kept. the gross lesions are a diffuse interstitial pneumonia with hemorrhagic foci, atelectasis, and interlobular edema and it also occurs in canada, europe, australia, and probably elsewhere. this disease has two major clinicopathologic forms: one involves the central nervous system of goat kids and young goats and is characterized by a nonsuppurative leukoencephalomyelitis; the other form involves the joints of adult goats and is characterized by a chronic, nonsuppurative arthritis-synovitis. in addition, infection with cae virus can cause chronic lymphocytic interstitial pneumonia. the lentivirus of cae, caprine arthritis and encephalitis virus (caev), is closely related to visna/maedi virus and, in fact, cross infection with cae virus in sheep has been achieved experimentally. similar to maedi, cae infection presumably occurs during the first weeks of life when the doe transmits the virus to her offspring through infected colostrum or milk. horizontal transmission between infected and susceptible goats via the respiratory route has also been described. after coming into contact with mucosal cells at the portal of entry, the virus is phagocytized by macrophages, which migrate to the regional lymph nodes. infected macrophages are disseminated hematogenously to the central nervous system, joints, lungs, and mammary glands. like maedi, there is some evidence that the recruitment of lymphocytic cells results from dysregulation of cytokine production by infected macrophages and lymphocytes in affected tissues. it can take several months before serum antibodies can be detected in infected goats. grossly, the interstitial pneumonia is diffuse and tends to be most severe in the caudal lobes. the lungs are gray-pink and firm in texture with numerous, -to -mm, gray-white foci on the cut surface. the tracheobronchial lymph nodes are consistently enlarged. microscopically, the alveolar walls are thickened by lymphocytes and conspicuous hyperplasia of type ii pneumonocytes ( fig. - ). one important difference between the pneumonias of cae and maedi is that in cae the alveoli are filled with proteinaceous eosinophilic material (alveolar proteinosis), which in electron micrographs has structural features of pulmonary surfactant. the pulmonary form of cae can be mistaken for parasitic pneumonia (muellerius capillaris) because these two diseases have lymphocytic interstitial pneumonia and can coexist in the same goat. explained otherwise. depending on the nature of the aspirated material, histopathologic evaluation generally reveals foreign particles such as vegetable cells, milk droplets, and large numbers of bacteria in bronchi, bronchioles, and alveoli (e- fig. - ). vegetable cells and milk typically induce an early neutrophilic response followed by a histiocytic reaction with "foreign body" multinucleated giant cells (see e- fig. - ). special stains are used for the microscopic confirmation of aspirated particles in the lung (e.g., pas for vegetable cells and oil red-o for oil or milk droplets). maedi (visna/maedi). maedi is an important, lifelong, and persistent viral disease of sheep and occurs in most countries, except australia and new zealand. maedi means "shortness of breath" in the icelandic language, and it is known as graaff-reinet disease in south africa, zwoegerziekte in the netherlands, la bouhite in france, and ovine progressive pneumonia (opp) in the united states. more recently, the disease has also been referred to as ovine lentivirusinduced lymphoid interstitial pneumonia or simply lymphoid interstitial pneumonia (lip). maedi is caused by visna/maedi virus (vmv), a nononcogenic small ruminant lentivirus (srlv) of the family retroviridae that is antigenically related to the lentivirus causing caprine arthritisencephalitis (cae). seroepidemiologic studies indicate that infection is widespread in the sheep population, yet the clinical disease seems to be rare. the pathogenesis is incompletely understood, but it is known that transmission occurs largely vertically, through ingestion of infected colostrum, and horizontally, via inhalation of infected respiratory secretions. once in the body, the ovine lentivirus causes lifelong infections within monocytes and macrophages, including alveolar and pulmonary intravascular macrophages; clinical signs do not develop until after a long incubation period of years or more. pulmonary lesions at the time of death are severe interstitial pneumonia and failure of the lungs to collapse when the thorax is opened. notable rib imprints, indicators of uncollapsed lungs, are often present on the pleural surface ( fig. - ). the lungs are pale, mottled, and typically heavy (two or three times normal weight), and the tracheobronchial lymph nodes are enlarged. microscopically, the interstitial pneumonia is characterized by balt hyperplasia and thickening of alveolar walls and peribronchial interstitial tissue by heavy infiltration of lymphocytes, largely t lymphocytes (see fig. - ). recruitment of mononuclear cells into the pulmonary interstitium is presumably the result of sustainable production of cytokines by retrovirus-infected pulmonary macrophages and lymphocytes. hyperplasia of type ii pneumonocytes is not a prominent feature of maedi, likely because in this disease there is no injury to type i pneumonocytes, but there is some alveolar fibrosis and smooth muscle hypertrophy in bronchioles. secondary bacterial infections often cause concomitant bronchopneumonia. enlargement of regional lymph nodes (tracheobronchial) is due to severe lymphoid hyperplasia, primarily of b lymphocytes. the virus can also infect many other tissues, causing nonsuppurative encephalitis (visna), lymphocytic arthritis, lymphofollicular mastitis, and vasculitis. maedi is clinically characterized by dyspnea and an insidious, slowly progressive emaciation despite good appetite. death is inevitable once clinical signs are present, but it may take many months. caprine arthritis-encephalitis. caprine arthritis-encephalitis (cae) is a retroviral disease of goats (small ruminant lentivirus) that has a pathogenesis remarkably similar to that of visna/maedi in sheep. it was first described in the united states in the s, but such as pasteurella multocida, pneumonia may progress to fibrinous or suppurative bronchopneumonia. one might expect some specific evidence pointing to the infectious agents (e.g., large intranuclear inclusion bodies in epithelial cells with adenoviral infection), but this is often not the case, either because examination is seldom done at the acute stage when the lesions are still present or because secondary bacterial infections mask the primary lesions. in the late stages, chronic enzootic pneumonia is characterized by hyperplastic bronchitis, atelectasis, alveolar and peribronchiolar fibrosis, and marked peribronchial lymphoid hyperplasia (cuffing pneumonia). ovine pneumonic mannheimiosis. ovine pneumonic mannheimiosis is one of the most common and economically significant diseases in most areas where sheep are raised. it is caused by mannheimia haemolytica and has a pathogenesis and lesions similar to those of pneumonic mannheimiosis of cattle. colonization and infection of lungs are facilitated by stressors such as changes in weather; handling; deworming; dipping; viral infections such as parainfluenza virus (piv ), respiratory syncytial virus (rsv), and adenovirus; and probably chlamydiae and bordetella parapertussis infections. lesions are characterized by a severe fibrinous bronchopneumonia (cranioventral) with pleuritis ( fig. - and e-fig. - ). subacute to chronic cases progress to purulent bronchopneumonia, and sequelae include abscesses and fibrous pleural adhesions. a similar form of pneumonic mannheimiosis has been reported with increased frequency in bighorn sheep. septicemic pasteurellosis. septicemic pasteurellosis, a common ovine disease, is caused by bibersteinia trehalosi (formerly pasteurella trehalosi or mannheimia haemolytica biotype t) in lambs months of age or older or by mannheimia haemolytica (biotype a) in lambs younger than months of age. both organisms are carried in the tonsils and oropharynx of clinically healthy sheep, and under abnormal circumstances (particularly under stress from dietary or environmental changes) bacteria can invade adjacent tissues, enter the bloodstream, and cause septicemia. gross lesions include a distinctive necrotizing pharyngitis and tonsillitis; ulcerative esophagitis (e- fig. - ) ; severe congestion and edema of the lungs; focal hepatic necrosis; and petechiae in the mucosa of the tongue, esophagus, and intestine and particularly in the lungs and pleura. clinically, goats are active and afebrile but progressively lose weight despite normal appetite. the encephalitic or arthritic signs tend to obscure the respiratory signs, which are only evident on exertion. secondary bacterial bronchopneumonia is common in affected animals. bacterial pneumonias. in the past, pasteurella haemolytica was incriminated in four major ovine diseases known as ( ) acute ovine pneumonic pasteurellosis (shipping fever), ( ) enzootic pneumonia (nonprogressive chronic pneumonia), ( ) fulminating septicemia, and ( ) mastitis. under the new nomenclature, mannheimia haemolytica is responsible for ovine pneumonia resembling shipping fever in cattle (ovine pneumonic mannheimiosis), septicemia in young lambs (younger than months of age), and ovine enzootic pneumonia and sporadic severe gangrenous mastitis in ewes. bibersteinia (pasteurella) trehalosi (formerly pasteurella haemolytica biotype t) is the agent incriminated in septicemia in lambs to months old. chronic enzootic pneumonia. in sheep, this entity is a multifactorial disease complex that, in contrast to ovine pneumonic mannheimiosis, causes only a mild to moderate pneumonia and it is rarely fatal. it generally affects animals younger than year of age. significant costs associated with chronic enzootic pneumonia include reduction of weight gain, labor costs, veterinary fees, and slaughterhouse waste. the modifier "chronic" is used here to avoid any confusion with pneumonic mannheimiosis ("acute enzootic pneumonia"). it is also sometimes called atypical pneumonia, chronic nonprogressive pneumonia, proliferative pneumonia, or other names. chronic enzootic pneumonia is a clinical epidemiologic term and does not imply a single causal agent but is the result of a combination of infectious, environmental, and managerial factors. the list of infectious agents involved in ovine enzootic pneumonia includes mannheimia haemolytica, pasteurella multocida, parainfluenza virus (pi- ), adenovirus, reovirus, respiratory syncytial virus (rsv), chlamydiae, and mycoplasmas (mycoplasma ovipneumoniae). in the early stages of enzootic pneumonia, a cranioventral bronchointerstitial pneumonia is characterized by moderate thickening of alveolar walls because of hyperplasia of type ii pneumonocytes. in some cases, when lungs are infected with secondary pathogens, viviparus of cattle. as seen in cattle with dictyocaulus viviparus, areas of atelectasis secondary to bronchiolar obstruction are present, particularly along the dorsal caudal aspects of the caudal lung lobes. microscopically, affected lungs are characterized by a catarrhal, eosinophilic bronchitis, with peribronchial lymphoid hyperplasia and smooth muscle hyperplasia of bronchi and bronchioles. bronchioles and alveoli can contain edematous fluid, eosinophils, and parasitic larvae and eggs. microscopic granulomas caused by aspirated eggs can be observed in the distal lung. the clinical signs (cough, moderate dyspnea, and loss of condition) and lesions relate mainly to obstruction of the small bronchi by adult worms and filaria. anemia of undetermined pathogenesis and secondary bacterial pneumonia are common in small ruminants with this parasitic disease. muellerius capillaris. muellerius capillaris, also called the nodular lungworm, occurs in sheep and goats in most areas of the world and is the most common lung parasite of sheep in europe and northern africa. it requires slugs or snails as intermediate hosts. the lesions in sheep are typically multifocal, subpleural nodules that tend to be most numerous in the dorsal areas of the caudal lung lobes ( fig. - , a) . these nodules are soft and hemorrhagic in the early stages but later become gray-green and hard or even calcified. microscopically, a focal, eosinophilic, and granulomatous reaction occurs in the microscopically, the hallmark lesion is a disseminated intravascular thrombosis often with bacterial colonies in the capillaries of affected tissues. the alveolar capillaries contain bacteria and microthrombi, and the alveolar lumens have fibrin and red blood cells. mannheimia haemolytica and bibersteinia trehalosi are readily isolated from many organs. affected animals usually die within a few hours of infection, and these animals only rarely have clinical signs such as dullness, recumbency, and dyspnea. contagious caprine pleuropneumonia. a number of mycoplasma spp., often referred to as the "mycoides cluster," can produce respiratory tract infections in goats; however, only mycoplasma capricolum ssp. capripneumoniae is considered to cause contagious caprine pleuropneumonia. this disease is the goat counterpart of contagious bovine pleuropneumonia in cattle; sheep do not have a corresponding disease. this oie-notifiable disease is important in africa, the middle east, and areas of asia, but it is also seen elsewhere. the gross lesions caused by mycoplasma capricolum ssp. capripneumoniae are similar to those of the bovine disease and consist of a severe, often unilateral fibrinous bronchopneumonia and pleuritis; however, distention of the interlobular septa (which are normally not as well developed in goats as in cattle) and formation of pulmonary sequestra are less obvious than in the bovine disease. clinically, contagious caprine pleuropneumonia is similar to contagious bovine pleuropneumonia, with high morbidity and mortality, fever, cough, dyspnea, and increasing distress and weakness. other small ruminant mycoplasmas. pneumonia, fibrinous polyarthritis, septicemia, meningitis, mastitis, peritonitis, and abortion are possible manifestations of disease caused by mycoplasma mycoides ssp. mycoides large colony type and mycoplasma mycoides ssp. capri. the pathogenicity of other mycoplasmas, such as mycoplasma ovipneumoniae, mycoplasma arginini, and mycoplasma capricolum ssp. capricolum, in sheep and goats is still being defined and specific description of the lesions would be premature. these organisms probably cause disease only in circumstances similar to those for enzootic pneumonia, where host, infectious, and environmental factors create a complex interaction in the pathogenesis of the disease. it has been suggested that igg antibodies directed against ovine mycoplasmal antigens cross-react with ciliary proteins, causing inflammation and ciliary dysfunction, a condition in lambs referred to as coughing syndrome. tuberculosis. although tuberculosis has generally been considered uncommon in sheep and goats, caprine tuberculosis has become a significant disease in areas of spain and europe. mycobacterium caprae (formerly mycobacterium bovis ssp. caprae/mycobacterium tuberculosis ssp. caprae) is the most common cause, but infection with mycobacterium bovis or with the mycobacterium avium complex does occur when the disease is prevalent in other species in the locality. the pulmonary form, similar to that seen in cattle, is characterized by a granulomatous pneumonia with multiple, large, caseous, calcified, and well-encapsulated granulomas scattered throughout the lungs. intralesional acid-fast organisms within macrophages are not as abundant as in bovine tuberculosis. staphylococcus aureus. young sheep ( to weeks old) are susceptible to staphylococcus aureus septicemia (tick pyemia). this bacterium causes disseminated inflammation and abscesses in the joints, heart, liver, kidneys, and cns, and in the lung it can also produce bronchopneumonia and pulmonary abscesses (e- fig. - ). dictyocaulus filaria. dictyocaulus filaria, also called the large lungworm, is a serious, worldwide, parasitic disease of the lungs, most commonly of lambs and goat kids but occurring in adults as well. the life cycle and lesions are similar to those of dictyocaulus epithelial cells spreads rapidly throughout the nasal, tracheal, and bronchial mucosa, with the more severe outbreaks reflecting more involvement of intrapulmonary airways and secondary infection with pasteurella multocida, trueperella (arcanobacterium) pyogenes, or haemophilus spp. although uncommon, human beings infected with swine influenza (h n ) can transmit the virus to pigs; therefore it is important that veterinarians or workers with influenza-like illness stay away from pig farms. natural transmission of h n and h n from human beings to ferrets (mustela putorius furo) and from human beings to cats and dogs has also been reported. pulmonary lesions caused by influenza virus alone are rarely seen in the postmortem room because this disease has a very low mortality rate unless complicated with secondary bacterial infections. grossly, a copious catarrhal to mucopurulent inflammation extends from the nasal passages to the bronchioles, with the volume of mucus being sufficient to plug small airways and cause a lobular or multilobular atelectasis in the cranioventral regions of the lungs. the appearance can be similar grossly, although not microscopically, to that of mycoplasma hyopneumoniae. fatal cases have severe alveolar and interstitial pulmonary edema. microscopically, the lesions in uncomplicated cases are typical of a virus-induced, necrotizing bronchitis-bronchiolitis, which in severe cases extends into the alveoli as bronchointerstitial pneumonia. it is characterized by necrosis of the bronchial/bronchiolar epithelium, thickening and infiltration of the alveolar wall with mononuclear cells and aggregates of macrophages, neutrophils, mucus, and some necrotic cells within the alveolar lumen. if these changes are extensive enough, the lumen of bronchioles can be occluded by exudate, causing lobular atelectasis. viral antigen can be demonstrated in infected epithelial cells by immunoperoxidase techniques. in the later stages of alveolar inflammation, neutrophils are progressively replaced by intraalveolar macrophages, unless the pneumonia is complicated by secondary bacterial infections. recent serologic surveys indicate that infection is also prevalent in wild pigs. clinically, a sudden onset of fever, nasal discharge, stiffness, labored breathing, weakness or even prostration, followed by painful and often paroxysmal coughing, is seen in animals of all age groups and may affect most of the herd. the outbreak subsides virtually without mortality within or weeks; the clinical appearance is much more alarming than the pathologic changes, unless the pigs have secondary infection with bacteria. infection can be confirmed using pcr in secretions collected with nasal swabs. the most important effect of most outbreaks of influenza is severe weight loss, but pregnant sows may abort or give birth to weak piglets. porcine reproductive and respiratory syndrome. a disease originally named mystery swine disease was first recognized in the united states in . in , it was seen in europe, and the disease now occurs worldwide in most major pig-raising countries. in , dutch investigators isolated a virus as the etiologic agent; porcine reproductive and respiratory syndrome virus (prrsv) is currently classified in the genus arterivirus of the family arteriviridae. as its name implies, prrs is characterized by late-term abortions and stillbirths and respiratory problems. the respiratory form is generally seen in nursery and grow/finish pigs. the pathogenesis has not been completely elucidated, but it is presumed that there is a mucosal portal of entry with virus replication in macrophages of the lymphoid tissue, followed by viremia and finally dissemination of infected macrophages to the lungs and other organs, such as the thymus, liver (kupffer cells), spleen, lymph nodes, and intestine. the pulmonary alveolar and intravascular macrophages are the major targets for prrs virus, which induces apoptosis of these cells. the virus also downregulates the innate immune response by subpleural alveoli where the adults, eggs, and coiled larvae reside ( fig. - , b) . clinical signs are usually not apparent. goats differ from sheep by having diffuse interstitial rather than focal lesions, and the reaction to the parasites seen microscopically varies from almost no lesions to a severe interstitial pneumonia with heavy infiltrates of mononuclear cells in alveolar walls resembling cae or mycoplasmal infections. secondary effects of muellerius capillaris infection in sheep and goats include decreased weight gain and possibly secondary bacterial infections. protostrongylus rufescens. protostrongylus rufescens is a worldwide parasite of sheep, goats, and wild ruminants. it requires an intermediate snail as a host. infection is usually subclinical, but protostrongylus rufescens can be pathogenic for lambs and goat kids and can cause anorexia, diarrhea, weight loss, and mucopurulent nasal discharge. the adult parasite lives in bronchioles as dictyocaulus spp., but it causes pulmonary nodules similar to those of muellerius capillaris. porcine pneumonias are unequivocally a major obstacle for the contemporary swine industry. the incidence, prevalence, and mortality rates of pneumonias in pigs depend on a series of complex, multifactorial interactions. among the most commonly recognized elements linked to porcine pneumonias are the following: • host (age, genetic makeup, immune status) • infectious agents (viruses, bacteria) • environmental determinants (humidity, temperature, ammonia concentrations) • management practices (crowding, mixing of animals, air quality, nutrition, stress) because of the nature of these multifactorial interactions, it will become obvious in the following paragraphs that more often than not a specific type of pneumonia frequently progresses to or coexists with another. the term porcine respiratory disease complex (prdc) has been introduced in clinical practice to describe pigs with signs of respiratory infection involving combined bacterial and viral infections. commonly implicated microbes include porcine reproductive and respiratory syndrome virus (prrsv), swine influenza virus (siv), porcine circovirus (pcv ), porcine respiratory coronavirus (prcov), mycoplasma hyopneumoniae, and pasteurella multocida. swine influenza (swine flu). swine influenza is a highly contagious acute respiratory viral disease of swine that is caused by swine influenza virus (siv), a type a influenza virus of the family orthomyxoviridae. it is generally accepted that swine influenza resulted from adaptation of the type a influenza virus that caused the human influenza pandemic during world war i. the most common subtypes of siv currently circulating in pigs are h n , h n , and h n . swine influenza is enzootic worldwide and is known to infect human beings who are in close contact with sick pigs. in , an outbreak of swine-human influenza (h n ), presumably transmitted from pigs to human beings, emerged in mexico and rapidly spread to many countries throughout the world. this new "pandemic" was attributed to a triple-reassortant of influenza a virus containing gene segments of swine, eurasian avian, and human strains. human infection with this novel strain affected mainly children and young adults, as well as individuals of any age with an underlying debilitating condition. transmission between influenza-infected and susceptible pigs occurs mainly by aerosol or oral route. siv attaches to and replicates within epithelial cells of the upper respiratory tract; the infection of similar inclusions are occasionally seen in bronchial glandular and renal epithelial cells. the lungs show thickening of the alveolar walls because of hyperplasia of type ii pneumonocytes and interstitial infiltrates of mononuclear cells, peribronchiolar fibrous hyperplasia, and necrotizing bronchitis/bronchiolitis. circovirus can be confirmed in affected tissue by immunohistochemical or pcr techniques. dual infections with pcv and prrsv frequently occur in pigs, and secondary infections with pneumocystis carinii are commonly seen in pigs with this coinfection. characteristically, alveoli are filled with a distinctive foamy exudate that contains the organism, which is not visible in h&e-stained sections but is easily demonstrated with gomori's methenamine silver stain (see fig. - ) . in human beings, pneumocystis (carinii) jirovecii pneumonia (pneumocystosis) is one of the most common and often fatal complications in aids patients. as in aids patients, abnormal populations of cd + and cd + t lymphocytes have been incriminated as the underlying mechanism leading to pneumocystosis in foals and pigs. nipah virus. nipah virus belongs to the paramyxoviridae family and shares a genus (henipavirus) with the closely related hendra virus (see section on pneumonias of horses). another emerging zoonotic disease, nipah virus caused a major epidemic with significant human mortality in southeast asia in and . people handling pigs were primarily affected. similar to hendra virus, fruit bats (flying foxes) act as natural reservoir and are involved in the transmission to pigs by poorly understood mechanisms. in pigs, this virus infects the respiratory system resulting in pneumonia with syncytial cells occurring in the vascular endothelium and in the respiratory epithelium at all levels of the lung. disease is spread to human beings via the respiratory route. human-to-human transmission of this virus has been reported in more recent outbreaks. other viral pneumonias of pigs. porcine respiratory coronavirus (prcov) is sporadically incriminated in pneumonia in pigs. this viral pneumonia is generally mild, and most pigs fully recover if the pneumonia is not complicated with other infections. lesions in the lung are those of bronchointerstitial pneumonia with necrotizing bronchiolitis. interestingly, infections with porcine and other respiratory coronaviruses have been used to investigate the pathogenesis of severe acute respiratory syndrome (sars), an emerging and highly contagious condition in human beings that is attributed to a novel human coronavirus (sars-cov). the relationship between sars-cov and animal coronavirus is still under investigation. other viruses rarely incriminated in porcine respiratory disease complex (prdc) include paramyxovirus, encephalomyocarditis virus, hemagglutinating encephalomyocarditis virus, and adenovirus. petechial hemorrhages in the lung and pulmonary edema may be seen with african swine fever, classical swine fever, and pseudorabies virus infections. porcine enzootic pneumonia. porcine enzootic pneumonia, a highly contagious disease of pigs caused by mycoplasma hyopneumoniae, is grossly characterized by suppurative or catarrhal bronchopneumonia ( fig. - and e- fig. - ). when its worldwide prevalence and deleterious effect on feed conversion are taken into account, this disease is probably the most economically significant respiratory disease of pigs. although an infectious disease, it is very much influenced by immune status and management factors, such as crowding (airspace and floor space), ventilation (air exchange rate), concentrations of noxious gases in the air (ammonia and hydrogen sulfide), relative humidity, temperature fluctuations, and mixing of stock from various sources. it has been demonstrated with inhibiting interferons and deregulates the adaptive immune response, thus interfering with the normal defense mechanisms predisposing pigs to septicemia and bacterial pneumonia. the most common opportunistic organisms are streptococcus suis, salmonella choleraesuis, mycoplasma hyopneumoniae, haemophilus parasuis, bordetella bronchiseptica, pasteurella multocida, and pneumocystis carinii. dual viral infections with prrsv and porcine circovirus (pcv ), siv, and porcine respiratory coronavirus (prcov) are commonly found in pigs, and such coinfections increase the severity of disease. on postmortem examination, pulmonary lesions vary from very mild changes characterized by failure of the lung to collapse when the thorax is opened and the presence of rib imprints (see fig. - ) to severe changes manifested by consolidation of the lung in cases that have been complicated with bacterial pneumonia. tracheobronchial and mediastinal lymph nodes are typically enlarged. microscopically, pulmonary changes are those of interstitial pneumonia characterized by thickening of alveolar walls by infiltrating macrophages and lymphocytes and mild hyperplasia of type ii pneumonocytes. necrotic cells are scattered in the alveolar lumens. unlike some other viral infections, bronchiolar epithelium does not appear to be affected. diagnosis of prrs in tissue collected at necropsy can be confirmed by immunohistochemistry and pcr techniques. infected pigs may become carriers and transmit the infection through body fluids and semen. clinically, prrs in nursery and young growing animals is characterized by sneezing, fever, anorexia, dyspnea, cough, and occasional death. some piglets develop severe cyanosis of the abdomen and ears, which explains why this syndrome was named blue ear disease when first described in europe. porcine circovirus-associated disease. another emerging porcine syndrome, characterized clinically by progressive emaciation in weaned pigs, was originally described in the s in canada, the united states, and europe. since then, it has disseminated to many countries, causing economic devastation in pig farms worldwide. because of the clinical signs and lesions in many organs, this syndrome was named postweaning multisystemic wasting syndrome (pmws). porcine circovirus (pcv ) has been incriminated as the etiologic agent and is a member of the circoviridae family. pcv has been associated with a number of syndromes in pigs, including systemic pcv infection (the preferred term for pmws because it may also affect mature pigs), pcv -associated pneumonia, pcv -associated enteritis, porcine dermatitis and nephropathy syndrome (pdns), pcv -associated reproductive failure, and, most recently, pcv -associated cerebellar vasculitis. the diseases caused by pcv are now collectively known as porcine circovirus-associated disease (pcvad); the most common manifestations are systemic pcv infection (pmws) and pcv -associated pneumonia as part of the porcine respiratory disease complex. all of these manifestations affect more than one organ, and there is substantial overlap between the syndromes. at necropsy, pigs with systemic pcv infection (pmws) and pcv -associated pneumonia are often in poor body condition, and the most remarkable changes, not considering other possible secondary infections, are enlargement of the superficial and visceral lymph nodes and a mild interstitial pneumonia characterized by failure of the lungs to collapse when the thorax is opened. jaundice is occasionally observed. microscopically, the lymphoid tissues show lymphoid depletion, histiocytic replacement of follicles, and notable proliferation of parafollicular histiocytes, some of which fuse and form syncytial cells (granulomatous lymphadenitis); necrosis of the lymphoid follicles is seen less often. in some cases, large basophilic inclusion bodies are present singly or as grapelike clusters (botryoid inclusions) within the cytoplasm of macrophages, particularly in peyer's patches, spleen, and lymph nodes (e- fig. - ). chapter respiratory system, mediastinum, and pleurae peribronchial, bronchiolar, and alveolar interstitium. additional virulence factors include the ability of mycoplasma hyopneumoniae to cause immunosuppression, reduce the phagocytic activity of neutrophils in the lung, and change the chemical composition of mucus. all of these functional alterations can predispose the lung to secondary bacterial infections. the lesions caused by mycoplasma hyopneumoniae start as a bronchointerstitial pneumonia and progress to a suppurative or mucopurulent bronchopneumonia once secondary pathogens are involved (commonly seen at necropsy). in most pigs, gross lesions affect only portions of the cranial lobes, but in more severely affected pigs, lesions involve % or more of the cranioventral portions of the lungs (see fig. - ). the affected lungs are dark red in the early stages but have a homogeneous pale-gray ("fish flesh") appearance in the more chronic stages of the disease. on cut surface, exudate can easily be expressed from airways, and depending on the stage of the lesions and secondary infections, the exudate varies from purulent to mucopurulent to mucoid. microscopic lesions are characterized by an influx of macrophages and neutrophils into the bronchi, bronchioles, and alveoli, and with time there is also notable balt hyperplasia (see fig. - , b) . in some cases, accumulation of exudate can be severe enough to cause occlusion of bronchioles and atelectasis of the corresponding lobules. the suppurative bronchopneumonia may be accompanied by a mild fibrinous pleuritis, which is often more severe if other organisms, such as mycoplasma hyorhinis, pasteurella multocida, or actinobacillus pleuropneumoniae, are also involved. abscesses and fibrous pleural adhesions are sequelae of chronic complicated infections. clinically, enzootic pneumonia occurs as a herd problem in two disease forms. a newly acquired infection of a previously clean herd causes disease in all age groups, resulting in acute respiratory distress and low mortality. in a chronically infected herd, the mature animals are immune and clinical signs are usually apparent only in growing pigs at times of particular stress such as at weaning. in such herds, coughing and reduced rate of weight gain are the most notable signs. porcine pasteurellosis. porcine pasteurellosis is an infectious disease complex with unclear pathogenesis that includes primary infections by pasteurella multocida alone (primary pasteurellosis) or, more frequently, after the defense mechanisms are impaired and a secondary bacterium colonizes the lung (porcine pneumonic pasteurellosis). in rare cases, pasteurella multocida causes acutely fatal septicemias in pigs (primary septicemic pasteurellosis). it is important to remember that pasteurella multocida serotypes a and d are both part of the normal nasal flora and are also causative agents of bronchopneumonia, pleuritis, and atrophic rhinitis in pigs. pasteurella multocida is one of the most common secondary pathogens isolated from the lungs of pigs with swine influenza virus (siv), porcine reproductive and respiratory syndrome virus (prrsv), porcine circovirus (pcv ), pseudorabies (suhv- ), classical swine fever (hog cholera), enzootic pneumonia, and porcine pleuropneumonia. secondary infections with pasteurella multocida notably change the early and mild bronchointerstitial reaction of enzootic and viral pneumonias into a severe suppurative bronchopneumonia with multiple abscesses and sometimes pleuritis. the other important role of pasteurella multocida in porcine pneumonias is as a cause of a fulminating, cranioventral, fibrinous bronchopneumonia (pleuropneumonia) after influenza virus infection or stress from inadequate ventilation resulting in high levels of ammonia in the air. the nature of the lesion and the predisposing factors of poor management or coexisting viral infections suggest that fulminating porcine pasteurellosis has a pathogenesis similar to that of pneumonic mannheimiosis of cattle. pharyngitis with subcutaneous cervical edema, fibrinohemorrhagic polyarthritis, and focal lymphocytic pcr that mycoplasma hyopneumoniae is present in the air of infected farms. the causative agent, mycoplasma hyopneumoniae, is a fastidious organism and very difficult to grow; thus the final diagnosis is frequently based on interpretation of lesions alone or supported by ancillary tests to detect this mycoplasma in affected lungs by immunohistochemistry, immunofluorescence, or pcr. the bronchopneumonic lesions of porcine enzootic pneumonia are in most cases mild to moderate, and thus mortality is low unless complicated with secondary pathogens, such as pasteurella multocida, trueperella (arcanobacterium) pyogenes, bordetella bronchiseptica, haemophilus spp., mycoplasma hyorhinis, and other mycoplasmas and ureaplasmas. although the pathogenesis of porcine enzootic pneumonia is not completely elucidated, it is known that mycoplasma hyopneumoniae first adheres to the cilia of the bronchi by means of a unique adhesive protein, produces ciliostasis, and finally colonizes the respiratory system by firmly attaching to the ciliated epithelial cells of the trachea and the bronchi of the cranioventral regions of the lungs. once attached to the respiratory epithelium, it provokes an influx of neutrophils into the tracheobronchial mucosa; causes extensive loss of cilia (deciliation); stimulates an intense hyperplasia of lymphocytes in the balt; and attracts mononuclear cells into the factors have been identified. these factors allow actinobacillus pleuropneumoniae to attach to cells; produce pores in cell membranes; damage capillaries and alveolar walls, resulting in vascular leakage and thrombosis; impair phagocytic function; and elicit failure of clearance mechanisms. the gross lesions in the acute form consist of a fibrinous bronchopneumonia characterized by severe consolidation and a fibrinous exudate on the pleural surface. although all lobes can be affected, a common site is the dorsal area of the caudal lobes. in fact, a large area of fibrinous pleuropneumonia involving the caudal lobe of a pig's lung is considered almost diagnostic for this disease (fig. - ) . on cut surface, consolidated lungs have notably dilated interlobular septa and irregular but well-circumscribed areas of necrosis caused by potent cytotoxins produced by actinobacillus pleuropneumoniae. except for the distribution, pulmonary lesions of porcine pleuropneumonia are identical to those of pneumonic mannheimiosis of cattle. the microscopic lesions are also very similar and include areas of coagulative necrosis surrounded by a thick cluster of "streaming (oat-shaped/oat cell) leukocytes" and notable distention of the interlobular septa because of severe edema and lymphatic thrombosis. bronchioles and alveoli are filled with edematous fluid, fibrin, neutrophils, and few macrophages (see fig. - ). pigs with the chronic form have multiple pulmonary abscesses and large ( to cm) pieces of necrotic lung encapsulated by connective tissue (sequestra)-changes frequently seen in slaughterhouses. interstitial nephritis are also present in porcine pneumonic pasteurellosis. sequelae of porcine pneumonic pasteurellosis include fibrous pleuritis and pericarditis, pulmonary abscesses, so-called sequestra, and usually death. in contrast to ruminants, mannheimia haemolytica is not a respiratory pathogen for pigs, but in some instances, it can cause abortion in sows. porcine pleuropneumonia. porcine pleuropneumonia is a highly contagious, worldwide disease of pigs caused by actinobacillus (haemophilus) pleuropneumoniae (app), which is characterized by a severe, often fatal, fibrinous bronchopneumonia with extensive pleuritis (pleuropneumonia). survivors generally develop notable residual lesions and become carriers of the organisms. porcine pleuropneumonia is an increasingly important cause of acute and chronic pneumonias, particularly in intensively raised pigs ( to months old). transmission of actinobacillus pleuropneumoniae occurs by the respiratory route, and the disease can be reproduced experimentally by intranasal inoculation of the bacterium. considered a primary pathogen, actinobacillus pleuropneumoniae can sporadically produce septicemia in young pigs and otitis media and otitis interna with vestibular syndrome in weaned pigs. two biovars and serotypes of the organism have been identified; all serotypes can cause the disease, but differences in virulence exist. the pathogenesis is not yet well understood, but specific virulence factors, such as rtx toxins (hemolytic/cytolytic toxins apx i to apx iv), capsular factors, fimbriae and adhesins, lipopolysaccharide, and permeability tuberculosis. tuberculosis is an important disease in domestic and wild pigs that has a much greater prevalence in pigs than in cattle or other domestic mammals in many countries. porcine tuberculosis is attributed to infection with mycobacterium bovis and porcine mycobacteriosis to infection with mycobacterium avium complex. a common scenario in small mixed-farming operations is the diagnosis of avian tuberculosis at the time that pigs are slaughtered, and the source is ingestion of tuberculous chickens or contaminated litter. as would be expected, granulomas are found in the mesenteric, mandibular, and retropharyngeal lymph nodes; to a lesser extent in the intestine, liver, and spleen; and only in rare cases in the lung. the route of infection in pulmonary tuberculosis and mycobacteriosis of pigs is most often hematogenous after oral exposure and intestinal infection. lung lesions are those of a granulomatous pneumonia. the microscopic lesions are basically those of tubercles (granulomas), but the degree of encapsulation, caseation, and calcification varies with the type of mycobacterium, age of the lesion, and host immune response. other bacterial pneumonias of pigs. septicemias in pigs often cause petechial hemorrhages in the lung and pulmonary edema. salmonellae, escherichia coli, and listeria monocytogenes can cause severe interstitial pneumonia in very young animals. salmonella choleraesuis causes a necrotizing fibrinous pneumonia similar to porcine pleuropneumonia, and salmonella typhisuis causes a chronic suppurative bronchopneumonia. in high health herds, actinobacillus suis may cause fibrinohemorrhagic pleuropneumonia and is easily confused with porcine pleuropneumonia. metastrongylosis. metastrongylus apri (elongatus), metastrongylus salmi, and metastrongylus pudendotectus (lungworms) of domestic and feral pigs occur throughout most of the world and require earthworms as intermediate hosts for transmission. the incidence of disease has therefore decreased with development of confinement housing. the importance of pig lungworms is mainly because infection results in growth retardation of the host. clinical signs include coughing because of parasitic bronchitis. the gross lesions, when noticeable, consist of small gray nodules, particularly along the ventral borders of the caudal lobes. the adult worms are grossly visible in bronchi, and microscopically, the parasites cause a catarrhal bronchitis with infiltration of eosinophils and lobular atelectasis ( fig. - ) . ascaris suum. the larvae of ascaris suum can cause edema, focal subpleural hemorrhages, and interstitial inflammation (see fig. - ). along their larval migration tracts, hemorrhages also occur in the liver and, after fibrosis, become the large white "milk spots" seen so frequently as incidental findings at necropsy. it has been reported that ascaris suum may cause immunosuppression in severely affected pigs. pigs can be killed if exposed to an overwhelming larval migration. other causes of pneumonia. foreign body granulomatous pneumonia occurs frequently in pigs after inhalation of vegetable material (starch pneumonia), presumably from dusty (nonpelleted) feed. lesions are clinically silent but are often mistaken for other pneumonic processes during inspection at slaughterhouses. microscopically, pulmonary changes are typical of foreign body granulomatous inflammation in which variably sized feed particles are surrounded by macrophages and neutrophils, and often have been phagocytosed by multinucleated giant cells. feed (vegetable) particles appear as thick-walled polygonal cells that stain positive with pas because of their rich carbohydrate (starch) content (see e- fig. - ) . clinically, porcine pleuropneumonia can vary from an acute form with unexpected death and blood-stained froth at the nostrils and mouth to a subacute form characterized by coughing and dyspnea accompanied by clinical signs of sepsis such as high fever, hypoxemia, anorexia, and lethargy (e- fig. - ) . a chronic form is characterized by decreased growth rate and persistent cough. animals that survive often carry the organism in the tonsils, shed the organism, and infect susceptible pigs. haemophilus pneumonia. in addition to glasser's disease characterized by polyserositis (pericarditis, pleuritis, peritonitis, polyarthritis, and meningitis) (e- fig. - ) , some serotypes of haemophilus parasuis (originally haemophilus influenzae suis) can also cause suppurative bronchopneumonia that in severe cases can be fatal. the causal organism, haemophilus parasuis, is usually carried in the nasopharynx of normal pigs and requires abnormal circumstances such as those following stress (weaning and cold weather) or viral infections (swine influenza or pcv ). specific pathogen-free (spf) pigs seem to be particularly susceptible to glasser's disease (arthritis and serositis) but not to pulmonary infection (bronchopneumonia). streptococcal pneumonia. streptococcus suis is a common cause of porcine disease worldwide and a serious zoonosis capable of causing death by septic shock or meningitis and residual deafness in butchers, veterinarians, and pig farmers. typically, streptococcus suis gains entrance to the susceptible young pig through the oropharyngeal mucosa and is carried in the tonsils, nasal mucosa, and mandibular lymph nodes of healthy animals, particularly in survivors of an outbreak. infected sows can abort or vertically transmit the infection to their offspring. some serotypes of streptococcus suis cause neonatal septicemia, and this can result in suppurative meningitis, otitis, arthritis, polyserositis, myocarditis, valvular endocarditis, and embolic pneumonia ( fig. - ). other serotypes of streptococcus suis can reach the lung by the aerogenous route and cause a suppurative bronchopneumonia, in combination with pasteurella multocida, escherichia coli, or mycoplasma hyopneumoniae, or in combination with actinobacillus pleuropneumoniae, which causes a fibrinous bronchopneumonia. coinfections of streptococcus suis with pcv and prrsv are also frequently seen in some farms. gross lesions in the acute stages include serous to catarrhal to mucopurulent nasopharyngitis and conjunctivitis. the lungs are edematous and have a diffuse interstitial pneumonia ( fig. - ) microscopically characterized by necrotizing bronchiolitis, necrosis and exfoliation of pneumonocytes, mild alveolar edema, and, several hours later, thickening of the alveolar walls because of interstitial mononuclear cell infiltrates and hyperplasia of type ii pneumonocytes. secondary infections with bordetella bronchiseptica and mycoplasmas are common and induce life-threatening suppurative bronchopneumonia. the thymus may be small relative to the age of the animal because of viral-induced lymphocytolysis. microscopically, eosinophilic inclusions are present in the epithelial cells of many tissues, in the nuclei or cytoplasm, or in both (see fig. - ). they appear early in the bronchiolar epithelium but are most prominent in the epithelium of the lung, stomach, renal pelvis, and urinary bladder, making these tissues good choices for diagnostic examination. viral inclusions are rarely seen in the later stages of this disease. the suppurative secondary bronchopneumonias often hinder the detection of viral lesions in the lung, particularly because bronchiolar cells containing inclusion bodies exfoliate and mix with the neutrophils recruited by the bacterial infection. distemper virus antigens can be readily demonstrated in infected cells by the immunoperoxidase technique (see fig. - ), which can also be used in skin biopsies for the antemortem diagnosis of canine distemper. distemper virus also has a tendency to affect developing tooth buds and ameloblasts, causing enamel hypoplasia in dogs that recover from infection. of all distemper lesions, demyelinating encephalomyelitis, which develops late, is the most devastating (see chapter ). sequelae to distemper include the nervous and pneumonic complications mentioned previously and various systemic infections, such as toxoplasmosis and sarcocystosis, because of depressed immunity. persistent viral infection occurs in some dogs that survive the disease, and they may become carriers and the source of infection for other susceptible animals. clinical signs consist of biphasic fever, diarrhea, vomiting, weight loss, mucopurulent oculonasal discharge, coughing, respiratory distress, and possible loss of vision. weeks later, hyperkeratosis of the foot pads ("hard pad") and the nose are observed, along with nervous signs, including ataxia, paralysis, convulsions, or residual myoclonus (muscle twitches, tremors, and "tics"). in general, inflammatory diseases of the lungs are less of a problem in dogs than in food-producing species and can be subdivided in two major groups, infectious and noninfectious pneumonias. "canine infectious respiratory disease" (cird) is the term currently used by clinicians to describe a heterogeneous group of respiratory infections in dogs; these diseases were previously clustered under the name of infectious tracheobronchitis or "kennel cough." cird is the canine counterpart of brd and prd complexes in cattle and pigs, respectively. the most common viruses in cird include canine parainfluenza virus (cpiv), canid herpesvirus (cahv- ), canine adenovirus- (cav- ), canine respiratory coronavirus (crcov), canine distemper virus (cdv), and canine influenza virus (civ). bordetella bronchiseptica, streptococcus equi ssp. zooepidemicus, and mycoplasma spp. are the most frequent bacterial isolates in cird. it has been recently recognized that animal shelters are an important source of viral and bacterial infections for dogs and cats. uremia and paraquat toxicity are perhaps the two most notable noninfectious causes of canine respiratory disease. canine distemper. canine distemper is an important and ubiquitous infectious disease of dogs, other canidae, wild felidae, mustelidae, and marine mammals throughout the world. it is caused by a morbillivirus that is antigenically related to the human measles, rinderpest (officially eradicated in ), "peste de petit ruminants," and phocine distemper viruses. canine distemper virus (cdv) is transmitted to susceptible puppies through infected body fluids. the virus invades through the upper respiratory tract and conjunctiva, proliferates in regional lymph nodes, becomes viremic, and in dogs with an inadequate antibody response, infects nearly all body tissues (pantropic), particularly the epithelial cells. distemper virus hampers the immune response, downregulates cytokine production, and persists for a long time in some tissues. cdv can target the lungs either directly as a viral pneumonia or indirectly by its immunosuppressive effects rendering the lungs susceptible to secondary bacterial and protozoal infections, or as a coinfection with other viruses such as canine adenovirus- and canid herpesvirus . : - , .) inclusion bodies occur within epithelial cells in early lesions. cahv- has also been identified as a cause of ulcerative keratoconjunctivitis in older dogs. canine influenza (canine flu). canine influenza is an emerging contagious respiratory infection of dogs that was first described in the united states and subsequently in other countries. it has a high morbidity (close to %), but the mortality, as with most other influenza infections, is relatively low (less than %). this disease, first diagnosed in greyhounds, is caused by a novel influenza-a virus (canine influenza virus or civ), a mutation from a previously recognized h n strain of equine influenza virus. dog-to-dog transmission does occur and therefore this infection must be distinguished from other viruses of the canine infectious respiratory disease (cird) group. pulmonary lesions are generally mild and transient, but infected dogs are susceptible to secondary bacterial bronchopneumonia. the most relevant lesions in dogs dying unexpectedly from canine influenza are pleural and pulmonary hemorrhages. microscopically, there is necrotizing tracheitis, bronchitis, and bronchiolitis with exudation of neutrophils and macrophages. in severe cases, hemorrhagic interstitial or bronchointerstitial pneumonia may be accompanied by vasculitis and thrombosis. influenza antigen can be demonstrated by immunohistochemistry in airway epithelium and alveolar macrophages. clinically, dogs with canine influenza are lethargic, inappetent, and hyperthermic and frequently cough and show nasal discharge. these signs resemble those seen in dogs with kennel cough or secondary bacterial pneumonia. in addition, there are confirmed cases of canine influenza caused by the porcine h n presumably transmitted from infected pet owners. bacterial pneumonias. dogs generally develop bacterial pneumonias when the pulmonary defense mechanisms have been impaired. pasteurella multocida, streptococcus spp., escherichia coli, klebsiella pneumoniae, and bordetella bronchiseptica can be involved in pneumonia secondary to distemper or after aspiration of gastric contents ( fig. - and e- fig. - ). streptococcus zooepidemicus can cause acute and fatal hemorrhagic pleuropneumonia with canine adenovirus type infection. cav- infection is a common but transient contagious disease of the respiratory tract of dogs, causing mild fever, oculonasal discharge, coughing, and poor weight gain. the portal of entry is generally by inhalation of infected aerosols followed by viral replication in the surface cells of the upper respiratory tract, mucous cells of the trachea and bronchi, nonciliated bronchiolar epithelial cells, and type ii pneumonocytes. pulmonary lesions are initially those of bronchointerstitial pneumonia, with necrosis and exfoliation of bronchiolar and alveolar epithelium, edema, and, a few days later, proliferation of type ii pneumonocytes, mild infiltration of neutrophils and lymphocytes in the alveolar interstitium, and hyperplastic bronchitis and bronchiolitis. large basophilic intranuclear viral inclusions are typically seen in bronchiolar and alveolar cells ( fig. - ) . infection with cav- is clinically mild unless complicated with a secondary bacterial infection or coinfections with other viruses such as distemper virus. experimental work suggests cav- reinfection may lead to hyperreactive airways, a nonspecific condition in which the bronchial mucosa becomes highly "responsive" to irritation such as that caused by cold air, gases, or cigarette smoke. however, it is not clear if this outcome is true in natural infections. canid herpesvirus . canid herpesvirus (cahv- ) can cause fatal systemic disease in newborn puppies and is probably a contributing factor in "fading puppy syndrome." hypothermia has been suggested as a pivotal component in the pathogenesis of fatal infections in puppies. many dogs are seropositive, suggesting that transient or subclinical infections are more common than realized; the virus remains latent in the trigeminal and other ganglia and can be reactivated after stress, resulting in asymptomatic transmission of cahv- virus to offspring via the placenta, thus resulting in abortion or stillbirths. in puppies, cahv- causes ulcerative tracheitis, interstitial pneumonia (e- fig. - ) , and focal necrosis and inflammation in the kidneys, liver, and brain. eosinophilic intranuclear aspiration pneumonia starts as an acute necrotizing bronchitis and bronchiolitis caused by aspiration of irritant materials such as gastric acid or a caustic material administered by mouth. the aspirate also contains potentially pathogenic bacteria, and because the mucociliary apparatus is damaged and these bacteria are not removed, they settle into the ventral portions of the lung (from gravity) and provoke a fibrinosuppurative and necrotizing bronchopneumonia. b, bronchoalveolar spaces are filled with neutrophils, macrophages, and bacteria (arrows). h&e stain. inset, large colonies of bacteria (arrows). h&e stain. (courtesy dr. a. lópez, atlantic veterinary college.) a b infection; thus it most frequently affects outdoor and hunting dogs. from the lung, infection is disseminated hematogenously to other organs, mainly bone, skin, brain, and eyes. pulmonary lesions are characterized by multifocal to coalescing pyogranulomatous pneumonia, generally with firm nodules scattered throughout the lungs (fig. - ) . microscopically, nodules are pyogranulomas with numerous macrophages (epithelioid cells), some neutrophils, multinucleated giant cells, and thick-walled yeasts (see fig. - , c). yeasts are to µm in diameter and are much better visualized when they are stained with pas reaction or gomori's methenamine silver stain. nodules can also be present in other tissues, chiefly lymph nodes, skin, spleen, liver, kidneys, bones, testes, prostate, and eyes. this fungus can be easily identified in properly prepared and stained transtracheal washes or lymph node aspirates. clinical signs can reflect involvement of virtually any body tissue; pulmonary effects include cough, decreased exercise tolerance, and terminal respiratory distress. coccidioidomycosis. coccidioidomycosis (san joaquin valley fever), caused by the dimorphic fungus coccidioides immitis, occurs mainly in animals living in arid regions of the southwestern united states, mexico, and central and south america. it is a primary respiratory tract (aerogenous) infection commonly seen at slaughterhouses in clinically normal feedlot cattle. in dogs, coccidioidomycosis also has an aerogenous portal of entry and then a b hemorrhagic pleural effusion in dogs. death is generally a consequence of severe sepsis and septic shock or from β-hemolytic streptococcal bacteremia causing emboli in the lungs, liver, brain, and lymph nodes. the primary source of the infection cannot be determined in most cases. dental disease in dogs may be a source of systemic and pulmonary infection, a concept wellrecognized in human medicine for many years. the role of mycoplasmas in canine pneumonia is still uncertain because these organisms are frequently isolated from normal nasopharyngeal flora. tuberculosis is uncommon in dogs because these animals appear to be quite resistant to infection; most cases occur in immunocompromised dogs or in dogs living with infected human beings. dogs are susceptible to the infection with mycobacterium tuberculosis, mycobacterium bovis, and mycobacterium avium complex, and therefore canine infection presupposes contact with human or animal tuberculosis. the clinicopathologic manifestation is pulmonary after inhalation or alimentary after oral exposure, but in most cases infection is disseminated to lymph nodes and visceral organs. the gross lesions are multifocal, firm nodules with necrotic centers, most often seen in the lungs, lymph nodes, kidneys, and liver. diffuse granulomatous pleuritis and pericarditis with copious serofibrinous or sanguineous effusion are common. microscopically, granulomas are formed by closely packed macrophages but with very little connective tissue. mycotic pneumonias. mycotic pneumonias are serious diseases seen commonly in animals in some regions. there are two main types: those caused by opportunistic fungi and those caused by a group of fungi associated with systemic "deep" mycoses. all of these fungi affect human beings and most domestic animals but are probably not transmitted between species. aspergillosis. opportunistic fungi, such as aspergillus spp. (particularly aspergillus fumigatus), are important in birds, but in domestic animals, they mainly affect immunosuppressed individuals or those on prolonged antibiotic therapy. the pulmonary lesion is a multifocal, nodular, pyogranulomatous, or granulomatous pneumonia. microscopically, there is necrosis and infiltrates of neutrophils, macrophages, and lymphocytes, with proliferation of fibroblasts eventually leading to encapsulation of the granuloma. fungal hyphae are generally visible in the core of the lesion and in the walls of blood vessels. systemic mycoses (dimorphic fungal infections) . systemic (deep) mycoses are caused by blastomyces dermatitidis, histoplasma capsulatum, coccidioides immitis, and cryptococcus neoformans/ cryptococcus gatti (see fig. - ). blastomycosis mainly affects dogs and is discussed here, whereas cryptococcosis is discussed in the section on pneumonias of cats. in contrast to other fungi, such as aspergillus spp., organisms of the systemic mycosis group are all primary pathogens of human beings and animals and thus do not necessarily require a preceding immunosuppression to cause disease. these fungi have virulence factors that favor hematogenous dissemination and evasion of immune and phagocytic responses. systemic dissemination is often exacerbated by the administration of immunosuppressant drugs such as corticosteroids. these fungi are usually detected by cytological evaluation of affected tissues. blastomycosis. blastomycosis occurs in many countries of the north american continent, africa, the middle east, and occasionally in europe. in the united states, it is most prevalent in the atlantic, st. lawrence, and ohio-mississippi river valley states, compared with the mountain-pacific region. blastomyces dermatitidis is a dimorphic fungus (mycelia-yeast) seen mainly in young dogs and occasionally in cats and horses. this fungus is present in the soil, and inhalation of spores is considered the principal route of from the alveolar interstitium associated with larvae or dead worms because little reaction develops to the live adults. crenosoma vulpis. crenosoma vulpis is a lungworm seen commonly in foxes and sporadically in dogs with access to the intermediate hosts-slugs and snails. the adult lungworms live in small bronchi and bronchioles in the caudal lobes, causing eosinophilic and catarrhal bronchitis manifested grossly as gray areas of inflammation and atelectasis. in some animals, crenosoma vulpis causes bronchiolar goblet cell metaplasia and mucous obstruction, resulting in lobular atelectasis due to the valve effect of the mucous plug. eucoleus aerophilus. eucoleus aerophilus (capillaria aerophila) is a nematode parasite typically found in the trachea and bronchi of wild and domestic carnivores. in some cases, this parasite may also involve the nasal passages and sinuses. although generally asymptomatic, some dogs cough because of the local irritation caused by the parasites on the tracheal or bronchial mucosa. paragonimus spp. paragonimus kellicotti in north america and paragonimus westermani in asia are generally asymptomatic fluke infections in fish-eating species. the life cycle involves two intermediate hosts, the first a freshwater snail and the second a freshwater crab or crayfish; in north america, cats and dogs acquire infection by eating crayfish. gross lesions include pleural hemorrhages where the metacercariae migrate into the lungs. later, multifocal eosinophilic pleuritis, and subpleural cysts up to mm long containing pairs of adult flukes, are found along with eosinophilic granulomas around clusters of eggs. like many other parasitic pneumonias, lesions and scars are more frequent in the caudal lobes. pneumothorax can occur if a cyst that communicates with an airway ruptures to the pleural surface. other parasitic infections. angiostrongylus vasorum and dirofilaria immitis are parasites of the pulmonary arteries and right ventricle and, depending on the stage, can produce different forms of pulmonary lesions. adult parasites can cause chronic arteritis that leads to pulmonary hypertension, pulmonary arterial thrombosis, interstitial (eosinophilic) granulomatous pneumonia, pulmonary interstitial fibrosis, congestive right-sided cardiac failure, and eventually caudal vena caval syndrome. other lesions include pleural petechial hemorrhages and, in later stages, diffuse pulmonary hemosiderosis and multifocal pulmonary infarcts. larvae and eggs also cause alveolar injury, thickening of the alveolar walls with eosinophils and lymphocytes (interstitial pneumonia), and multifocal or coalescing granulomas with giant cells (parasitic granulomas). pneumocystis carinii has been reported as a sporadic cause of chronic interstitial pneumonia in dogs with a compromised immune system (see pneumonias of horses; also see fig. - ). aspiration pneumonia. aspiration pneumonia is an important form of pneumonia that occurs in dogs when vomit or regurgitated materials are aspirated into the lungs, or when drugs or radiographic contrast media are accidentally introduced into the airways (e- fig. - ) . as in other animal species, aspiration pneumonia may be unilateral or may more often affect the right cranial lobe ( fig. - ). the severity of lesions depends very much on the chemical and microbiologic composition of the aspirated material. in general, aspiration in monogastric animals, particularly in dogs and cats, is more severe because of the low ph of the gastric contents (chemical pneumonitis). in severe cases, dogs and cats die rapidly from septic shock and ards (see fig. - ), which is microscopically characterized by diffuse alveolar damage, protein-rich pulmonary edema, neutrophilic alveolitis, and formation of typical hyaline membranes along the alveolar walls (see fig. - ). in animals that survive the acute stages of aspiration, pulmonary lesions progress to bronchopneumonia. aspiration pneumonia is a common sequela to disseminates systemically to other organs. clinical signs relate to the location of lesions, so there can be respiratory distress, lameness, generalized lymphadenopathy, or cutaneous lesions, among others. the lesions caused by coccidioides immitis consist of focal granulomas or pyogranulomas that can have suppurative or caseated centers. the fungal organisms are readily seen in histologic or cytologic preparation as large ( to µm in diameter), double-walled, and highly refractile spherules containing numerous endospores (see fig. - , d) . histoplasmosis. histoplasmosis is a systemic infection that results from inhalation and, in dogs, possibly ingestion of another dimorphic fungus, histoplasma capsulatum. histoplasmosis occurs sporadically in dogs and human beings and, to a lesser extent, in cats and horses. bats often eliminate histoplasma capsulatum in the feces, and droppings from bats and birds, particularly pigeons, heavily promote the growth and survival of this fungus in the soil of enzootic areas. pulmonary lesions are grossly characterized by variably sized, firm, poorly encapsulated granulomas and, sometimes, more diffuse involvement of the lungs. microscopically, granulomatous lesions typically have many macrophages filled with small ( to µm), punctiform, intracytoplasmic, dark oval bodies (yeasts) (see fig. - , a) that are best demonstrated with pas reaction or gomori's methenamine silver stain. similar nodules or diffuse involvement can be present in other tissues, chiefly lymph nodes, spleen, intestine, and liver. toxoplasmosis. toxoplasmosis is a worldwide disease caused by the obligate intracellular, protozoal parasite toxoplasma gondii. cats and other felidae are the definitive hosts in which the mature parasite divides sexually in the intestinal mucosa. human beings, dogs, cats, and many wild mammals can become intermediate hosts after accidental ingestion of fertile oocysts shed in cat feces or ingestion of undercooked or raw meat containing tissue cysts, and fetuses can be infected transplacentally from an infected dam. in most instances, the parasite infects many cells of different tissues and induces an antibody response (seropositive animals) but does not cause clinical disease. toxoplasmosis is often triggered by immunosuppression, such as that caused by canine distemper virus. toxoplasmosis is characterized by focal necrosis around the protozoan. pulmonary lesions are severe, multifocal necrotizing interstitial pneumonia with notable proliferation of type ii pneumonocytes and infiltrates of macrophages and neutrophils. other lesions in disseminated toxoplasmosis include multifocal necrotizing hepatitis, myocarditis, splenitis, myositis, encephalitis, and ophthalmitis. the parasites appear microscopically as small ( to µm) basophilic cysts that can be found free in affected tissues or within the cytoplasm of many epithelial cells and macrophages (see e- fig. - ) . similar findings can be seen sporadically in dogs infected with neospora caninum and sarcocystis canis, and immunohistochemistry would be required to differentiate those protozoal organisms from toxoplasma gondii. filaroides hirthi. filaroides hirthi, a lungworm of the alveoli and bronchioles of dogs, has long been known as a cause of mild subclinical infection in large colonies of beagle dogs in the united states. however, it can on occasion cause severe and even fatal disease in individual pets, presumably as a result of immunosuppression. clinical signs may include coughing and terminal respiratory distress. grossly, the lesions are multifocal subpleural nodules, often with a green hue because of eosinophils, scattered throughout the lungs. microscopically, these nodules are eosinophilic granulomas arising .e chapter respiratory system, mediastinum, and pleurae other pneumonias. idiopathic pulmonary fibrosis is a rare condition of uncertain etiology reported in the west highland white terrier breed that shares similarities with human and feline idiopathic pulmonary fibrosis. microscopically, there is diffuse interstitial pneumonia and progressive alveolar fibrosis with capillary obliteration, hyperplasia of type ii cells, some of which exhibit cellular atypia, and finally hypertrophy and hyperplasia of smooth muscle. the interstitial fibrosis eventually spills over alveolar spaces causing conspicuous intraalveolar fibrosis. although upper respiratory tract infections are common and important in cats, pneumonias are uncommon except when there is immunosuppression or aspiration of gastric contents. viral infections such as feline rhinotracheitis and calicivirus may cause lesions in the lungs, but unless there is secondary invasion by bacteria, they do not usually cause a fatal pneumonia. feline rhinotracheitis. feline rhinotracheitis is an important viral disease of cats caused by the ubiquitous felid herpesvirus (fehv- ). this infection affects primarily young or debilitated cats causing inflammation in the nasal, ocular, and tracheal mucosa and, to a much lesser extent, the lung (see species-specific diseases of the nasal cavity and paranasal sinuses). when lungs are affected, fehv- causes bronchointerstitial pneumonia with necrosis of bronchiolar and alveolar epithelium, thickening of the alveolar walls, and extensive permeability edema. eosinophilic intranuclear inclusion bodies may be seen in infected epithelial cells early in infection. feline calicivirus. feline calicivirus (fcv) causes upper respiratory disease, stomatitis, conjunctivitis, and, to a lesser extent, interstitial pneumonia. microscopically, affected lungs exhibit the typical pattern of bronchointerstitial pneumonia with necrotizing bronchiolitis, thickening of alveolar walls, occasionally hyaline membranes, hyperplasia of type ii pneumonocytes, and macrophages admixed with cellular debris in the alveolar lumens. because pulmonary lesions are similar to those caused by fehv- , isolation or in situ detection is required for final diagnosis. feline infectious peritonitis. feline infectious peritonitis (fip) is caused by fip virus (fipv), a mutated form of feline enteric cleft palate, and in dogs with megaesophagus secondary to either myasthenia gravis or persistent right aortic arch. it is also an important complication of general anesthesia or neurologic diseases affecting laryngeal function. paraquat. paraquat, a broad-spectrum herbicide widely used in gardening and agriculture, can cause severe and often fatal toxic interstitial pneumonia (pneumonitis) in dogs, cats, human beings, and other species. after ingestion or inhalation, this herbicide selectively accumulates in the lung where paraquat toxic metabolites are produced by club (clara) cells. these metabolites promote local release of free radicals in the lung, which causes extensive injury to club cells and to the blood-air barrier, presumably through lipid peroxidation of type i and ii pneumonocytes and alveolar endothelial cells (see fig. - ). paraquat toxicity has been used experimentally as a model of oxidant-induced alveolar injury and pulmonary fibrosis. soon after poisoning, the lungs are heavy, edematous, and hemorrhagic because of extensive necrosis of epithelial and endothelial cells in the alveolar walls. the lungs of animals that survive acute paraquat toxicosis are pale, fail to collapse when the thorax is opened, and have interstitial emphysema, bullous emphysema, and occasionally pneumomediastinum. microscopic findings in the acute and subacute phases include necrosis of type i pneumonocytes, interstitial and alveolar edema, intraalveolar hemorrhages, and proliferation of type ii pneumonocytes. in the chronic stages ( to weeks later), the lesions are typically characterized by severe interstitial and intraalveolar fibrosis. uremic pneumopathy. uremic pneumonopathy (pneumonitis) is one of the many extrarenal lesions seen in dogs with chronic uremia. lesions are characterized by a combination of pulmonary edema and calcification of vascular smooth muscle and alveolar basement membranes. in severe cases, alveolar calcification prevents lung collapse when the thorax is opened. in the more advanced cases, the lungs appear diffusely distended, pale red or brown in color, and show a rough pleural surface with rib imprints (see fig. - ). on palpation, the pulmonary parenchyma has a typical "gritty" texture because of mineralization of the alveolar and vascular walls, which are best visualized microscopically by using special stains such as von kossa (see fig. - ). because this is not primarily an inflammatory lesion, the term pneumonitis should not be used. fig. - ) . a, note that the lungs did not collapse when the thorax was opened (loss of negative pressure) and as a result fill almost the entire thoracic cavity. the cranioventral aspects of the lung are consolidated with hemorrhage. b, alveolar capillary congestion, thick hyaline membranes along the alveolar septa (arrows), and intraalveolar hemorrhage. these microscopic changes are typical of the diffuse alveolar damage seen in lungs with ards. h&e stain. (courtesy dr. a. lópez, atlantic veterinary college.) feline calicivirus has removed chlamydophila felis from its previously overstated importance as a lung pathogen. tuberculosis. cats are susceptible to three types of mycobacterial infections: classic tuberculosis, feline leprosy, and atypical mycobacteriosis. classic tuberculosis in cats is rare and generally caused by mycobacterium bovis and mycobacterium microti but also, to a lesser extent, by mycobacterium tuberculosis. nosocomial tuberculosis (mycobacterium bovis) in cats has been reported with increased frequency. the usual route of infection for feline tuberculosis is oral, through infected rodents/meat or unpasteurized milk, so the granulomatous lesions are mainly in the intestine and mesenteric lymph nodes where they may disseminate through infected phagocytes to other organs. the solid and noncaseated appearance of tuberculous nodules is grossly similar to that of neoplasms, so they must be differentiated from pulmonary neoplasms (e.g., lymphoma). classic tuberculosis with dermal lesions in cats should be differentiated from feline leprosy (localized skin granulomas) caused by mycobacterium lepraemurium and other nonculturable species of acid-fast bacilli. atypical mycobacteriosis is caused by contamination of a skin wound with saprophytic and nonsaprophytic mycobacteria such as those of the mycobacterium avium complex. advances in pcr techniques have notably reduced the time required for etiologic diagnosis of mycobacteriosis in veterinary diagnostic laboratories. cryptococcosis. cryptococcosis (pulmonary cryptococcus neoformans or cryptococcus gatti) is the most frequent systemic mycosis in cats, and lesions are akin to those discussed in the section on mycotic pneumonias of dogs. it occurs worldwide in all species but is diagnosed most frequently in cats, horses, dogs, and human beings. some healthy dogs and cats harbor cryptococcus in the nasal cavity and become asymptomatic carriers. clinical infection may occur in immunocompetent cats and in cats that are immunologically compromised, such as by felv, fiv, malnutrition, or corticosteroid treatment. lesions can occur in nearly any tissue, resulting in a wide c coronavirus (fecv), and is one of a few viral infections of domestic animals that result in pyogranulomatous pneumonia. this disease is microscopically characterized by a vasculitis affecting many tissues and organs ( fig. - ) . other viral pneumonias. other viruses sporadically incriminated in feline interstitial pneumonia are cowpox virus (cpxv) and influenza a h n . pasteurellae. bacteria from the nasal flora such as pasteurella multocida and pasteurella-like organisms are occasionally associated with secondary bronchopneumonia in cats ( fig. - ) . pasteurella multocida also causes otitis media and meningitis, but its role as a respiratory pathogen is mainly associated with pyothorax. interestingly, there are reports of pasteurella multocida pneumonia in older or immunosuppressed human beings acquired through contact with domestic cats. mycoplasmas. mycoplasmas are often isolated from the lungs of cats with pulmonary lesions but are not definitively established as primary pathogens in feline pneumonias. feline pneumonitis. the term feline pneumonitis is a misnomer because the major lesions caused by chlamydophila felis (formerly chlamydia psittaci) are severe conjunctivitis and rhinitis (see species-specific diseases of the nasal cavity and paranasal sinuses). the elucidation of the importance of feline viral rhinotracheitis and organism infects erythrocytes in the erythrocytic stage of disease and multiplies in intravascular macrophages/monocytes, including those in the alveolar capillaries (e- fig. - ) , during the leukocytic stage of disease. aspiration pneumonia. aspiration pneumonias are common in cats as a result of vomiting, regurgitation, dysphagia, or anesthetic complication or after accidental administration of food, oral medicaments, or contrast media into the trachea (iatrogenic). pulmonary lesions are similar to those described for dogs, and the type of lung lesion depends on the chemical and bacterial composition of the aspirated material (see the section on aspiration pneumonia of dogs). feline idiopathic pulmonary fibrosis. feline idiopathic pulmonary fibrosis is a rare, progressive, and fatal disease of cats of uncertain etiology characterized by multifocal fibrotic nodules subpleurally and randomly in the lung making the pleural surface resemble nodular cirrhosis of the liver ( fig. - ) . microscopically, the affected alveolar and peribronchiolar interstitium is thickened by excessive fibrosis, abundant deposition of extracellular matrix, and hypertrophy of smooth muscle. some investigators suggest an intrinsic cellular defect in type ii pneumonocytes as the underlying cause. the alveolar walls are diffusely lined by cuboidal hyperplastic type ii pneumonocytes, and the alveolar lumens often contain exfoliated cells and necrotic debris. this feline condition has morphologic features similar to "equine multinodular pulmonary fibrosis" and "cryptogenic pulmonary fibrosis" in human beings. fetal pneumonias. pneumonia is one of the most frequent lesions found in fetuses submitted for postmortem examination, particularly in foals and food-producing animals. because of autolysis, lack of inflation, and the lungs being at various stages of development, fetal lesions are often missed or misdiagnosed. in the nonaerated fetal lung, the bronchoalveolar spaces are filled with a viscous, locally produced fluid known as lung fluid or lung liquid. it has been estimated that an ovine fetus produces approximately . ml of "lung fluid" per kilogram of body weight per hour. in the variety of clinical signs. however, granulomatous rhinitis, sinusitis, otitis media and interna, pneumonia, ulcerative dermatitis, and meningoencephalitis are most common. the pulmonary lesion in cryptococcosis is a multifocal granulomatous pneumonia and, like those occurring in other internal organs, they are small, gelatinous, white foci. the gelatinous appearance is due to the broad mucous capsule around the yeast (see fig. - , b) . microscopically, lesions contain great numbers of fungal organisms ( to µm in diameter without the capsule) and only a few macrophages, lymphocytes, and multinucleated giant cells. this thick polysaccharide capsule does not stain well with h&e, and thus there is a large empty space or halo around the yeast. feline lungworm. aelurostrongylus abstrusus, known as feline lungworm, is a parasite that occurs in cats wherever the necessary slug and snail intermediate hosts are found. it can cause chronic respiratory disease with coughing and weight loss and, sometimes, severe dyspnea and death, particularly if there are secondary bacterial infections. the gross lesions are multifocal, amber, and subpleural granulomatous nodules up to cm in diameter throughout the lungs. on incision, these nodules may contain viscous exudate. microscopically, the adult parasites, eggs, and coiled larvae are in the bronchioles and alveoli, where they cause catarrhal bronchiolitis, hyperplasia of submucosal glands, and, later, granulomatous alveolitis, alveolar fibrosis, and fibromuscular hyperplasia ( fig. - ) . during routine examination of feline lungs, it is quite common to find fibromuscular hyperplasia in bronchioles and arterioles in otherwise healthy cats. it was alleged in the past that this fibromuscular hyperplasia was a long-term sequela of subclinical infection with aelurostrongylus abstrusus. however, this view has been challenged; thus the pathogenesis and significance of pulmonary fibromuscular hyperplasia in healthy cats remains uncertain. in severe cases, fibromuscular hyperplasia is grossly visible in the lungs as white subpleural nodules. other parasitic pneumonias. toxoplasma gondii, paragonimus kellicotti, and dirofilaria immitis can also affect cats (see the section on parasitic pneumonias of dogs). cytauxzoon felis is an apicomplexan hemoparasite that affects domestic and wild felidae. the diseases that cause fetal pneumonia in farm animals. gross lesions in the lungs are generally undetected, but microscopic lesions include focal necrotizing interstitial pneumonia and focal necrosis in the liver, spleen, or brain. fetal bronchointerstitial pneumonia also occurs in some viral abortions, such as those caused by infectious bovine rhinotracheitis (ibr) virus and bovine parainfluenza virus (bpiv- ) in cattle and equine viral rhinopneumonitis (evr) in horses. fetal pneumonias in dogs and cats are infrequently described, perhaps because aborted puppies and kittens are rarely submitted for postmortem examination. with advancements in molecular biology techniques, the etiologic diagnosis of abortions and their association with pulmonary fetal lesions is rapidly improving. neonatal pneumonias and septicemias. these entities are rather common in newborn animals lacking passive immunity because of the lack of either ingestion or absorption of maternal colostrum (failure of passive transfer or hypogammaglobulinemia). in addition to septicemias causing interstitial pneumonia, farm animals with hypogammaglobulinemia can develop bronchopneumonia by inhalation of bacterial pathogens. these include histophilus somni and pasteurella multocida in calves; streptococcus spp. in foals; and escherichia coli, listeria monocytogenes, and streptococcus suis in pigs. meconium aspiration syndrome. meconium aspiration syndrome (mas) is an important but preventable condition in human babies that originates when amniotic fluid contaminated with meconium is aspirated during labor or immediately after birth. the pathogenesis of mas is basically the same as in those of fetal bronchopneumonia (see fig. - ) . fetal hypoxia, a common event during dystocia or prolonged parturition, causes the fetus to relax the anal sphincter and release meconium into the amniotic fluid. aspiration of meconium can occur directly from aspirating contaminated amniotic fluid before delivery (respiratory movements with an open glottis) or immediately after delivery when the meconium lodged in the nasopharynx is carried into the lung with the first breath of air. this latter form of aspiration is prevented in delivery rooms by routine suction of the nasopharynx in meconium-stained babies. mas is well known in human babies, but the occurrence and significance in animals remains largely unknown. mas has been reported in calves, foals, piglets, and puppies. although pulmonary lesions are generally mild and transient, aspiration of meconium can be life-threatening for newborn babies and animals because it typically occurs in compromised neonates already suffering from intrauterine hypoxia and acidosis. neonatal acidosis is known to impair colostrum absorption in calves. common mas sequelae are lobular atelectasis, pulmonary hypertension, and possibly airway hyperreactivity. in the most severe cases of mas, focal (patchy) atelectasis can be observed grossly in the lung, indicating failure of the lungs to be fully aerated because of the mechanical obstruction and the chemical effect of meconium on pulmonary surfactant (see fig. - ). microscopically, meconium and keratin exfoliated from skin of the fetus into the amniotic fluid are present in bronchi, bronchioles, and alveoli and accompanied by mild alveolitis characterized by infiltration of leukocytes followed by alveolar macrophages and occasional giant cells (e- fig. - ) . lung cancer in animals is rare, unlike in human beings, in which the incidence is alarming and continues to be the number one cause of death due to cancer in canada, the united states, and europe. interestingly, prostatic and breast cancers, so much feared by men fetus, this fluid normally moves along the tracheobronchial tree, reaching the oropharynx, where a fraction is swallowed into the gastrointestinal tract, and a small portion is released into the amniotic fluid. at the time of birth, the lung fluid is rapidly reabsorbed from the lungs by alveolar absorption and lymphatic drainage. aspiration of amniotic fluid contaminated with meconium and bacteria from placentitis is the most common route by which microbial pathogens reach the fetal lungs. this form of pneumonia is secondary to fetal hypoxia and acidosis ("fetal distress"), which cause the fetus to relax the anal sphincter, release meconium into the amniotic fluid, and, in the terminal stages, inspire deeply with open glottis, resulting in the aspiration of contaminated fluid ( fig. - ). gross lesions are only occasionally recognized, but microscopic changes are similar to those of a bronchopneumonia. microscopically, bronchoalveolar spaces contain variable numbers of neutrophils, macrophages, epidermal squames, and pieces of meconium that appear as bright yellow material because of its bile content. in contrast to postnatal bronchopneumonia, lesions in fetuses are not restricted to the cranioventral aspects of the lungs but typically involve all pulmonary lobes. in cattle, brucella abortus and trueperella (arcanobacterium) pyogenes are two of the most common bacteria isolated from the lungs of aborted fetuses. these bacteria are usually present in large numbers in the amniotic fluid of cows with bacterial placentitis. inflammation of the placenta interferes with oxygen exchange between fetal and maternal tissue, and the resultant fetal hypoxia induces the fetus to "breathe" with an open glottis and aspirate the amniotic fluid. aspergillus spp. (mycotic abortion) and ureaplasma diversum cause sporadic cases of placentitis, which results in fetal pneumonia and abortion. in addition to the respiratory route (aspiration), pathogens, such as bacteria and viruses, can also reach the lungs via fetal blood and cause interstitial pneumonia. listeriosis (listeria monocytogenes), salmonellosis (salmonella spp.), and chlamydiosis (chlamydophila abortus [c. psittaci]) are the best known examples of blood-borne primary benign neoplasms of the lungs, such as pulmonary adenomas, are highly unusual in domestic animals. most primary neoplasms are malignant and appear as solitary masses of variable size that, with time, can metastasize to other areas of the lungs and to distant organs. it is sometimes difficult on gross and microscopic examination to differentiate primary lung cancer from pulmonary metastasis resulting from malignant neoplasms elsewhere in the body. it is often difficult to determine the precise topographic origin of a neoplasm within the lungs-for example, whether it originates in the conducting system (bronchogenic carcinoma), transitional system (bronchiolar carcinoma), exchange system (alveolar carcinoma), or bronchial glands (bronchial gland carcinoma). according to the literature, pulmonary carcinomas in animals arise generally from club (clara) cells or type ii pneumonocytes of the bronchioloalveolar region, in contrast to those in human beings, which are mostly bronchogenic. tumors located at the hilus generally arise from major bronchi and tend to be a solitary large mass with occasional small metastasis to the periphery of the lung. in contrast, tumors arising from the bronchioloalveolar region are often multicentric with numerous peripheral metastases in the lung parenchyma. because of histologic architecture and irrespective of the site of origin, many malignant epithelial neoplasms are classified by the all-encompassing term of pulmonary adenocarcinomas. dogs and cats are the species most frequently affected with primary pulmonary neoplasms, largely carcinomas, generally in older animals. the mean age for primary lung tumors is years for dogs and years for cats. pulmonary carcinomas in other domestic animals, except for retrovirus-induced pulmonary carcinoma in sheep, are less common, possibly because fewer farm animals are allowed to reach their natural life span. these neoplasms can be invasive or expansive, vary in color (white, tan, or gray) and texture (soft or firm), and often have areas of necrosis and hemorrhage, which result in a "craterous" or "umbilicate" appearance. this umbilicate appearance is frequently seen in rapidly growing carcinomas in which the center of the tumoral mass undergoes necrosis as a result of ischemia. some lung neoplasms resemble pulmonary consolidation or large granulomas. cats with moderately differentiated neoplasms had significantly longer survival time (median, days) than cats with poorly differentiated neoplasms (median, days). dogs with primary lung neoplasms, grades i, ii, and iii, had survival times of , , and days, respectively. ovine pulmonary adenocarcinoma (ovine pulmonary carcinoma). ovine pulmonary adenocarcinoma, also known as pulmonary adenomatosis and jaagsiekte (from the south african afrikaans word for "driving sickness"), is a transmissible, retrovirus-induced neoplasia of ovine lungs caused by jaagsiekte sheep retrovirus (jsrv). it occurs in sheep throughout the world, with the notable exception of australia and new zealand; its incidence is high in scotland, south africa, and peru and unknown but probably low in north america. this pulmonary carcinoma behaves very much like a chronic pneumonia, and jsrv shares many epidemiologic similarities with the ovine lentivirus responsible for maedi and the retrovirus responsible for enzootic nasal carcinoma in small ruminants. pulmonary adenomatosis has been transmitted to goats experimentally but is not known to be a spontaneous disease in that species. ovine pulmonary adenocarcinoma affects mainly mature sheep but can occasionally affect young stock. intensive husbandry probably facilitates horizontal transmission by the copious nasal discharge and explains why the disease occurs as devastating epizootics with % to % mortality when first introduced into a flock. differential diagnosis between maedi and pulmonary adenomatosis can prove difficult because both diseases often coexist in the same flock and women, are a distant second. to say that cigarette smoking is responsible for this epidemic of lung cancer is unnecessary. although dogs have been proposed as valuable "sentinels" for environmental hazards, such as exposure to passive smoking, asbestos, dyes, and insecticides, it is not known if the prevalence of canine lung tumors has increased in geographical areas with high contamination. alterations in genes (oncogenes) and chromosomes and changes in biologically active molecules have been linked to lung cancer in recent years. as with many other forms of cancer, epidemiologic studies indicate that the incidence of pulmonary neoplasms increases with age, but there are still insufficient data to confirm that particular canine or feline breeds have a higher predisposition to spontaneous lung neoplasms. a standard nomenclature of pulmonary neoplasms in domestic animals is lacking, and as a consequence, multiplicity of names and synonyms occur in the veterinary literature. some classifications are based on the primary site, whereas others emphasize more the histomorphologic type. the most common types of benign and malignant pulmonary neoplasms in domestic mammals are listed in box - . clinically, the signs of pulmonary neoplasia vary with the degree of invasiveness, the amount of parenchyma involved, and locations of metastases. signs may be vague, such as cough, lethargy, anorexia, weight loss, and perhaps dyspnea. in addition, paraneoplastic syndromes, such as hypercalcemia, endocrinopathies, and pulmonary hypertrophic osteoarthropathy, have been associated with pulmonary neoplasms. primary neoplasms of the lungs. primary neoplasms of the lungs arise from cells normally present in the pulmonary tissue and can be epithelial or mesenchymal, although the latter are rare. any malignant tumor metastatic from another body location (e.g., osteosarcoma in dogs, uterine carcinoma in cows, and malignant melanoma in horses) box - classification of pulmonary neoplasms .e chapter respiratory system, mediastinum, and pleurae abundant cytoplasm containing numerous acidophilic granules, which are positive for pas and for s- protein using immunohistochemistry. although this tumor can cause bronchial obstruction and respiratory signs, in most cases, it is an incidental finding in older horses submitted for postmortem examination. lymphomatoid granulomatosis. lymphomatoid granulomatosis is a rare but interesting pulmonary disease of human beings, dogs, cats, and possibly horses and donkeys characterized by nodules or large solid masses in one or more lung lobes. these frequently metastasize to lymph nodes, kidneys, and liver. microscopically, tumors are formed by large pleomorphic mononuclear (lymphomatoid) cells with a high mitotic rate and frequent formation of binucleated or multinucleated cells. tumor cells have a distinct tendency to grow around blood vessels and invade and destroy the vascular walls. lymphomatoid granulomatosis has some resemblance to lymphoma and is therefore also referred to as angiocentric lymphoma; phenotypic marking confirms that neoplastic cells are a mixed population of plasma cells, b and t lymphocytes, and histiocytes. cerebral and cutaneous forms of lymphomatoid granulomatosis have also reported in human beings, dogs, and cats. secondary neoplasms of the lungs. secondary neoplasms of the lungs are all malignant by definition because they are the result of metastasis to the lungs from malignant neoplasms elsewhere. because the pulmonary capillaries are the first filter met by tumor emboli released into the vena cava or pulmonary arteries, secondary neoplasms in the lung are relatively common in comparison to primary ones. also, secondary tumors can be epithelial or mesenchymal in origin. common metastatic tumors of epithelial origin are mammary, thyroid ( fig. - ) , and uterine carcinomas. tumors of mesenchymal origin are osteosarcoma ( fig. - , a) ; hemangiosarcoma ( fig. - , b) ; malignant melanoma in dogs; lymphoma in cows, pigs, dogs, and cats ( fig. - ) ; and vaccineassociated sarcoma in cats. usually, secondary pulmonary neoplasms are multiple; scattered throughout all pulmonary lobes (hematogenous dissemination); of variable size; and, according to the growth pattern, can be nodular, diffuse, or radiating (e- fig. - ) . the appearance of metastatic neoplasms differs according to the type of neoplasm. for example, dark red cystic nodules containing blood indicate hemangiosarcoma, dark black solid nodules indicate melanoma, and hard solid nodules (white, yellow, or tan color) with bone spicules indicate osteosarcoma. the gross appearances of or in the same animal. death is inevitable after several months of the initial onset of respiratory signs, and a specific humoral immune response to jsrv is undetectable in affected sheep. during the early stages of ovine pulmonary carcinoma, the lungs are enlarged, heavy, and wet and have several firm, gray, variably sized nodules that in some cases can be located in the cranioventral lobes mimicking a bronchopneumonic lesion ( fig. - , a) . in the later stages, the nodules become confluent, and large segments of both lungs are diffusely, but not symmetrically, infiltrated by neoplastic cells. on cross section, edematous fluid and a copious mucoid secretion are present in the trachea and bronchi ( fig. - , b) . microscopically, the nodules consist of cuboidal or columnar epithelial cells lining airways and alveoli and forming papillary or acinar (glandlike) structures (see fig. - , a) . because the cells have been identified ultrastructurally as originating from both type ii alveolar epithelial cells and club (clara) cells, the neoplasm is considered a "bronchioloalveolar" carcinoma. sequelae often include secondary bronchopneumonia, abscesses, and fibrous pleural adhesions. metastases occur to tracheobronchial and mediastinal lymph nodes and, to a lesser extent, to other tissues such as pleura, muscle, liver, and kidneys. neoplastic cells stain strongly positive for jsrv using immunohistochemistry. clinically, ovine pulmonary adenocarcinoma is characterized by a gradual loss of condition, coughing, and respiratory distress, especially after exercise (e.g., herding or "driving"). appetite and temperature are normal, unless there are secondary bacterial infections. an important differentiating feature from maedi (interstitial pneumonia) can be observed if animals with pulmonary adenomatosis are raised by their hind limbs; copious, thin, mucoid fluid, produced by neoplastic cells in the lungs, pours from the nostrils of some animals. carcinoid (neuroendocrine) tumor of the lungs. carcinoid tumor of the lungs is a neoplasm presumably arising from neuroendocrine cells and is sporadically seen in dogs as multiple, large, firm pulmonary masses close to the mainstem bronchi. it has also been reported in the nasal cavity of horses. tumor cells are generally polygonal with finely granular, pale, or slightly eosinophilic cytoplasm. nuclei are small, and mitotic figures are absent or rare. granular cell tumor. granular cell tumor is a rare and locally invasive tumor that has been reported mainly in human beings and older horses. the cell origin of this tumor was thought to be the myoblast, but it is currently presumed to be schwann cells, which are normally present in the bronchovascular bundles of the lung. microscopically, neoplastic cells are large, polyhedron-shaped with metastatic carcinomas are generally similar to the primary neoplasm and sometimes have umbilicated centers. proper diagnoses of pulmonary neoplasms in live animals require history, clinical signs, radiographs, cytologic analysis of bal fluid, and, when necessary, a lung biopsy. identification of a specific lineage of neoplastic cells in biopsy or postmortem specimens is often difficult and requires electron microscopy or immunohistochemical techniques. electron microscopy allows identification of distinctive cellular components such as osmiophilic lamellar phospholipid nephritic bodies in alveolar type ii epithelial cells or melanosomes in melanomas. immunohistochemical staining is also helpful in identifying tumor cells. the thoracic wall, diaphragm, and mediastinum are lined by the parietal pleura, which reflects onto the lungs at the hilum and continues as the visceral pleura, covering the entire surface of the lungs, except at the hilus where the bronchi and blood vessels enter. the space between the parietal and visceral pleura (pleural space) is only minimal and under normal conditions contains only traces of clear fluid, which is a lubricant, and a few exfoliated cells. samples of this fluid are obtained by thoracocentesis, a simple procedure in which a needle is passed into the pleural cavity. volumetric, biochemical, and cytologic changes in this fluid are routinely used in veterinary diagnostics. anomalies congenital defects are rare and generally of little clinical significance. cysts within the mediastinum of dogs and, less often, cats in severe cases, the amount of fluid present in the thoracic cavity can be considerable. for instance, a medium-size dog can have l of fluid, and a cow may accumulate l or more. excessive fluid in the thorax causes compressive atelectasis resulting in respiratory distress (see fig. - ). hydrothorax is most commonly seen in cattle with right-sided heart failure or cor pulmonale (hydrostatic) (e- fig. - ); dogs with congestive heart failure (hydrostatic), chronic hepatic disease (hepatic hydrothorax) ( fig. - ) , or nephrotic syndrome (hypoproteinemia); pigs with mulberry heart disease (increased vascular permeability); and horses with african horse sickness (increased vascular permeability). hemothorax. blood in the thoracic cavity is called hemothorax, but the term has been used for exudate with a sanguineous component. causes include rupture of a major blood vessel as a result of severe thoracic trauma (e.g., hit by car); erosion of a vascular wall by malignant cells or inflammation (e.g., aortitis caused by spirocerca lupi); ruptured aortic aneurysms; clotting defects, including coagulopathies; warfarin toxicity; disseminated intravascular coagulation (consumption coagulopathy); and thrombocytopenia. hemothorax is generally acute and fatal. on gross examination, the thoracic cavity can be filled with blood, and the lungs are partially or completely atelectatic ( fig. - ). chylothorax. the accumulation of chyle (lymph rich in triglycerides) in the thoracic cavity ( fig. - ) is a result of the rupture of major lymph vessels, usually the thoracic duct or the right lymphatic duct. the clinical and pathologic effects of chylothorax are similar to those of the other pleural effusions. causes include thoracic neoplasia (the most common cause in human beings but a distant second to idiopathic cases in dogs), trauma, congenital lymph vessel anomalies, lymphangitis, dirofilariasis, and iatrogenic rupture of the thoracic duct during surgery. the source of the leakage of chyle is rarely found at necropsy. when the leakage of chyle occurs in the abdominal cavity, the condition is referred to as chyloabdomen. cytologic and biochemical examination of fluid collected by thoracocentesis typically reveals large numbers of lymphocytes, lipid droplets, few neutrophils in chronic cases, and high triglyceride content. can be large enough to compromise pulmonary function or mimic neoplasia in thoracic radiographs. these cysts may arise from the thymus (thymic branchial cysts), bronchi (bronchogenic cysts), ectopic thyroid tissue (thyroglossal duct cysts), or from remnants of the branchial pouches, and they are generally lined by epithelium and surrounded by a capsule of stromal tissue. anomalies of the thoracic duct cause some cases of chylothorax. pleural calcification. pleural calcification is commonly found in dogs and less often in cats with chronic uremia. lesions appear as linear white streaks in parietal pleura, mainly over the intercostal muscles of the cranial part of the thoracic cavity. the lesions are not functionally significant but indicate a severe underlying renal problem. vitamin d toxicity (hypervitaminosis d) and ingestion of hypercalcemic substances, such as vitamin d analogs, can also cause calcification of the pleura and other organs. pneumothorax. pneumothorax is the presence of air in the thoracic cavity where there should normally be negative pressure to facilitate inspiration. human beings have a complete and strong mediastinum so that pneumothorax is generally unilateral and thus not a serious problem. in dogs, the barrier varies, but in general it is not complete, so often some communication exists between left and right sides. there are two main forms of pneumothorax. in spontaneous (idiopathic) pneumothorax, air leaking into the pleural cavity from the lungs occurs without any known underlying disease or trauma. in secondary pneumothorax, movement of air into the pleural cavity results from underlying pulmonary or thoracic wall disease. the most common causes of secondary pneumothorax in veterinary medicine are penetrating wounds to the thoracic wall, perforated esophagus, iatrogenic trauma to the thorax and lung during a transthoracic lung biopsy or thoracoscopy, tracheal rupture from improper intubation, and rupture of emphysematous bullae or parasitic pulmonary cysts (paragonimus spp.) that communicate with the thoracic cavity. pneumothorax and pneumomediastinum caused by high air pressure (barotrauma) are also well documented in cats after equipment failure during anesthesia. clinical signs of pneumothorax include respiratory distress, and the lesion is simply a collapsed, atelectatic lung. the air is readily reabsorbed from the cavity if the site of entry is sealed. pleural effusion. pleural effusion is a general term used to describe accumulation of any fluid (transudate, modified transudate, exudate, blood, lymph, or chyle) in the thoracic cavity. cytologic and biochemical evaluations of pleural effusions taken by thoracocentesis are helpful in determining the type of effusion and possible pathogenesis. based on protein concentration and total numbers of nucleated cells, pleural effusions are cytologically divided into transudates, modified transudates, and exudates. hydrothorax. when the fluid is serous, clear, and odorless and fails to coagulate when exposed to air, the condition is referred to as hydrothorax (transudate). causes of hydrothorax are the same as those involved in edema formation in other organs: increased hydrostatic pressure (heart failure), decreased oncotic pressure (hypoproteinemia, as in liver disease), alterations in vascular permeability (inflammation), or obstruction of lymph drainage (neoplasia). in cases in which the leakage is corrected, if the fluid is a transudate, it is rapidly reabsorbed. if the fluid persists, it irritates the pleura and causes mesothelial hyperplasia and fibrosis, which thickens the pleura. from a perforated esophagus. chronic injury typically results in serosal fibrosis and tight adhesions between visceral and parietal pleurae (see fig. - ). when extensive, these adhesions can obliterate the pleural space. pleuritis or pleurisy. inflammation of the visceral or parietal pleurae is called pleuritis, and according to the type of exudate, it can be fibrinous, suppurative, granulomatous, hemorrhagic, or a combination of exudates. acute fibrinous pleuritis can progress with time to pleural fibrosis ( fig. - ). when suppurative pleuritis results in accumulation of purulent exudate in the cavity, the lesion is called pyothorax or thoracic empyema ( fig. - ) . clinically, pleuritis causes considerable pain, and in addition, empyema can result in severe toxemia. pleural fibrous adhesions (between parietal and visceral pleura) and fibrosis are the most common sequelae of chronic pleuritis and can significantly interfere with inflation of the lungs. pleuritis can occur as an extension of pneumonia, particularly in fibrinous bronchopneumonias (pleuropneumonia), or it can occur alone, without pulmonary involvement ( fig. - ). bovine and ovine pneumonic mannheimiosis and porcine and bovine pleuropneumonia are good examples of pleuritis associated with fibrinous bronchopneumonias. polyserositis in pigs and pleural empyema, particularly in cats and horses, are examples of pleural inflammation in pleural tissue is readily susceptible to injury caused by direct implantation of an organism through a penetrating thoracic or diaphragmatic wound; by hematogenous dissemination of infectious organisms in septicemias; or by direct extension from an adjacent inflammatory process, such as in fibrinous bronchopneumonia or in contrast to those with the effusive ("wet") form, in which thoracic involvement is primarily that of a pleural effusion. cytologic evaluation of the effusion typically shows a low to moderate cellularity with degenerated leukocytes, lymphocytes, macrophages, and mesothelial cells, and a pink granular background as a result of the high protein content. pleuritis is also an important problem in horses. nocardia spp. can cause fibrinopurulent pneumonia and pyothorax with characteristic sulfur granules. although mycoplasma felis can be isolated from the respiratory tract of normal horses, it is also isolated from horses with pleuritis and pleural effusion, particularly during the early stages of infection. the portal of entry of this infection is presumably aerogenous, first to the lung and subsequently to the pleura. the pleural surface of the lung is often involved in neoplasms that have metastasized from other organs to the pulmonary parenchyma and ruptured the visceral pleura to seed the pleural cavity. mesothelioma is the only primary neoplasm of the pleura. which involvement of the lungs may not accompany the pleuritis. pleural inflammation is most frequently caused by bacteria, which cause polyserositis reaching the pleura hematogenously. these bacteria include haemophilus parasuis (glasser's disease) (see , streptococcus suis, and some strains of pasteurella multocida in pigs; streptococcus equi ssp. equi and streptococcus equi ssp. zooepidemicus in horses; escherichia coli in calves; and mycoplasma spp. and haemophilus spp. in sheep and goats. contamination of pleural surfaces can be the result of extension of a septic process (e.g., puncture wounds of the thoracic wall and, in cattle, traumatic reticulopericarditis) and ruptured pulmonary abscesses (e.g., trueperella pyogenes). in dogs and cats, bacteria (e.g., nocardia, actinomyces, and bacteroides) can cause pyogranulomatous pleuritis, characterized by accumulation of blood-stained pus ("tomato soup") in the thoracic cavity. this exudate usually contains yellowish flecks called sulfur granules ( fig. - ), although these are less common in nocardial empyema in cats. many species of bacteria, such as escherichia coli, trueperella pyogenes, pasteurella multocida, and fusobacterium necrophorum, can be present in pyothorax of dogs and cats. these bacteria occur alone or in mixed infections. the pathogenesis of pleural empyema in cats is still debatable, but bite wounds or penetration of foreign material (migrating grass awns) are likely. pyogranulomatous pleuritis with empyema occurs occasionally in dogs, presumably associated with inhaled small plant material and penetrating (migrating) grass awns. because of their physical shape (barbed) and assisted by the respiratory movement, aspirated grass awns can penetrate airways, move through the pulmonary parenchyma, and eventually perforate the visceral pleura causing pyogranulomatous pleuritis. cats with the noneffusive ("dry") form of feline infectious peritonitis (fip) frequently have focal pyogranulomatous pleuritis, mesothelioma is a rare neoplasm of the thoracic, pericardial, and peritoneal mesothelium of human beings that is seen most commonly in calves, in which it can be congenital. in human beings, it has long been associated with inhalation of certain types of asbestos fibers (asbestos mining and ship building) alone or with cigarette smoking as a probable cocarcinogen; no convincing association between the incidence of mesothelioma and exposure to asbestos has been made in domestic animals. in animals, there may be pleural effusion with resulting respiratory distress, cough, and weight loss. mesothelioma initially causes a thoracic effusion, but cytologic diagnosis can be difficult because of the morphologic resemblance of malignant and reactive mesothelial cells. during inflammation, mesothelial cells become reactive and not only increase in number but also become pleomorphic and form multinucleated cells that may be cytologically mistaken for those of a carcinoma. grossly, mesothelioma appears as multiple, discrete nodules or arborescent, spreading growths on the pleural surface ( fig. - ) . microscopically, either the mesothelial covering cells or the supporting tissue can be the predominant malignant component, so the neoplasm can microscopically resemble a carcinoma or a sarcoma. figure - nocardiosis. a, chronic pleuritis (nocardia asteroides), pleural cavity, cat. the pleural cavity is covered with abundant red-brown ("tomato soup") exudate" (syringe). once considered to be pathognomonic of nocardia spp. infection, it is no longer regarded as being diagnostic of nocardiosis. the fluid contains abundant protein, erythrocytes, granulomatous inflammatory cells, and sulfur granules. b, chronic pleuritis (nocardia asteroides), visceral pleura, dog. the thickened pleura has a granular pink-gray appearance because of granulomatous inflammation and the proliferation of fibrovascular tissue of the pleura. c, chronic pleuritis (nocardia asteroides), dog. the pleura has been thrown up into villous-like projections composed of abundant fibrovascular tissue and granulomatous inflammation. leakage from the neocapillaries of the fibrovascular tissue is responsible for the hemorrhagic appearance of the pleural exudate. h&e stain. d, chronic pleuritis (nocardia asteroides), thoracic cage, parietal pleura, cat. large pieces of exudate, which contain yellow sulfur granules, are present on the thickened pleura. although considered malignant, mesotheliomas rarely metastasize to distant organs. secondary neoplasms of the pleura. secondary tumors may also spread into the visceral and parietal pleura. thymomas are rare neoplasms that grow in the cranial mediastinum of adult or aged dogs, cats, pigs, cattle, and sheep. thymomas are composed of thymic epithelium and lymphocytes (see chapter ). old age, both in human beings and in animals, is known to be a risk factor for pulmonary infections, but the precise mechanisms involved in this increased susceptibility are still under investigation. some studies have shown that in aged individuals the antibacterial properties provided by surfactant proteins, proinflammatory cytokines, and complement are altered. pulmonary hyperinflation (often referred to as senile emphysema) has been reported as an age-related change in human and canine lungs. other age-related changes described in canine lungs include mineralization of bronchial cartilage, pleural and alveolar fibrosis, and heterotopic bone formation (so-called "pulmonary osteomas"). we thank all pathologists at the atlantic veterinary college, university of prince edward island for providing case material. suggested readings are available at www.expertconsult.com. lung section showing a distended and partially occluded blood vessel (center of figure) containing large granular cells. these large cells are macrophages, and their cytoplasm is filled with myriad merozoites isolation of porcine circoviruslike viruses from pigs with a wasting disease in the usa and europe exercise-induced pulmonary hemorrhage effect of mucociliary transport relies on efficient regulation of ciliary beating epidemiology, diagnosis, and treatment of blastomycosis in dogs and cats canine h n influenza virus infection in dogs and mice failure of respiratory defenses in the pathogenesis of bacterial pneumonia in cattle the respiratory system advances in diagnosis of respiratory diseases of small ruminants canine nasal disease transmission of equine influenza virus to dogs dear jd: bacterial pneumonia in dogs and cats acute respiratory distress syndrome in dogs and cats: a review of clinical findings and pathophysiology inflammatory response to infectious pulmonary injury laryngeal paralysis: a study of cases in a mixed-breed population of horses stem cells of the respiratory tract exudative pleural disease in small animals bovine respiratory disease research pulmonary thromboembolism coccidioidomycosis in dogs and cats: a review cousens c: pathology and pathogenesis of ovine pulmonary adenocarcinoma prognosis factors for survival in cats after removal of a primary lung tumor: cases ( - ) the acute respiratory distress syndrome: from mechanism to translation endogenous lipid pneumonia in cats: cases ( - ) retroviral infections in sheep and goats: small ruminant lentiviruses and host interaction canine and feline nasal neoplasia the acute respiratory distress syndrome equine respiratory medicine and surgery canine pleural and mediastinal effusions: a retrospective study of cases a review of histiocytic diseases of dogs and cats estimation of nasal shedding and seroprevalence of organisms known to be associated with bovine respiratory disease in australian live export cattle polymicrobial respiratory disease in pigs current state of knowledge on porcine circovirus type -associated lesions common and emerging infectious diseases in the animal shelter chronic rhinitis in the cat advances in the understanding of pathogenesis, and diagnosis and therapeutics of feline allergic asthma mannheimia haemolytica and bovine respiratory disease detection of respiratory viruses and bordetella bronchiseptica in dogs with acute respiratory tract infections current perspectives on the diagnosis and epidemiology of mycoplasma hyopneumoniae infection mannheimia haemolytica: bacterialhost interactions in bovine pneumonia acute lung injury review rhodococcus equi: the many facets of a pathogenic actinomycete tumors of the respiratory system key: cord- -cc j j authors: marcus, benjamin a.; achenbach, peter; ziegler, anette-gabriele title: typ- -diabetes: früherkennung und ansätze zur prävention: update date: - - journal: diabetologe doi: . /s - - -x sha: doc_id: cord_uid: cc j j the incidence of type diabetes is increasing, especially in young children. early diagnosis is possible in the asymptomatic stage of islet autoimmunity. screening is offered to high-risk families, but also feasible and useful in the general population, in studies such as fr da(plus) in bavaria (germany). complications at clinical manifestation can be prevented by early diagnosis. participation in experimental interventions to delay stage progression is possible. numerous approaches to secondary prevention are being pursued. treatment with the monoclonal antibody teplizumab successfully delayed progression to clinical diabetes in patients in stage . infants at high risk for developing type diabetes can be identified by genetic screening. primary prevention pursues, among others, the goal of preventing the onset of the autoimmune reaction. the point trial aims to improve immune tolerance to insulin by oral exposure in high-risk children and to delay or prevent the onset of autoimmunity. following up on the focus issue “early detection and preventive treatment of type diabetes” published in this journal in , this article gives an update on selected developments over the past years. große prospektive geburtskohorten haben unser verständnis für die entstehung des typ- -diabetes und den natürlichen verlauf dieser chronischen autoimmunerkrankung entscheidend vorangebracht, und tun dies ist auch weiterhin. wir kennen die genetischen faktoren, die das auftreten der erkrankung begünstigen. wir können einen typ- -diabetes heute durch den nachweis von inselantikörpern diagnostizieren, lange bevor es zu veränderungen des glukosestoffwechsels oder gar zu symptomen kommt. in einer bahnbrechenden präventionsstudie konnten die klinische manifestation bereits um mehrere jahre hinausgezögert und die betazellfunktion stabilisiert werden, was auch die suche nach ei-ner kausalen therapie befruchten könnte. das immer bessere verständnis der komplexen vorgänge, die zur fehlleitung des immunsystems und zur fortschreitenden zerstörung von betazellen führen, eröffnet darüber hinaus verschiedene ansatzpunkte, das entstehen von autoimmunität zu verhindern und den prozess aufzuhalten. im themenheft dieser zeitschrift früherkennung und präventive behandlung des typ- -diabetes -weichenstellungen für die zukunft [ ] vom juni wurden diese bereiche bereits ausführlich dargestellt. in diesem beitrag sollen als update ausgewählte neue ergebnisse und entwicklungen der letzten jahre vorgestellt werden. der nachweis von gegen unterschiedliche betazellantigene gerichteten autoantikörpern im blut ist der etablierte und derzeit wichtigste marker für den autoimmunprozess, der den typ- -diabetes charakterisiert. es kommt zu einem untergang der insulinproduzierenden zellen der bauchspeicheldrüse durch autoreaktive t-zellen mit zunächst langsam, dann kurz vor der manifestation rasch abnehmender insulinproduktion. eine gestörte glukosetoleranz lässt sich erst feststellen, wenn bereits ein großteil der betazellen ihrer funktion nicht mehr nachkommen kann. die wesentlichen autoantikörper sind insulinautoantikörper (iaa), glutamatdekarboxylaseautoantikörper (gada), antikörper ge-gen das insulinomassoziierte antigen (ia- a) und zinktransporter- -autoantikörper (znt a). autoantikörpern markiert das frühstadium des typ- -diabetes der nachweis von oder mehr dieser autoantikörper beim asymptomatischen kind ohne gestörten glukosestoffwechsel ist inzwischen als eines der frühstadien des typ- -diabetes (stadium ) anerkannt. beim stadium liegen zudem mäßig erhöhte nüchternglukosewerte und/oder eine gestörte glukosetoleranz vor. der (neu) manifestierte typ- -diabetes nach gültigen klinischen und laborchemischen kriterien ist das stadium (. tab. ). anhand in der covid- -pandemie leistet die fr da plus -studie einen über die typ- -diabetes-früherkennung hinausgehenden beitrag zur epidemiologischen forschung. mit einem von italienischen wissenschaftlern entwickelten, nichtkommerziellen verfahren werden die kapillarblutproben auch auf igg-antikörper (igg: immunglobulin g) gegen die rezeptorbindungsdomäne des s-proteins von sars-cov- ("severe acute respiratory syndrome coronavirus ") untersucht. der luciferaseimmunopräzipitationstest (lips) funktioniert nach einem ähnlichen prinzip wie der nachweis von iaa [ ] . dabei wird auch auf anonymisierte fr da-proben seit august zurückgegriffen. so kann die immunitätslage von kindern im stark betroffenen bayern vor und im gesamten verlauf der pandemie ermittelt werden [ ] . the incidence of type diabetes is increasing, especially in young children. early diagnosis is possible in the asymptomatic stage of islet autoimmunity. screening is offered to highrisk families, but also feasible and useful in the general population, in studies such as fr da plus in bavaria (germany). complications at clinical manifestation can be prevented by early diagnosis. participation in experimental interventions to delay stage progression is possible. numerous approaches to secondary prevention are being pursued. treatment with the monoclonal antibody teplizumab successfully delayed progression to clinical diabetes in patients in stage . infants at high risk for developing type diabetes can be identified by genetic screening. primary prevention pursues, among others, the goal of preventing the onset of the autoimmune reaction. the point trial aims to improve immune tolerance to insulin by oral exposure in high-risk children and to delay or prevent the onset of autoimmunity. following up on the focus issue "early detection and preventive treatment of type diabetes" published in this journal in , this article gives an update on selected developments over the past years. um zu prüfen, ob teplizumab die klinische manifestation verhindern kann, wurden in einer trialnet-studie angehörige von personen mit typ- -diabetes behandelt, die selbst bereits ein frühstadium mit multiplen inselautoantikörpern und eine dysglykämie oder gestörte glukosetoleranz (stadium ) entwickelt hatten. die ergebnisse dieser untersuchung, die für mehr als die hälfte der behandelten eine verdoppelung der zeit bis zur klinischen erkrankung auf jahre ergaben, wurden im new england journal of medicine (nejm) publiziert [ ] . somit konnte erstmals die manifestation der erkrankung wirksam hinausgezögert werden, was einen durchbruch für die präventive therapie des typ- -diabetes darstellt. in die doppelblinde, randomisierte und plazebokontrollierte phase- -studie wurden verwandte von patienten mit typ- -diabetes, oder mehr autoantikörpern und dysglykämie eingeschlossen, mehrheitlich kinder ab jahren und jugendliche. sie erhielten wochen lang -mal täglich infusionen mit teplizumab oder kochsalz. im followup erfolgten mindestens halbjährlich glukosetoleranztests, die nachbeobachtungszeit betrug im median etwas über jahre, % der probanden konnten über mehr als jahre nachverfolgt werden. bei ( %) der mit teplizumab behandelten teilnehmenden und ( %) von in der plazebogruppe wurde ein klinischer typ- -diabetes diagnostiziert. im ersten jahr nach der behandlung war der effekt besonders ausgeprägt: hier manifestierte sich die erkrankung nur bei ( %) teilneh-mern in der teplizumab-vs. ( %) in der plazebogruppe. jährlich erkrankten , % auf den diesjährigen "scientific sessions" der "american diabetes association" (ada) wurden daten aus dem erweiterten follow-up der studie vorgestellt -und bei der medianen hinauszögerung noch ein weiteres, ., jahr hinzugefügt. zudem war teplizumab in der lage, den c-peptid-abfall nach manifestation nicht nur zu bremsen, sondern signifikant umzukehren. dies könnte bedeuten, dass nicht nur die zerstörung der betazellen gestoppt, sondern auch die insulinproduktion in dysfunktionalen zellen teilweise wiederhergestellt wurde [ ] . auch wenn derzeit noch "nur" davon ausgegangen werden sollte, dass sich für die behandelten patienten der beginn der erkrankung weiter in die zukunft verschiebt und der effekt bei einer dauerhaft therapiebedürftigen erkrankung moderat erscheinen mag, wirkt sich gerade bei kindern jedes gewonnene klinisch gesunde jahr noch weit mehr aus als im erwachsenenalter. für eine individualisierte sekundärprävention interessant wird die tatsache, dass anhand von biomarkern -hla-merkmalen (hla: humanes leukozytenantigen) und dem fehlen von znt a -abgeschätzt werden kann, bei welchen patienten ein ansprechen auf die anti-cd -behandlung bessere erfolgschancen hat [ ] . inzwischen wurden mehr als genloci identifiziert, die in unterschiedlichem ausmaß für das entstehen eines typ- -diabetes prädisponieren. die meisten davon sind mit der immunantwort so-wie der entwicklung und dem erhalt von toleranz gegenüber antigenen assoziiert [ ] . die genetische empfänglichkeitalleinführtaberwahrscheinlichnicht zum entstehen der inselautoimmunität. es wird angenommen, dass umweltfaktoren, die auf eine genetische prädisposition treffen, entscheidend mit zur initiierung des autoimmunprozesses beitragen. dabei liegt es nahe, dass dem frühen säuglingsalter, in dem dieser prozess noch nicht in gang gekommen ist, eine besondere bedeutung zukommt [ ] . nichtpharmakologische interventionen zielen auf die zusammensetzung der ernährung und ggf. gezielte supplementationen ab, auch das darmmikrobiom rückt immer mehr in den fokus [ ] . in der interventionellen präventionsstudie babydiet wurde gezeigt , dass -obwohl die zu frühe einführung von gluten mit der beikost, vor dem . lebensmonat, mit einem höheren risiko einhergeht -eine darüber hinausgehende glutenfreie ernährung das entstehen der inselautoimmunität nicht verhindert. auch durch elimination anderer potenziell antigener proteine durch die verwendung stark hydrolysierter säuglingsmilch konnte in der prospektiven trigr-studie die diabetesinzidenz nicht gesenkt werden [ , ] . es lassen sich weiterhin keine über die allgemeingültigen empfehlungen zu einer gesunden säuglingsernährung hinausgehenden ernährungsmaßnahmen zur senkung des typ- -diabetes-risikos ableiten. die identifikation von neugeborenen und säuglingen mit einem hohen genetischen typ- -diabetes-risiko ist heute einfach und kostengünstig möglich, sodass sie auch in bevölkerungsweiten studien angeboten werden kann. die "global platform for the prevention of autoimmune diabetes" (gppad), ein netzwerk kooperierender wissenschaftler und institutionen [ ] , führt dies seit regional in europäischen ländern durch. in deutschland wird das virusinfekte werden mit dem typ- -diabetes in verbindung gebracht, insbesondere virale atemwegsinfekte in der frühen kindheit und infektionen mit enteroviren und durchfallerregern. hier könnten zukünftig entsprechende impfungen, z. b. gegen coxsackie-viren [ ] , oder antivirale therapien zur senkung des erkrankungsrisikos beitragen [ ] . dervorfast jahreneingeführten rotavirenimpfung wird ein kürzlich festgestellter geringer rückgang der diabetesinzidenz bei vollständig geimpften kindern zugeschrieben. hinweise hierzu lieferten retrospektive auswertungen aus australien und den usa [ , ] , wobei die ergebnisse einer weiteren amerikanischen analyse dies zuletzt wieder in frage stellten [ ] . zumindest lässt sich sicher sagen, dass auch die rotavirenimpfungso wie alle anderen empfohlenen schutzimpfungen -sich keinesfalls negativ auf das typ- -diabetes-risiko auswirkt. die erstmalige, wirksame verzögerung der manifestation mit teplizumab allein ist schon bemerkenswert [ ] , die mögliche reaktivierung zuvor nicht sezernierender betazellen könnte auch die entwicklung kausaler therapien mit vorantreiben. in größeren studien mit mehreren behandlungszyklen und in der weite-ren langfristigen nachbeobachtung der studienpatienten wird das potenzial der substanz weiter untersucht werden. die kombination mit anderen, schon im frühen stadium in klinischen studien eingesetzten, vielversprechenden und verträglichen immunmodulatoren wie z. b. abatacept oder niedrig dosiertem antithymozytenglobulin, die an anderen stellen der pathogenese des typ- -diabetes angreifen [ , ] , sowie mit weiteren aufkommenden betazellregenerativen therapien wird bereits geplant. studien zur früherkennung stoßen bei eltern auf zuspruch, und auch familien, die bisher nicht von typ- -diabetes betroffen waren, sind am angebot primärer und sekundärer präventionsstudien sehr interessiert. die möglichkeit, komplikationen bei der manifestation zu verhindern und erste erfolge in der medikamentösen sekundärprävention werden die schon länger geführte debatte um eine verstetigung und ausweitung von typ- -diabetes-früherkennungsuntersuchungen, z. b. als zusätzliches angebot bei den gesetzlichen vorsorgeuntersuchungen, sicher befruchten [ ] . früherkennung und präventive behandlung des typ- -diabetes typ- -diabetes im asymptomatischen frühstadium classification and diagnosis of diabetes: standards of medical care in diabetes- type diabetes the challenge of modulating β-cell autoimmunity in type diabetes type diabetes trialnet: a multifaceted approach to bringing disease-modifying therapy to clinical use in type diabetes predicting type diabetes using biomarkers birth and coming of age of islet autoantibodies a future for cd antibodies in immunotherapy of type diabetes rationale for enteroviral vaccination and antiviral therapies in human type diabetes association betweenrotavirusvaccinationandtype diabetes in children teplizumab preserves c-peptide in recent-onset type diabetes: two-year results from the randomized, placebo-controlled protégé trial etablierte früherkennungsstudie zu typ- -diabetes testet nun tausende kinder auch auf antikörper gegen sars-cov- an anti-cd antibody, teplizumab, in relatives at risk for type diabetes landmark models to define the age-adjusted risk of developing stage type diabetes across childhood and adolescence prevention strategies for type diabetes: a story of promising efforts and unmet expectations recruiting young pre-symptomatic children for a clinical trial in type diabetes: insights from the fr da insulin intervention study effect of hydrolyzed infant formula vs conventional formula on risk of type diabetes: the trigr randomized clinical trial ketoacidosis at onset of type diabetes in children up to years of age and the changes over a period of years in saxony, eastern-germany: a population based register study immunological biomarkers for the development and progression of type diabetes screening for type diabetes: are we nearly there yet? type diabetes-early life origins and changing epidemiology association of rotavirus vaccination with the incidence of type diabetes in children treatment of type diabetes with teplizumab: clinical and immunological followup after years from diagnosis provention bio's teplizumab continued to significantly delay the onset of insulin-dependenttype diabetes(t d)inpresymptomatic patients lower incidence rate of type diabetes after receipt of the rotavirus vaccine in the united states traveling down the long road to type diabetes mellitus prevention who is enrolling? the path to monitoring in type diabetestrialnet'spathwaytoprevention continuous glucose monitoring predicts progression to diabetes in autoantibody positive children a hexavalent coxsackievirus b vaccine is highly immunogenic and has a strong protective capacity in mice and nonhuman primates delaying diabetes onset the human gut microbiome in early-onset type diabetes from the teddy study früherkennung des typ- -diabetes in der fr da-studie identification of infants with increased type diabetes genetic risk for enrollment into primary prevention trials-gppad- study design and first results why is the presence of autoantibodies against gad associated with a relatively slow progression to clinical diabetes? oral insulin therapy for primary prevention of type diabetes in infants with high genetic risk: the gppad-point (global platform for the prevention of autoimmune diabetes primary oral insulin trial) study protocol yield of a public health screening of children for islet autoantibodies in bavaria key: cord- -x tleomb authors: dodiuk-gad, roni p.; chung, wen-hung; shear, neil h. title: adverse medication reactions date: - - journal: clinical and basic immunodermatology doi: . / - - - - _ sha: doc_id: cord_uid: x tleomb cutaneous adverse drug reactions (adrs) are among the most frequent adverse reactions in patients receiving drug therapy. they have a broad spectrum of clinical manifestations, are caused by various drugs, and result from different pathophysiological mechanisms. hence, their diagnosis and management is challenging. severe cutaneous adrs comprise a group of diseases with major morbidity and mortality, reaching % mortality rate in cases of toxic epidermal necrolysis. this chapter covers the terminology, epidemiology, pathogenesis and classification of cutaneous adr, describes the severe cutaneous adrs and the clinical and laboratory approach to the patient with cutaneous adr and presents the translation of laboratory-based discoveries on the genetic predisposition and pathogenesis of cutaneous adrs to clinical management guidelines. the world health organization defined an adverse drug reaction (adr) in as "a response to a drug that is noxious and unintended and occurs at doses normally used in man" [ ] . edwards and aronson [ ] proposed a different definition in : "an appreciably harmful or unpleasant reaction, resulting from an intervention related to the use of a medicinal product, which predicts hazard from future administration and warrants prevention or specific treatment, or alteration of the dosage regimen, or withdrawal of the product." the terms 'adverse reaction' and 'adverse effect' are interchangeable, except that an adverse reaction is seen from the point of view of the patient and adverse effect is seen from the point of view of the drug. however, both terms must be distinguished from 'adverse event'. an adverse event is an adverse outcome that occurs while a patient is taking a drug, but is not or not necessarily attributable to it [ ] . differentiating between serious adr and severe adr is imperative. serious adr is a legal term applied to any untoward medical occurrence that at any dose results in death, is life-threatening, requires inpatient hospitalization or prolongation of existing hospitalization, results in persistent or significant disability/incapacity, or is a congenital anomaly/birth defect [ ] . conversely, the term 'severe' is a clinical term used to describe the intensity (severity) of a medical event, as in the grading 'mild', 'moderate', and 'severe'; thus, a severe skin reaction need not be serious [ ] . adrs are associated with significant morbidity and mortality and have considerable economic implications. clinical manifestations of an adr are variable and may include cutaneous and or systemic features [ ] . when analyzing the type of adrs most encountered, two major groups emerge; common-mild reactions and raresevere reactions. common-severe reactions are not approved for clinical usage and rare-mild reactions are usually not noticed or reported. cutaneous adrs are among the most frequent adverse reactions in patients receiving drug therapy [ ] . they accounted for % of all reported adrs in a -year retrospective study in taiwan [ ] . the prevalence and incidence of cutaneous adrs vary greatly among different populations [ ] [ ] [ ] [ ] [ ] . in the usa, a -year prospective study found that the prevalence of cutaneous adrs was . % in hospitalized patients [ ] ; and an -year retrospective study found the annual incidence of cutaneous adrs to be . per , persons [ ] . in denmark, in a -year cross-sectional study the prevalence of cutaneous adrs was . % in in-patients and . % in outpatients [ ] . in southern china, in an -year retrospective study, the prevalence of cutaneous adrs was . % in hospitalized patients [ ] . in india, in a -month prospective study, the primary incidence of cutaneous adrs was . per , persons [ ] . the need to survey adrs in clinical practice is universally recognized. various methods may be employed: spontaneous surveillance, prescription-event monitoring (pem), linkage analysis, case-control surveillance and cohort studies [ ] . in , the th world health assembly reaffirmed the need for early detection and rapid dissemination of information on adverse reactions due to medications. this affirmation led to the creation of the world health organization (who) programme for international drug monitoring, under whose auspices systems have been created in member states for the collection and evaluation of individual case safety reports (icsrs) [ ] . in the who set up its international drug monitoring programme in sweden at the uppsala monitoring centre (umc) http://www.who-umc. org. the us food and drug administration (fda) provides several options for reporting adverse events. one such option is medwatch, the fda safety information and adverse event reporting program http://www.fda.gov/safety/ medwatch/default.htm, founded in as a system for both consumers and healthcare professionals to report adverse events. medwatch is intended to detect safety hazard signals for medical products; in the event a signal is detected, the fda can issue medical product safety alerts or order product recalls, withdrawals, or labelling changes to protect the public health [ ] . a number of international research groups are investigating severe cutaneous adrs (scars): the regiscar network, an international registry of scar established in , the japanese research committee, j-scar, the asian scar consisting of japan and taiwan scar groups (j-scar and t-scar) established in , and the southeast asia network, sea-scar, with ten member countries: brunei, cambodia, indonesia, laos, malaysia, myanmar, philippines, thailand, singapore, and vietnam. the international serious adverse event consortium (isaec), a non-profit organization formed in , is a pharmaceutical industry-and fda-led international consortium that focuses on identifying and validating dna variants useful in predicting the risk of rare drug-induced serious adverse events [ ] . mechanisms of adverse drug reactions (adrs) can be classified into immunologic and non-immunologic etiologies. there are two common types of immune-mediated drug reactions: immediate-type hypersensitivity (type i hypersensitivity) and delayed-type hypersensitivity (type iv hypersensitivity). . immediate-type drug hypersensitivity: immediate-type drug hypersensitivity reactions usually occur minutes to hours after drug exposure, with clinical manifestations including pruritus, urticaria, angioedema, and bronchospasm to anaphylaxis. the reaction is mediated mainly by drug-specific ige, the most common causative agents being penicillins, cephalosporins and neuromuscular blocking agents. ige-mediated reactions to drugs are usually thought to be an immune response to a hapten/carrier complex. in the primary drug sensitization, drug-specific ige is formed when plasma cells transformed from activated b cells interact with t cells. in an allergic reaction, drug allergens bind to mast cells with high-affinity fc receptor, to which drug-specific ige is bound, causing mast cells to release mediators, such as histamine, leukotrienes, prostaglandins and cytokines [ ] . . delayed-type drug hypersensitivity: delayed-type drug hypersensitivity reactions usually take several days to weeks following drug exposure, with variable clinical presentations that may include maculopapular eruption (mpe), fixed drug eruption (fde), acute generalized exanthematous pustulosis (agep), stevens-johnson syndrome (sjs), toxic epidermal necrolysis (ten) and drug reaction with eosinophilia and systemic symptoms (dress). t cell receptor (tcr), cd + and cd + t cells are involved in different delayed-type drug hypersensitivity reactions [ ] . drugs are low molecular weight and usually considered not able to bind to tcrs to activate adaptive immunity. in the case of drug allergy, drug interactions with tcrs may involve a drug-peptide complex presented by human leucocyte antigen (hla) molecules of antigen-presenting cells (apcs). this process is known as the hapten concept; an example is β-lactams that covalently bind to lysine residues [ ] . drugs can also interact directly with tcrs without binding to the peptide/hla of the apc in what is known as the p-i concept (pharmacological interaction of drugs with immune receptors) [ ] . for example, carbamazepine is not able to bind covalently to peptides or proteins, but can associate with low affinity to tcrs and provoke t cell activation [ ] . the immunohistologic characteristics of delayed type drug hypersensitivity are summarized in table . . the skin of mpe is infiltrated by numerous mononuclear cells (cd and cd t cells, monocyte/macrophages) and some eosinophils. typically, interface dermatitis is seen with a predominance of cd + t cells. these cells are located mainly in the perivascular dermis, and both cd + and cd + t cells are located at the dermoepidermal junction [ ] . skin manifestations of dress may vary from mpe-like to exfoliative dermatitis and are characterized by a heavy infiltration of cd + and cd + t cells, monocyte/macro-phages and eosinophils [ ] . mpe and dress share many pathological features, but dress exhibits more severe dyskeratosis (keratinocyte death in epidermis) and a greater extent of systemic involvement and eosinophilia [ ] . immunohistology of skin lesions in agep reveals intraepidermal pustules with infiltration of neutrophils surrounded by il- producing t cells [ ] . despite very diverse clinical presentations, constant features of delayed-type drug hypersensitivity are the presence of high numbers of drug-specific cd + cytotoxic t cells and low numbers of innate nk lymphocytes [ , , ] . cd + t cells of cutaneous adrs have classic cytotoxic functions: lysis of autologous lymphocytes or keratinocytes in an mhc class i-restricted and drug-dependent manner [ ] . cytotoxic immune cells in sjs/ten drug-induced sjs and ten are severe cutaneous adrs in which cytotoxic t lymphocytes (ctls) and natural killer (nk) cells are activated, and subsequently carry out the cellular immune reactions directed at keratinocytes in a major histocompatibility class (mhc) i-restricted manner. upon activation of these immunocytes, various cytotoxic signals, including granulysin, perforin/granzyme b, fas/fas ligand, and cytokines/chemokines, are relayed to the skin lesions to mediate the disseminated keratinocyte death [ ] [ ] [ ] . it is noteworthy that the number of granulysin-positive cells in fixed drug eruptions was found to be similar to that observed in sjs/ten [ ] . non-immune-mediated hypersensitivity is commonly referred to as pseudoallergic reactions because they do not involve a specific immune mechanism -neither ige-mediated (type i) nor delayed (type iv) hypersensitivity. clinical manifestations, which range from milder erythematous to urticarial reactions to severe lethal anaphylaxis, may be indistinguishable from immune system-mediated hypersensitivity reactions. common non-immune-mediated hypersensitivity can be caused by contrast media, vancomycin, non-steroidal anti-inflammatory drugs (nsaids), opiates, plasma expanders, and drugs used in general anesthesia [ ] . nsaids-induced pseudoallergic reactions have been attributed to cyclooxygenase- inhibition and overproduction of leukotrienes, and may require higher drug doses than are needed for true ige-mediated reactions [ ] . mast cell drug-specific t cells mediate skin inflammation in variable clinical presentations of delayed-type drug hypersensitivity through the release and induction of different cytokines and chemokines (table . ) [ ] . the heterogeneous cytokines include th cytokines (interferon-γ) and th cytokines (il- , il- ) [ ] . increased expression of il- , which is a key cytokine for activation of eosinophils, is commonly seen in delayed-type drug hypersensitivity [ ] . the activation of eosinophils can be further enhanced by the chemokines eotaxin and rantes [ ] . thymus and activation-regulated chemokine (tarc/ccl ) has been reported to be a dress specific cytokine [ ] . in addition to th and th cytokines, a recent study demonstrated the involvement of il- aproducing th in dress and sjs/ten [ ] . elevated expression of the neutrophil-attracting il- has been known to be the key cytokine involved in agep. there are several cytokines involved in sjs/ ten. numerous studies have shown tumor necrosis factor alpha (tnf-α) strongly expressed in sjs/ten lesions and correlated with disease severity [ , , ] . tnf-α is a potent cytokine that induces cell apoptosis, cell activation, differentiation, and inflammatory processes [ , ] . interferon gamma (ifn-γ) is a common cytokine involved in delayed-type drug hypersensitivity, including sjs/ ten. ifn-γ was intensely expressed in the superficial dermis and epidermis of sjs/ten lesions [ , ] . ifn-γ is also known to promote antigen presentation and thus stimulate the cell-mediated immunity by upregulation of mhc molecules [ ] [ ] [ ] . in addition to tnf-α and ifn-γ, several cytokines and chemokine receptors that are responsible for trafficking, proliferation, and activation of t-cells and other immune cells have been found elevated in the skin lesions, blister fluids, blister cells, pbmcs, or plasma of sjs/ten patients . these cytokines/chemokines include il- , il- , il- , il- , il- , il- , il- , il- , ccr , cxcr , cxcr , and ccr [ , , , [ ] [ ] [ ] . the central hypothesis proposed to explain the severe mucocutaneous lesions of sjs/ten is the cd + cytotoxic t cell and natural killer (nk) cell-mediated cytotoxic immune reactions. three major cytotoxic signals from cytotoxic cells are reported to be involved in the extensive skin necrosis of sjs/ten, including the fas-fasl interaction, perforin/granzyme b, and granulysin, which can induce keratinocyte apoptosis [ , , ] . granulysin is not only a cytotoxic protein; it is also a chemoattractant and proinflammatory activator that can promote monocyte expression of ccl [ ] , and is capable of promoting antigen-presenting (dendritic) cells and leukocyte recruitment, and activating specific immune responses, such as il- b,il- , il- , tnf-a [ ] . reports on the familial occurrence of severe drug hypersensitivity and cases occurring in identical twins suggest genetic links [ ] [ ] [ ] [ ] . the hla genes show strong association with drug hypersensitivity. examples of strong associations of hla alleles with specific drug-induced hypersensitivity reactions include abacavir, nevirapine, carbamazepine, and allopurinol (table . ). the view that hla alleles are the main genetic determinants of sjs/ten was first proposed by roujeau et al. [ ] , who reported the weak associations of hla-a , b , and mpe maculopapular drug eruption, dress drug reaction with eosinophilia and systemic symptoms, sjs/ten stevens-johnson syndrome/toxic epidermal necrolysis dr in sulfonamide-related ten, and hla-a , b in oxicam-related ten in europeans [ ] . following the immunological hypothesis, the most striking evidence of genetic susceptibility to sjs/ten was provided by the findings that hla-b* : is strongly associated with carbamazepine-induced sjs/ten [ ] , hla-b* : with allopurinol-induced sjs/ten or dress [ ] , and hla-b* with abacavir hypersensitivity [ ] . the hla association to specific drug-induced hypersensitivity can be ethnic and phenotype-specific. the strength of hla associations with specific drug-induced hypersensitivity in different populations has been found related to the prevalence of the susceptibility allele in the ethnic population. the association of hla-b* : with carbamazepineinduced sjs/ten was replicated in other asian countries, including thailand, hong kong, malaysia, china, vietnam, cambodia, reunion, philippines and indian ethnicities, which carry high hla-b* : allele frequency, but not in europeans, which carry low hla-b* : allele frequency (< %) [ ] . in contrast, the strong association of hla-b* : with allopurinol-induced sjs/ten is more universal, being found in han chinese in china, thai populations, korean, japanese, and european populations; hla-b* : is the allele common to all these populations [ ] . the phenotype-specific characteristics are exemplified by carbamazepine hypersensitivity. while hla-b* : is strongly associated with carbamazepine-induced sjs/ten, it is not associated with carbamazepine-induced dress; in an international study, hla-a* : was strongly associated with carbamazepine-induced dress, but not with carbamazepineinduced sjs/ten [ ] . phenytoin -an aromatic antiepileptic drug structurally related to carbamazepine -also frequently causes sjs/ten and dress [ , ] . hla-b* : has been associated with phenytoin-related sjs/ten in asians, although the association is much weaker than that found for carbamazepine-related sjs/ten [ ] . a recent genome-wide association study by chung wh et al. turned up cytochrome (cyp) cvariants, including cyp c * , that showed a strong association with phenytoin-related scar. the significant association between cyp c * and phenytoin-related severe cutaneous ards was replicated in different asian populations [ ] . similar to delayed-type drug hypersensitivity, genetic predisposing factors have been reported in immediate-type drug hypersensitivity. β-lactam allergy was reported associated with gene variants of il , il , and il ra [ ] [ ] [ ] [ ] . several genetic predisposing factors, including gene polymorphisms in cysteinyl leukotriene receptor type (cysltr ) and leukotriene c synthase (ltc s) [ ] and high-affinity ige receptor (fcepsilonr ) [ ] , were associated with aspirin. cutaneous adrs may be classified in terms of their presumed mechanism, severity of the reaction, histological findings, and cutaneous morphological manifestations. the modern pharmacological classification of adrs differentiates two basic types of reactions; type a, predictable reactions, and type b, unpredictable or idiosyncratic reactions. type a reactions ('augmented') are dose-dependent, common and predictable based on the pharmacology of the drug; about % of all adrs are type a. type b reactions ('bizarre') do not occur at any dose in most patients, but may be dose dependent in susceptible individuals. they are uncommon, affecting a small number of patients based on an individual predisposition that depends on both genetic and environmental factors [ , ] . the pathogenesis of type a reaction was described in the sixteenth century by paracelsus, the swiss german renaissance physician who founded the discipline of toxicology: "all things are poison, and nothing is without poison; only the dose permits something not to be poisonous" [ ] . the pathogenesis of type b reaction was designated in the first century bc didactic poem, de rerum natura (on the nature of things), by the roman poet and philosopher lucretius: "one man's meat is another man's poison" [ ] . type b reactions can be categorized into different subtypes according to gell and coombs' classification system [ ] . the effector phase of the allergic reaction is classified into four types: type i mediated by drug-specific ige antibodies, types ii and iii mediated by drug specific igg or igm or iga antibodies, and type iv induced by drug-specifc t lymphocytes [ ] . this classification system may be helpful in daily clinical practice as a guide to diagnostic and therapeutic decisions. in addition to the basic classification of type a and b reactions, further types of reactions were subsequently added; type c-dose and time-related, 'chronic'; type dtime-related. 'delayed'; type e-withdrawal effects, 'end of use'; and type f-unexpected failure of therapy, 'failure' [ ] . the diagnosis of a cutaneous adr must be followed by differentiation between a simple reaction involving only the skin and a complex reaction that includes systemic involvement of organs in addition to the skin [ ] . systemic involvement should be explored even in a mild cutaneous eruption due to a drug since the severity of skin manifestation does not necessarily mirror the severity of the systemic involvement. systemic involvement is evaluated by assessing the patient's symptoms, including fever, facial edema, malaise, chills, dyspnea, cough, palpitations, nausea, vomiting, diarrhea, sore throat and arthralgia. further investigation is based on the patient's symptoms. basic laboratory screen, conducted in cases of suspected systemic involvement, includes a full blood count, liver and renal function tests, and urine analysis [ ] . skin biopsy is an invaluable diagnostic modality in the assessment of drug eruptions. histologically, drug eruptions can elicit a variety of inflammatory disease patterns in the skin and panniculus, and overlapping reaction patterns. ackerman et al.'s basic patterns of inflammatory skin diseases [ ] (table . ) are a helpful guide. the most common pattern of drug eruptions is the perivascular type, while psoriasiform and granulomatous patterns are rarely reported [ ] . drug eruptions may also mimic specific skin diseases such as lupus, lichen planus or lymphoma [ ] . a single drug may cause a wide range of reaction patterns and no reaction pattern is specific for a particular drug [ ] . while the histological changes are not distinctive in many cases of drug eruption, a few important histopathological clues may aid in the diagnosis: ( ) overlapping histological patterns in one specimen (e.g., lichenoid and spongiotic). ( ) presence of eosinophils (although not mandatory); although eosinophils are an important tell-tale sign of a drug-induced reaction, they may also be conspicuous in skin rashes devoid of a drug association and sparse or absent in some drug exanthems. ( ) apoptotic keratinocytes. ( ) mismatch between clinical and histomorphological features [ , , ] . in a study assessing the histological pattern of cases of diagnosed drug eruption during a -year period in one institution [ ] , the majority of the cases ( %) were morbilliform-type rashes. the most common histological pattern was superficial perivascular and interstitial with interface changes. eosinophils were present in only % of cases, and approximately half ( %) of the cases exhibited epidermal-dermal interface changes [ ] . in view of the large diversity of cutaneous drug reactions, it is helpful to approach them as clinicopathologic entities and to base the diagnosis on a combination of clinical, histo- logical and disease course data [ ] . heightened awareness of the possible mimicry of other skin diseases and of the suspicious histopathological clues pointing to drug etiology are key elements to the appropriate histological diagnosis of drug reactions in the skin [ , , ] . a widely accepted approach to diagnosing the type of drug eruption is a simplified method based on the morphology of the primary lesions. the four main categories are maculopapular, urticarial, pustular and blistering [ ] . the diagnosis of the drug eruption can be challenging since the same cutaneous morphology can be manifested in a simple reaction involving only the skin and in a complex reaction including systemic involvement in addition to the skin. therefore, there are two major steps in diagnosing drug eruptions: determine the morphology and assess systemic involvement [ ] . terminology the term 'maculopapular' is descriptive. morbilliform means measles-like, the rash of measles consisting of macules and papules that tends to confluence. the etymon of 'exanthema' is the greek 'exanthema', which means 'a breaking out'. thus exanthema merely means 'rash', and 'exanthematous rash' literally means 'rash-like rash'. therefore, the terminology is redundant [ ] . polymorphous pink-to-red macules and or papules usually in a symmetric distribution that may coalesce to form plaques ( fig. . ) [ ] . the eruption begins on the trunk and upper extremities and progressively becomes confluent. in addition, purpuric lesions may appear on the ankles and feet [ ] . the drug eruption can also manifest in a scarlatiniform pattern of pinpoint-sized pink-red papules coalescing and giving the skin the texture of sandpaper [ ] . frequency the most common drug-induced eruptions, occurring in - % of first-time users of most drugs [ ] . lag period - days [ ] . symptoms pruritus and low-grade fever are common [ ] . the eruption usually begins on the trunk and becomes generalized. palms and soles are often involved; mucous membranes are usually spared [ ] . histology nonspecific changes consisting of mostly superficial but also deep perivascular and interstitial infiltrate of lymphocytes. eosinophils and epidermal-dermal interface changes appear in approximately half the cases [ ] . differential diagnosis viral exanthems, scarlet fever, toxic shock syndrome, acute graft versus host disease (gvhd), kawasaki disease, juvenile idiopathic arthritis [ ] . treatment identifying and discontinuing the causative drug are the most important steps in management. symptomatic treatment with antipruritic agents and potent topical glucocorticoids may be helpful [ ] . a decision can be made to continue the drug and offer symptomatic treatment if the drug is of paramount importance, but the risk: benefit ratio of this option has to be carefully weighed, and the evolution of the eruption must be meticulously monitored [ ] . prognosis the eruption often fades within - days of discontinuation of the offending drug and scaling and desquamation may follow. re-challenge may lead to reappearance of the reaction within a few days [ ] . offending drugs the most common classes of drugs implicated are penicillins, sulfonamides, cephalosporins, and antiepileptics [ ] . terminology the term 'urticaria', first introduced by william cullen in the eighteenth century, is derived from urtica urens (common european stinging nettle). one of the earliest descriptions of urticaria comes from china, and is more than erythematous macules and papules coalescent into illdefined plaques on the trunk -maculopapular morphology of cutaneous adr , years old. in the huangdi neijing, written around bc, urticaria is referred to as feng yin zheng ('wind type concealed rash'). in ancient latin medical literature, urticaria was called 'uredo' (urere means 'to burn'), and in the old persian medical texts, 'essera' (meaning 'elevation') [ ] . skin signs urticaria is induced by superficial dermal swelling due to plasma leakage and vasodilation triggered by activation of mast cells. the skin manifestations of this process include erythematous and edematous papules and plaques (wheals) of various sizes that may coalesce to form large plaques [ ] . wheals may be characterized by pink or pale center and assume a figurate or polycyclic configuration. linear lesions can be seen with dermatographism [ , ] . frequency drug-induced urticarial eruptions are the second most common type of cutaneous drug eruption and account for approximately % of all cutaneous drug eruptions [ ] . lag period urticaria occurs within minutes to days of drug administration [ ] . symptoms a major clinical feature is pruritus, the lack of which should put the diagnosis in doubt. the lesions can also be painful if they occur on the soles, over joints, or in areas where the skin is tightly adhered to subcutaneous tissue [ ] . a single lesion lasts less than h and upon resolution leaves normal skin. however, new lesions may continue to arise for various periods of time. acute urticaria is defined when a bout of hives lasts less than weeks; when it lasts longer, it is defined as chronic urticaria [ ] . urticaria may be associated with angioedema [ ] . angioedema is defined as a deep, dermal, subcutaneous and/or mucous swelling that may involve the intestinal lining and the upper respiratory tract. symptoms include slight heat, burning, pain and sensation of pressure or tightness. however, pruritus is minimal or absent. swelling of gastrointestinal tract mucosa can induce abdominal pain, vomiting and diarrhea. edema of the respiratory tract may induce various symptoms including life-threatening asphyxia. drug-induced angioedema is associated with urticaria in approximately % of cases. some drugs may induce angioedema without urticaria [ ] . common sites of involvement lesions of urticaria can appear anywhere on the skin, including the palms, soles and scalp, but not on mucosal surfaces [ ] . angioedema most commonly occurs in the head, neck and hands, but can occur anywhere and frequently involves mucosal tissue. swelling may be more prominent in areas of looser skin, such as the scrotum, labia, lips, and eyelids [ ] . histology urticarial drug reactions are characterised by dermal edema and a superficial and deep perivascular and interstitial dermatitis. the mixed inflammatory infiltrate comprises lymphocytes, histiocytes, mast cells, eosinophils and neutrophils. the presence of neutrophils and deep vascular plexus involvement may be a clue to the drug-induced nature of the urticaria [ ] . the wheals with central red halo of urticaria may resemble the target lesions of erythema multiforme. four clinical signs of urticaria can help distinguish it from erythema multiforme: ( ) the central zone consists of normal skin, whereas in erythema multiforme, skin is dusty, bullous or crusted. ( ) each lesion is transient, lasting less than h, whereas erythema multiforme lesions are 'fixed' for a few days. ( ) new lesions appear daily and in erythema multiforme all lesions appear within the first h. ( ) there may be associated swelling of face, hands and feet and in erythema multiforme there is no edema [ ] . differential diagnosis of urticaria includes also bullous pemphigoid, urticarial vasculitis and serum sickness-like reaction (sslr). drug-induced urticaria needs to be differentiated from cases of urticaria induced by other etiologies, such as food, environmental allergens, insects, systemic illness, physical stimuli, genetic and idiopathic [ ] . urticaria and angioedema are the most common symptoms of anaphylaxis ( % of cases), and are one of the clinical criteria of the national institute of allergy and infectious disease (niaid) and the food allergy and anaphylaxis network (faan) for the diagnosis of anaphylaxis [ ] . therefore, all cases of sudden acute urticaria and angioedema should be evaluated for indications of the anaphylactic type of reaction: presence of respiratory compromise, decreased blood pressure, and end-organ dysfunction (collapse, syncope, incontinence) [ ] . treatment the most important step in the management of drug induced urticaria with or without angioedema is withdrawal of the causative agent. in most cases of acute urticaria, when the trigger is removed the rash quickly resolves. h -receptor blockers are the mainstay of treatment for patients with only cutaneous symptoms. systemic glucocorticoids are indicated in all cases with upper airway edema and should be considered in cases with extensive cutaneous involvement. epinephrine is reserved for angioedema with upper airway involvement [ ] . the presence or absence of any airway involvement should be specifically investigated. prognosis both urticaria and angioedema fade without visible sequelae. following resolution, there should be no residual pigmentary changes unless excoriated [ ] . offending drugs many drugs can induce acute urticaria, and do so by both immunologic and non-immunolgic mechanisms. the major drugs responsible for immunologically based urticaria are antibiotics, especially penicillins and cephalosporins [ ] . the major drugs triggering mast cell release (non-immunolgic mechanisms) are aspirin, nonsteroidal anti-inflammatory drugs (nsaids), opioids and radiocontrast media [ ] . viral infections or connective tissue diseases may induce or augment urticarial drug reactions [ ] . the national institute of allergy and infectious diseases (niaid) and the food allergy and anaphylaxis network (faan) defined anaphylaxis as a systemic reaction resulting from the sudden release of multiple mediators from mast cells and basophils, often life threatening, and usually unexpected. the world allergy organization (wao) has divided anaphylaxis into immunologic (further divided into immunoglobulin e [ige]-mediated and non-ige-mediated), non-immunologic, and idiopathic causes. drugs are the second most common cause of anaphylaxis after food, which constitutes % of triggers [ ] . common medications associated with anaphylaxis include penicillins, nsaids, and biologic response modifiers [ ] . the niaid/ faan definition of anaphylaxis has been translated into clinical diagnostic criteria that include an acute onset of illness (minutes to hours) and involvement of the dermatologic, respiratory, cardiovascular, or gastrointestinal systems [ ] . epinephrine is the only first-line treatment for anaphylaxis and is the sole effective treatment for an acute reaction. delays in administration have been associated with fatalities. supportive treatment with oxygen, fluids and additional drugs are also necessary according to the cardiopulmonary resuscitation (cpr) anaphylaxis algorithm [ ] . • serum sickness-like reaction (sslr) -see severe cutaneous adverse drug reactions. terminology the term pustule originates in classical latin in which pustule means a blister [ ] . skin signs pustular drug eruptions are characterized by monomorphic eruption consisting of erythematous papules (mostly follicular) and pustules at the same location lacking comedones. acneiform drug eruptions (acne medicamentosa) the term acneiform is applied to eruptions that resemble acne vulgaris. frequency varies, depending on the drug. the highest incidence involves epidermal growth factor receptor inhibitors (egfris), affecting - % of patients [ ] . lag period the eruption begins after a variable delay; corticosteroids may induce an acneiform eruption from shortly after their introduction ( - weeks) to several months [ ] . acneiform eruptions induced by egfris usually appear after - weeks of treatment but can also occur after only a few days [ ] . symptoms pruritus, tenderness and pain may occur. in cases of chemotherapy-related side effects, their appearance and severity are part of the criteria used for the classification of the adr [ ] . common sites of involvement lesions may be located in and beyond the seborrheic areas, such as the arms, trunk, lower back and genitalia [ ] . histology drug-induced acneiform eruptions show histopathologic features similar to acne vulgaris. early lesions most commonly have a corneocytic plug within a widened infundibulum, accompanied by infundibular spongiosis, perifollicular edema, with sparse perivascular and periinfundibular infiltrates of neutrophils and lymphocytes. larger older lesions show similar findings but the infiltrate is denser, with more neutrophils around the involved follicles, and infundibular rupture [ , ] . in a review of the histological findings of acneiform eruptions induced by egfris [ ] , all ten cases showed a superficial, predominantly neutrophilic suppurative folliculitis with ectatic infundibula and a rupture of the epithelial lining. differential diagnosis the main differential diagnosis is acne. the following clinical characteristics of acneiform drug eruptions may aid in differentiating between the two entities: ( ) clinical presentation: monomorphic pattern, lack of comedones and cysts and localization on areas beyond the seborrheic area. ( ) patient characteristics: age of onset before or after the teens, and absence of past history of acne. ( ) resistance to conventional acne therapy. ( ) time relationship: onset after recent drug introduction, improvement after drug withdrawal, and recurrence after drug reintroduction [ ] . the differential diagnosis also includes folliculitis, rosacea, perioral dermatitis, demodicosis, acne cosmetic, acne mechanica, chloracne, acne necrotica and acneiform presentation of cutaneous lymphomas [ ] . treatment the main treatment is withdrawal of the offending drug and the application of topical treatments as needed (benzoyl peroxide topical antibiotics and topical retinoids) [ ] . the management of acneiform eruptions associated with chemotherapy differs from all other types of acneiform drug euptions, as acneiform eruption is an expected outcome and discontinuation of the medication is not an option in a patient who is responding to therapy [ , , , ] . in fact, continuation of egfri therapy in these patients may be especially favourable in view of studies that have shown an increased survival with increasing severity of rash [ ] . the cutaneous reaction serves as an important clinical tool for determining tumor response and survival [ ] . the national cancer institute developed a scale for defining the degree of rash and laid down management guidelines for each stage [ ] . other management protocols were suggested by bachet et al. [ ] , who recommended that unless contraindicated, a tetracycline should be routinely prescribed for the prevention of acneiform eruption in patients treated with an egfri for more than weeks. chiang et al. [ ] reported successful treatment with isotretinoin for high grade and refractory cases. prognosis in most patients with acneiform drug eruption, the rash resolves upon discontinuation of the offending drug and the use of topical treatment. in egfri-induced acneiform eruption, prophylactic administration of a tetracycline was associated with significantly lower incidence of grade - folliculitis and improved quality of life of patients [ ] . few cases of drug-induced eosinophilic pustular folliculitis have been reported [ , [ ] [ ] [ ] [ ] . drugs reported include chemotherapy (cyclophosphamide, methotrexate, and -fluorouracil) [ ] , minocycline [ ] , carbamazepine [ ] , and allopurinol with timedium bromide [ ] . clinical presentation includes pruritic follicular papules and pustules on the face, scalp, trunk and arms [ ] . histological findings include spongiosis of the follicular epithelium, and an intraand perifollicular lymphohistiocytic infiltrate with numerous eosinophils that form microabscesses within the follicular epithelium [ ] . topical steroids are the first line of treatment [ ] . acute generalized exanthematous pustulosis (agep) -see severe cutaneous adverse drug reactions. terminology the term pseudoporphyria was coined in by korting to describe patients with chronic renal failure and a bullous disease resembling porphyria cutanea tarda (pct) [ ] . frequency the incidence of pseudoporphyria is unknown. however, in a -month prospective study, % ( / ) of children taking naproxen for juvenile idiopathic arthritis developed pseudoporphyria [ ] . lag period the skin lesions appear following drug intake combined with exposure to light. various time durations were reported, weeks to months [ ] [ ] [ ] . the clinical features of pseudoporphyria may be identical to those of pct; both exhibit vesicles, bullae, milia, and scarring on sun-exposed skin. in contrast to pct, however, hypertrichosis, hyperpigmentation, sclerodermoid changes, and dystrophic calcification are rarely reported in pseudoporphyria [ ] . often, fragility and bruising may be the only clinical signs [ ] . in children, facial scarring resembling erythropoietic protoporphyria (epp) may be found [ ] . symptoms skin fragility and photosensitivity [ ] . the lesions appear on sunexposed skin, particularly the hands and feet, but also on the face and extensor surfaces of legs [ ] . histology the histological features are identical to those seen in pct. the blisters are subepidermal and the floor of the blister is typically lined by well-preserved dermal papillae (festooning). there is usually no significant inflammatory component although a light perivascular lymphocytic infiltrate may occasionally be seen in the superficial dermis. thickening of the superficial vessels (highlighted by a pas stain) and dermal sclerosis with elastosis may be apparent. in both pseudoporphyria and pct, direct immunofluorescence reveals granular deposits of igg and c at the basement membrane zone and in the perivascular region [ ] . differential diagnosis while pseudoporphyria and pct share clinical and histologic features, they can be differentiated by several features. most important, by definition, biochemical porphyrin abnormalities are absent in pseudoporphyria. epidemiologically, pseudoporphyria affects mainly women while there is a male predilection in pct. clinically, hypertrichosis, hyperpigmentation, sclerodermoid changes, and dystrophic calcification are frequently evident in pct and conspicuously absent in pseudoporphyria [ ] . the differential diagnosis also includes other types of cutaneous porphyria that manifest with blistering, epidermolysis bullosa acquisita, polymorphous light eruption, and other photosensitive dermatosis [ ] . treatment treatment entails discontinuation of suspected agents and sun protection, especially against uva wavelengths, for several months following withdrawal of the drug [ ] . prognosis blisters may continue to appear for weeksmonths after discontinuation of the offending drug [ ] . offending drugs the most common group of drugs causing pseudoporphyria are nsaids [ ] . other groups are antibiotics, diuretics and retinoids. additional culprits are hemodialysis, renal failure, tanning beds and excessive sun exposure [ ] . terminology fixed drug eruption (fde) was first reported by boums in [ ] , and the term was coined by brocq in [ ] . frequency the incidence is not known, but is suspected to vary greatly by geographic region [ ] . lag period after initial use of the offending agent, a variable refractory period of weeks, months or years may pass before the lesions first appear on the skin of a sensitized individual [ ] . repeated exposure to the agent typically results in acute lesions within min to h. a refractory phase may occur following an acute flare in which exposure to the offending drug will not exacerbate the lesion for weeks to months [ ] . in its classical form, fde typically presents round or oval, sharply demarcated, red to livid, slightly elevated plaques ranging from several millimeters to over cm in diameter. vesicles or even blisters can develop [ ] . usually only a single lesion appears. sometimes, multiple lesions are present and even lead to generalized fde characterized by multiple, sharply defined, deep red macules distributed bilaterally and often symmetrically. generalized bullous fde is characterized by flaccid blisters arising on these macules. mucosal lesions are usually bullous and may appear with or without involvement of other areas of the skin [ ] . symptoms patients often complain of burning and itching in the lesions. general symptoms such as fever, nausea, dysuria, abdominal cramps and diarrhea are rare [ ] . pruritus and burning may be the only manifestations of reactivation in a postinflammatory hyperpigmentation lesion [ ] . common sites of involvement the eruption can occur anywhere on the body, but the lips, palms, soles, genitalia (especially male genitalia), groin and occasionally oral mucosa are favored sites [ ] . the diagnostic hallmark of fde is the reappearance of the lesions precisely over the previously affected sites. studies investigating the predilection areas indicate that some specific kind of drugs cause fde predominantly at specific sites: examples are tetracycline and location on the male genital area, and naproxen and fde on the lips [ ] . in rare cases, fde manifests in old trauma sites such as bcg vaccination, burn scar, venipuncture site or insect bite. with each recurrence, additional sites may be affected. the presence of numerous lesions is referred to as generalized fde [ ] . histology histologically, the acute phase is characterized by marked basal cell hydropic degeneration, with lymphocyte tagging along the dermoepidermal junction and individual keratinocyte necrosis. marked pigmentary incontinence is typical, and may be the sole histological finding in late lesions [ ] . differential diagnosis skin lesions can imitate various dermatoses, including lichen planus, erythema multiforme, erythema annulare centrifugum, and pityriasis rosea. in generalized fde, residual pigmentation in healed lesions may be reminiscent of erythema dyschromicum perstans. involvement of oral and genital mucosa raises the possibility of herpes simplex, pemphigus vulgaris, aphthous stomatitis, behçet syndrome, and erosive lichen planus [ ] . generalized bullous fde may resemble sjs/ten. the following typical clinical features of generalized bullous fde may aid in differentiating between conditions: ( ) blistering usually affects only a small percentage of body surface area, and between the large blisters there are sizable areas of intact skin. ( ) erosive mucosal involvement is rare, and when it does occur is rather mild. ( ) patients usually do not feel sick or have fever, and generally are in much better overall health than those with sjs/ten. ( ) most patients report a history of a similar, often local reaction [ ] . treatment for mild lesions, topical corticosteroids usually suffice. in severe involvement, especially generalized bullous fde, systemic corticosteroids may be indicated. strict avoidance of the causative drug and cross-reacting substances is essential for prophylaxis. successful desensitization was reported [ ] . prognosis the prognosis of localized fde is good and the lesions fade within a few days to leave a post-inflammatory brown pigmentation [ ] . generalized bullous fde does not have this benign nature and the mortality rate was % in a recent case control study of patients [ ] . offending drugs the most common groups of drugs implicated are antibiotics, analgesics, antiphlogistics and hypnotics [ ] . there is usually only one causative drug (monosensitivity), but sometimes several drugs can induce fde in the same patient (multisensitivity). it has also been claimed that recurrences of fde can be induced in nonspecific fashion by mast cell degranulators such as food, acetylsalicylic acid, bacterial toxins, or physical stimuli [ ] . • drug-induced/triggered autoimmune blistering dermatosis (pemphigus, bullous pemphigoid (bp)) and linear iga bullous dermatosis (labd) [ ] . cases of autoimmune blistering dermatosis resulting from exposure to drugs present clinical, histologic and immunopathologic features identical or very similar to those seen in idiopathic disease, but are induced by systemic ingestion or local use of certain drugs. there appear to be two main types: drug-induced autoimmune blistering dermatosis proper, the acute and self-limiting type with rapid resolution after withdrawal of the offending agent; and drug-triggered autoimmune blistering dermatosis in which the role played by the drug is only secondary to hereditary and immunologic factors. the drug stimulates a predisposition (hidden susceptibility) to develop the disease and is considered the chronic type in which the disease persists despite withdrawal of the offending agent [ , ] . frequency unknown. [ , , ] . of note, drug-induced labd patients tend to be older than idopathic type patients [ , ] . the polymorphic nature of the eruption may mimic other bullous diseases and or drug-induced bullous diseases such as sjs, ten, and fde [ ] . treatment treatment consists of discontinuing the offending agent, and, depending on the severity of the disease, systemic immunosuppressive treatment [ ] . prognosis drug-induced autoimmune blistering dermatosis remits after the offending drug is withdrawn, while drugtriggered autoimmune blistering dermatosis may persist despite withdrawal of the offending agent and chronic immunosuppressive treatment may be required [ , ] . two major groups of chemical structures were found in the drugs or their metabolites implicated in pemphigus: sulfhydryl radical drugs (thiol drugs or sh drugs) such as penicillamine, and phenol drugs such as aspirin [ , , ] . bp many drugs were reported [ , , ] , the most frequent being nsaids, cardiovascular agents and penicillin-derived antibiotics [ ] . in addition, external use of skin and mucous membrane preparations has been documented to provoke cases of either bp or cicatricial pemphigoid [ ] . labd of the various drugs reported, vancomycin is the most common [ , , ] . reactions. the incidence of dress remains to be determined because of variable presentations and lack of universally accepted diagnostic criteria [ ] . the estimated risk at first or second prescription of an aromatic antiepileptic drug was - . in , [ ] . a slight female predominance was found in the regiscar study (male/female . ) [ ] . the drugs most commonly inducing dress are anti-convulsants (mainly aromatic anti-convulsants such as carbamazepine), allopurinol, sulfonamides (the anti-infective sulfamethaxazole-trimethoprim, and the anti-inflammatory sulfasalazine), and antibiotics (such as vancomycin and minocycline) [ ] . numerous other drugs have been reported [ , , ] . the role of human herpesvirus (hhv) reactivation in the development of this adverse drug reaction is well recognized, especially hhv- [ ] . hhv- reactivation is among the diagnostic criteria of the japanese consensus group for dress/drug-induced hypersensitivity syndrome [ ] . the reactivation of other herpesviruses, including hhv- , cytomegalovirus (cmv), epstein-barr virus (ebv), and human herpes simplex virus was also reported [ ] . dress is considered to result from complex interactions between genetic predisposition, exposure to drug and viral reactivation [ ] . delayed onset of - weeks after drug administration followed by a stepwise development of manifestations. rechallenge can result in a reaction within hours to days [ ] . the lag period differs between drugs; carbamazepine tended to show a longer latency (median days) than allopurinol (median days) in the regiscar study [ ] . dress has multi-organ involvement with cutaneous, mucosal, hematological and solid organ manifestations. the cutaneous involvement in dress is typically extensive and symptomatic (pruritus, burning and pain) [ , ] . various dermatological features were reported. walsh et al. [ ] proposed a classification system based on four distinct patterns: ( ) urticated papular exanthema, the most common, ( ) morbilliform erythema, ( ) exfoliative erythroderma, and ( ) erythema multiforme-like (em-like), which was prognostic of more severe hepatic involvement. the extent of skin involvement varies between studies: it exceeded % of the body surface area in most of the patients ( %) according to the regiscar study [ ] ; head and neck edema observed in most patients [ , ] ; and pustules reported in various studies, predominantly in a facial distribution of the edema [ , ] . [ ] . most frequent were oral lesions including lips, oral cavity and throat [ ] . the manifestations of oral lesions in dress include cheilitis, erosions and dysphagia that may appear before skin lesions, and oropharaynx is considered the first site of herpesvirus reactivation in dress [ ] . involvement of eyes and genitalia were also reported in the regiscar study [ ] . multi-organ involvement is common in dress and may include a wide variety of systems. highgrade fever ( - °c) is a typical early manifestation that may last for several weeks; it often precedes the cutaneous eruption by several days [ ] . lymphadenopathy is common and has two distinct types: a benign pattern of lymphoid hyperplasia and a pseudolymphoma pattern [ ] . hematologic abnormalities are frequent and diverse, the most common being marked leukocytosis, eosinophilia and atypical lymphocytes [ ] . however, neutrophilia, monocytosis, thrombocytopenia, anemia, pancytopenia and hemophagocytic syndrome were also reported [ , , , ] . hypereosinophilia and activated neutrophils, if persistent, can contribute to organ damage [ ] . the liver is the most frequently affected visceral organ in dress; hepatitis with isolated elevation of liver enzymes is common and usually anicteric and without cholangitis. however, severe acute hepatitis with liver failure may result and is the primary cause of mortality in dress [ ] . renal involvement is common [ ] . involvement of the following organs was also reported: lungs, muscle, heart, pancreas, colon, thyroid, joints, parotid gland and brain [ ] . the type of organs involved was found to be related to the eliciting drug [ ] . the most common pathological changes found in a study of patients with dress were basket-weave hyperkeratosis ( %), dyskeratosis ( %), lymphocytic exocytosis ( %), spongiosis ( %), papillary edema ( %), perivascular lymphocytic infiltration ( %), eosinophilic infiltration ( %), and interface vacuolization in the dermoepidermal junction ( %) [ ] . the presence of severe dyskeratosis was correlated with a greater extent of systemic involvement [ ] . in a different study assessing the histological findings of cases with dress [ ] , the predominant pathological pattern was spongiotic dermatitis with superficial lymphocytic infiltrate ( %); necrotic keratinocytes were noted in % of cases, and were associated with a worse hepatic involvement [ ] . the diverse presentations in dress have hampered efforts to define diagnostic criteria. three diagnostic criteria have been proposed: bacquet et al. [ ] , the japanese study group of severe cutaneous adverse reactions to drugs (j-scar) [ ] , and the regiscar network [ ] . the first step in the management is immediate withdrawal of the culprit drug. the treatment is tailored according to the severity and extent of systemic involvement, and the diagnosis of viral reactivation of herpesviruses (mostly hhv- ) [ , , ] . management protocol for dress based on the consensus of experts was designed by the french society of dermatology [ ] , and includes four visceral involvement severity categories and corresponding treatment: counselling both the patient and his family members about drug avoidance is necessary. first-degree relatives have a higher risk of developing the same drug reactions [ ] . increased knowledge of hla susceptibility genes enables screening patients with dress for several high risk drugs [ , ] . symptoms are usually present for several weeks even after discontinuation of the offending agent and appropriate treatment [ ] . late complications include the appearance of autoimmune diseases such as lupus erythematosus and autoimmune thyroiditis, with laboratory evidence of autoantibodies [ ] . systemic corticosteroids were found beneficial in the prevention of autoimmune disease. however, this effect needs to be counterbalanced against the higher risk of viral reactivation and infection. [ ] . in a -year follow-up study of affected patients with dress in taiwan, the overall cumulative incidence of long-term sequelae was . %; four developed autoimmune diseases (graves disease, type diabetes mellitus and autoimmune hemolytic anemia); and the other two developed renal failure and required lifelong hemodialysis. the author concluded that the sequelae of dress can be divided into two major types that appear in different age groups: young patients tend to develop autoimmune diseases; elderly patients are more vulnerable to end-organ failure [ ] . mortality in dress has been estimated at %, with most patients dying from liver failure [ ] . pancytopenia, leukocytosis, tachycardia, tachypnea, coagulopathy, gastrointestinal bleeding and systemic inflammatory response syndrome were associated with a poor outcome in dress patients [ , ] . the incidence of sslr is unknown. epidemiology studies in children suggest that the overall frequency induced by cefaclor is . - . % per course of the drug [ ] . most reactions were reported in children under years old, mainly during the second and third courses of therapy [ ] . cefaclor is the most common cause of sslr in children, inducing . % of cases [ ] . other drugs implicated include other cephalosporins, [ ] penicillins, [ ] minocycline, [ ] insulin, [ ] and infliximab [ ] . usually - days (range - days) [ , ] . skin the skin is the most frequent finding in sslr, including erythema that progresses to urticarial lesions (pruritic and migratory), urticarial wheals with dusty to purple centers ('purple urticaria') that morphologically resemble erythema multiforme (em) [ ] and other cutaneous manifestations including morbilliform or scarlatiniform eruptions [ ] . mucous membranes are not involved [ ] . systemic involvement joint involvement may be prominent, presenting with edema, decreased range of motion, warmth, pain, and difficulty walking. polyarticular involvement is often observed, with involvement mainly of the wrists, ankles, hips and knees [ ] . some authors suggested that joint involvement may be related in part to increased fluid in the skin around affected joints due to urticarial eruption rather than arthritis [ ] . fever, malaise, myalgia and lymphadenopathy were also reported. neurologic involvement, gastrointestinal symptoms and renal complications were rarely documented [ ] . notable laboratory abnormalities include elevated erythrocyte sedimentation rate (esr), c-reactive protein (crp) and leukocytosis [ , ] . the histological findings of sslr appear to be in the spectrum of urticaria with no vasculitis [ ] . histology can be helpful in differentiating sslr from acute hemorrhagic edema of infancy, which is characterized by vasculitis [ ] . there are no diagnostic criteria. the diagnosis is based on clinical findings [ ] . withdrawal of the offending agent and symptomatic treatment with oral antihistamines and topical corticosteroids are usually sufficient. a short course of oral corticosteroids may be required in patients with severe symptoms [ ] . the disease course is benign and resolves in a few days. however, a few cases lasting several weeks have been described [ ] . no long-term morbidity has been reported [ ] . the estimated incidence of agep is - cases per million per year [ ] . female predominance was reported in several studies [ ] [ ] [ ] . the majority of cases appear to be related to drugs (> %), mainly antibacterials [ ] . in a large multinational casecontrol study (the euroscar study), the following agents were highly suspected drugs for agep: prestinomycin, ampicillin/amoxicillin, quinolones, (hydroxy)chloroquine, anti-infective sulfonamides, terbinafine and diltiazem [ ] . latent periods fall into two categories, according to the offending drug: median duration of day, associated with antibiotics (including sulphonamides), and median duration of days for all other associated drugs [ ] . longer periods of months were reported in a few agep cases with an underlying malignancy [ ] . agep is a severe acute pustular cutaneous reaction characterized by a rapid clinical course [ ] . skin the typical morphology of agep is an acute edematous erythema with burning and or itching sensation, followed by dozens to hundreds of small (pinhead sized) non-follicular sterile pustules with a predilection for the big folds, or with widespread distribution (fig. . ) . sometimes confluence of pustules may mimic a positive nikolsky's sign [ , ] . additional cutaneous manifestations include marked edema of the face, purpura, blisters and target-like lesions [ , , ] , all of which overlap with manifestation of agep and ten [ , ] , and acute localized exanthematous pustulosis (alep) [ , ] . mild, nonerosive mucous membrane involvement of one location (mostly oral) occurs in about % of cases [ ] . systemic involvement fever (above °c) and leukocytosis with neutrophilia are almost always apparent. lymphadenopathy, myalgia, headache, mild eosinophilia, elevated crp, slight reduction of creatinine clearance, and mild elevation of aminotransferases were also reported [ , ] . a -year retrospective review of patients with agep [ ] turned up patients ( %) with at least one systemic involvement in the acute phase, with abnormal hepatic function test, with renal insufficiency, two with acute respiratory distress and one patient with agranulocytosis. mean peripheral neutrophil counts and mean c-reactive protein levels were elevated significantly in patients with systemic involvement [ ] . biopsy specimen should be obtained from an early pustular lesion [ ] . a histopathological study of agep cases [ ] found the following histopathological features: ( ) all cases demonstrated pustules (sub/intracorneal and or intraepidermal). the agep validation score developed by the euro-scar study group is a standardized scoring system made up of data related to clinical features (morphology and clinical course) and histopathology. based on this score, agep cases can be categorized as no agep, possible agep, probable agep, and definite agep [ ] . treatment consists of discontinuation of the causative drug and supportive treatment. although, specific treatment is generally unnecessary, topical and systemic steroids were reported [ , ] . the treatment of overlapping agep and ten cases is not yet established [ ] , although successful treatment with infliximab was documented [ ] . after elimination of the causative drug, pustules usually spontaneously disappear in a few days with desquamation, and the reaction fully resolves within days [ ] . the overall prognosis is good, although high fever or superinfection of skin lesions can sometimes lead to life-threatening situations in patients of old age or poor general condition [ ] . the mortality rate is about % [ ] . the annual incidence of sjs and ten is . - and . - . per million individuals, respectively [ , ] . the annual incidence of sjs and/or ten in hiv patients is estimated at - per individuals, approximately -fold higher than that of the general population [ ] . the incidence of sjs/ten increases with age; children less than years of age account for only % of the samples in most studies [ ] . women are two times more likely to be affected by sjs/ten than men in the adult population, while the male to female ratio is about equal in children [ ] . drug exposure is the most common cause of sjs/ten [ ] , with more than drugs identified [ ] . the groups of medications associated with high risk of inducing sjs/ fig. . multiple, pin-head sized, non-follicular pustules on erythematous skin on the trunk in a patient with agep ten vary according to the population. in the general population in europe, high risk drugs for sjs/ten include allopurinol, carbamazepine, cotrimoxazole and other anti-infective sulfonamides, lamotrigine, nevirapine, oxicam-nsaids, phenytoin, phenobarbital and sulfasalazine [ ] . in the pediatric population in europe, they include anti-infective sulfonamides, phenobarbital, carbamazepine and lamotrogone [ ] . in africa, they include antibacterial sulfonamides, nevirapine, tuberculosis drugs, nsaids, antiepileptics, aminopenicillin, analgesics and allopurinol [ ] . non-medication triggers, implicated mainly in sjs, include infections, contrast media and vaccinations [ ] [ ] [ ] . alden is an algorithm for the assessment of drug causality in sjs/ten developed by the regiscar study group and consists of parameters according to which the drug causality is classified as very unlikely, unlikely, possible, probable and very probable [ ] . usually - days. the median latency was longer (above weeks) for drugs with no associated risk [ ] . sjs and ten represent different degrees of a severe, acute and life-threatening mucocutaneous reaction. we will refer to this disease spectrum as a single entity, namely sjs/ ten. the classification of sjs/ten, defined by bastuji-garin et al. [ ] , is based on the extent of epidermal detachment and the findings of characteristic skin lesions (table . ). it should be emphasized that only necrotic skin, which is already detached (e.g., blisters, erosions), or detach-able skin (positive nikolsky sign whereby slight rubbing of the skin results in exfoliation of the outermost layer) should be included in the evaluation of the extent of epidermal detachment [ ] . the characteristic skin morphology of sjs/ten consists of 'flat, atypical target lesions' and 'spots/macules', which are defined as follows. flat, atypical target lesions are round lesions, with only two zones and/or a poorly defined border, nonpalpable with the exception of potential central blister. 'spots/macules' are nonpalpable, erythematous or purpuric macules with irregular shape and size, often confluent [ ] . epidermal necrosis, the hallmark process of sjs/ten, induces flaccid blisters with positive asboe-hansen sign (lateral extension of bullae with pressure), erosions, positive nikolsky sign, and in severe cases extensive skin sloughing [ ] . at least % of epidermal detachment is required for the diagnosis of sjs/ten [ ] . in rare instances, extensive epidermal necrosis occurs with only widespread erythema and no evidence of 'flat, atypical target lesions' or 'spots/macules'; these cases were classified as 'ten without spots' (table . ). a characteristic sign of sjs/ten is severe pain and tenderness of the skin [ ] . mucosal involvement is evident in most of the cases with erythema, erosions and ulceration, due to necrosis of the epithelial lining [ ] . sjs/ten involve more than mucosal sites in - % of cases [ ] . most common sites are oral (fig. . ) , ocular and genital mucous membranes, although any mucous membrane may be involved, such as respiratory, gastrointestinal and urethral [ ] . fuchs syndrome is a unique type of sjs that involves the mucosa without skin lesions and was reported to be associated with mycoplasma pneumoniae, mostly in children and adolescents [ ] . table . classification of erythema multiforme major (emm), stevens-johnson syndrome (sjs) and toxic epidermal necrolysis (ten) according to bastuji-garin et al. [ ] emm sjs a sjs-ten overlap ten ten with spots ten without spots/ten with widespread erythema systemic involvement systemic findings in sjs/ten include: ( ) flu-like symptoms (malaise, fever, anorexia) that are usually the initial signs of the disease in the prodromal phase prior to the cutaneous involvement. ( ) epidermal barrier breakdown-related symptoms including hypothermia, dehydration and sepsis. ( ) organ involvement induced by necrosis of epithelial lining, including respiratory distress syndrome, colitis, hepatitis and nephritis [ ] . characteristic histologic features include extensive keratinocyte destruction via apoptosis with separation of the epidermis from the dermis at the dermoepidermal junction. a paucicellular, dermal mononuclear infiltrate has been commonly described. lymphocytes cross the dermoepidermal junction with moderate infiltration of the epidermis. em and sjs often demonstrate less keratinocyte destruction on a background of extensive dermal mononuclear inflammation [ ] . in a retrospective analysis of the clinical records and histologic material of patients with ten, the histologic spectrum ranged from sparse to extensive dermal mononuclear inflammation, the extent of which predicts clinical outcome approximately as well as scorten. increased inflammation correlated with a worse prognosis; a mean cell count of dermal mononuclear > cells per high-power field predicted a worse prognosis ( %) vs % mortality in those with < cells in patients with % or more total body surface area sloughing [ ] . however, in a retrospective study analyzing clinical records and skin biopsy of patients with sjs, sjs/ten overlap and ten, dermal infiltrate severity was not associated with day- scorten or hospital death, but full-thickness epidermal necrosis was associated with mortality [ ] . diagnostic criteria based on integration of the major clinical characteristics of skin and mucous membrane findings, pathology assessment, lag period and systemic signs remain to be defined. the management of sjs/ten consists of a multidisciplinary approach that includes the following important aspects: . identification and withdrawal of the culprit drug: documenting the medication history during the previous months and withdrawal of all suspected and unessential medications [ ] . . transfer of the patient to intensive care, burn unit or other specialty unit: supportive care including thermoregulation, fluid replacement, nutritional support, monitoring for infection, sedation and pain management, and psychological support [ ] . . assessment of skin, mucous membranes and systemic involvement and the scorten score: type of lesions in the skin, extent of epidermal detachment, and mucous membranes and systemic involvement. all patients should be evaluated by an ophthalmologist promptly following the diagnosis and at regular follow-up intervals to minimize potential long-term ocular sequelae [ ] . possible acute manifestations include the eyelids, conjunctiva and cornea, and result in the classification of ocular involvement as mild, moderate or severe [ ] . bringing other specialists in on the patient's care is decided in accordance with the relevant findings. the scorten system, a severity-of-illness score for toxic epidermal necrolysis, developed to stratify severity of illness and predict mortality in patients with ten, includes seven independent risk factors: age, malignancy, tachycardia, initial body surface area of epidermal detachment, serum urea, serum glucose, and bicarbonate [ ] . . skin treatment: there are no clinical guidelines for the skin care of patients with sjs/ten. debridement of the necrotic epidermis was recommended in past publications [ , ] . recent publications advise avoiding debridement, which may cause hypertrophic scars, and recommend considering the detached epidermis as a natural biological dressing that favors reepithelialization [ , , ] . various topical treatments reported include bioactive skin substitutes, semi-synthetic and synthetic dressings, and topical antimicrobials [ , ] . a recent report on the management of sjs/ten in an experienced french referral center described the following treatment; wound care once a day with minimal manipulation to prevent skin detachment, including a bath containing a solution of chlorhexidine / (morphine is given prior to the bath and/or equimolar mix of oxygen and nitrogen monoxide during the bath); if bathing is not possible, the chlorhexidine solution is sprayed - times daily on the skin, blister fluid is aspirated while maintaining the blister roof, vaseline is systemically applied over all detached skin areas, topical sulfa-containing medications are avoided, and hydrocellular or absorbent nonadhesive dressings are applied at least once daily to cover pressure points [ ] . . mucous membranes treatment: specialized care is essential to prevent lifelong complications [ ] . although there is no standardized care for ocular management, the following supportive local treatment is advised: tear replacement solutions, removal of pseudomembranes, lysis of symblepharon, debridement of loosened epithelium, topical antibiotics to prevent secondary infection, topical corticosteroid to prevent scar formation, and cycloplegic drops to relieve pain, photophobia and ciliary spasm [ ] . amniotic membrane transplantation was found effective in the acute and chronic stages of sjs/ten [ , ] . a 'triple-ten' protocol for severe ocular cases was recently reported [ ] , comprised of the following: ( ) subconjunctival triamcinolone (kenalog mg) administered into each of the fornices to curb the local inflammatory response without compromising systemic immunity. ( ) placement of amniotic membrane tissue mounted on a polycarbonate skirt (prokera) over the corneal and limbal regions to facilitate reepithelialization of the ocular surface. ( ) insertion of a steeply curved acrylic scleral shell spacer (technovent, sc ) to vault the lids away from the globe and provide a barrier to symblephara formation. this treatment offers an effective therapeutic option, without the need for microsurgical equipment, microscope, or sutures in the critical care setting. oral-the mouth should be rinsed several times a day with an antiseptic or anifungal solution and the lips lubricated with an ointment such as dexpanthenol [ ] . genital-wet dressings or sitz baths and lubrication with emollient are recommended to avoid adhesions and strictures of genital erosions in females [ , ] . a specialist is required in case of involvement of other mucous membranes: respiratory, gastrointestinal and/or urethral. . systemic immunomodulatory treatment: the optimal therapeutic regimen has yet to be established, but according to recent publications, the following conclusions can be drawn: the use of ivig does not yield survival benefits in sjs/ten [ ] ; cyclosporine decreased the death rate and the progression of detachment (dosage of mg/kg/ day for days) [ ] ; systemic corticosteroids were associated with clinical benefit according to the euroscarstudy [ ] and were reported to be the most common treatment for sjs/ten in a recent survey of drug hypersensitivity experts from countries [ ] . one of the suggested protocols is iv dexamethasone . mg/kg pulse therapy (given for - min) for consecutive days [ ] . treatment with anti-tnf biologic treatment was reported to be beneficial [ ] [ ] [ ] . a prospective, randomized, open-label trial currently underway in taiwan [ ] comparing etanercept versus systemic corticosteroids in patients with sjs/ten, reported that the average duration to reach maximal skin detachment and complete skin healing was shorter in the etanercept group. in vitro investigations demonstrated that etanercept, steroids or thalidomide significantly decreased granulysin expression of blister cells. etanercept did not, however, increase the cytotoxic effect to keratinocytes found with thalidomide [ ] . . causality assessment and communication with the patient and his/her family, health-care providers and regulatory agencies: recent discoveries of specific hlas that predict genetic susceptibility to sjs/ten offer a simple, fast, safe and reliable method for establishing clear causality between a drug and a disease [ ] . the hlas are specific to a drug and an ethnic background [ ] . since these tests are available only for certain drugs and a negative test does not exclude the drug as the offending agent, additional clinical and laboratory methods are available for assessing causality. the mortality rates of sjs/ten are variable. that of ten may approach % [ ] , and that of children with sjs/ten is approximately - . % [ ] . in a large-scale, populationbased, -year follow-up study of sjs/ten patients, the -week in-hospital mortality rate was %, and the death rate from weeks to year was % [ ] . the mortality rate at year in this study was % for sjs, % for sjs and ten overlap, and % for ten. several factors were found to affect mortality: age, severity of reaction, recent malignancy, preexisting severe kidney or liver disorder, and recent infection. the last two factors were recognized for the first time in this study as being independent risk factors for death. all other factors are part of the scorten [ ] . the severity of the reaction was a major risk factor for death in the first few weeks, and severe co-morbidities and older age had major impact on mortality after weeks [ ] . early and late physical complications are common among patients who survive sjs/ten [ ] , with some % experiencing long-term sequelae [ ] . complications may affect multiple organ systems including skin, nails, hair, oral and genital mucosal sufaces, eyes, kidneys, gastrointestinal tract, and respiratory system [ ] . ocular complications, which can lead to blindness, are the major long-term morbidity [ ] . a few studies have dealt with the quality of life of patients surviving sjs/ten [ ] [ ] [ ] , which was found to be lower in every domain from before hospitalization to follow-up and a low rate of return to previous employment was documented [ ] . patients reported concerns about social interactions, fear of taking medications, and fear of contracting an illness necessitating medication [ ] . insufficient information and support for patients surviving sjs/ ten was also documented [ ] [ ] [ ] . unfortunately, because of the rarity of sjs/ten, most physicians are not aware of the long-term complications of the diseases [ ] . there are several methods to approach a patient with a cutaneous adr. the following is the authors' protocol: clinical assessment of drug-induced skin injury: ds by dr. shear a cutaneous eruption in a patient taking a medication should immediately raise the suspicion of a cutaneous adr. the physician must then determine whether the patient's clinical symptoms are signs of a cutaneous adr or of another skin disease not related to a drug. the diagnosis of a cutaneous adr is based on three key clinical elements (fig. . ) : ( ) appearancethe morphology of the cutaneous eruption according to four main categories of the primary lesion: maculopapular, urticarial, bullous and pustular (see section "morphological classification of cutaneous adrs"). ( ) systemicextra-cutaneous signs (fever, dyspnea, lymphadenopathy, etc.) that distinguish between a simple reaction involving only the skin and a complex reaction that includes systemic involvement in addition to the skin (see section "severity of cutaneous adrs: skin only (simple) versus skin and systemic involvement (complex)") and ( ) histologyhistopathology and direct immunofluorescence studies of skin biopsies to confirm the clinical impression and to distinguish between a drug-induced eruption and other skin diseases (see section "histological classification of cutaneous adrs"). establishing a differential diagnosis that takes into account all possible diagnoses is essential. ranking the approximate likelihood of each condition is encouraged. all medications, regardless of route of administration, must be considered, especially new drugs taken in the weeks prior to the skin reaction. drugs taken intermittently, such as vitamins, sedatives, pain relievers, laxatives and natural products, must also be considered. assessment of the lag period -the time between initiation of the drug and onset of the cutaneous reaction -is crucial in view of the different lag times for different cutaneous drug reactions. a recommended method for drug exposure analysis is to chart a timeline in order to visualize the chronology and facilitate comprehension of the event. the timeline includes the relevant information (starting day, dosage, and discontinuing day) for each drug and the signs and symptoms throughout the period in question [ ] . the most important challenge in assessing drug-induced skin injury is establishing whether there is a causal relationship between the suspected drug and the untoward clinical event. the following methods are helpful: ( ) patient history: the patient should be questioned about previous cutaneous reactions to drugs, and whether rechallenge with the drug improved the eruption [ ] . these data should also be part of the above timeline. ( ) good communication strategies will aid in the interactions with the patient and family following a cutaneous adr and decrease the likelihood of lawsuits, especially in cases of severe reactions such as sjs/ten. physicians are advised to follow these steps: ( ) express empathy and say "sorry" according to the "apology laws" in an honest and respectful fashion and in a way that protects the physician from having an apology used against him in case of legal action. http:// www.sorryworks.net/. ( ) provide disclosure in a "disclosure meeting" planned according to the acronym cones: context -arrange the setting for a quiet, uninterrupted meeting and decide on the participants; opening shot -the first sentence in the meeting explains the aim of the conversation; narrative -lay out the facts; it is advised to avoid using the words "error" and "mistake" since the adr is a result of multiple factors, particularly when the facts are not completely known; emotions -provide an empathic environment; summary ( ) provide the patient with clear information on his cutaneous adr, the name of the offending drug, potential cross-reacting drugs, and drugs which can be safely taken as an alternative to the offending drug. in addition, advise the patient to wear a medic-alert bracelet. ( ) family counselling is part of the management plan since the predisposition to some cutaneous adrs may be genetic [ ] . information on the adverse event must be provided to the family physician and entered in the patient's records. report the cutaneous adr to the manufacturer and regulatory agencies [ ] . the strong associations found between hla alleles and specific drug-induced hypersensitivity reactions have fostered pharmacogenetic testing to prevent the development of lifethreatening drug-induced hypersensitivity reactions, such as sjs/ten and dress. the usefulness of such testing is dependent on a number of factors, including the incidence and severity of the adverse event, the sensitivity and specificity of the predictive markers, and the availability of equally effective, alternative medications for individuals who test positive. although the incidence of sjs/ten is relatively low, it is life-threatening and many patients who survive have longterm sequelae, such as ocular complications. hla-b* is a useful and strong predictive marker with high sensitivity and specificity for carbamazepine-induced sjs/ten in asian populations. this genetic association is strong enough that it prompted the usfda and many countries to relabel the genetic information for carbamazepine, and to recommend screening for hla-b* before prescribing the drug for subjects of asian descent. the hla-b* test for carbamazepine-induced sjs/ten has very high sensitivity (near %) and specificity ( %). with the . % prevalence rate of carbamazepine-induced sjs/ten among chinese, the hla-b* test has a . % positive predictive value and % negative predictive value for detecting [ ] . in view of the serious consequences of sjs/ten and the availability of alternative drugs, withholding carbamazepine from screened patients who test positive for hla-b* and switching to alternative antiepileptic drugs is reasonable and feasible in the high risk populations, including chinese and south-east asians. abacavir is used in the treatment of hiv infection, and has been associated with drug hypersensitivity syndrome in % of patients [ ] . hla-b* is a strong and useful predictive marker with high sensitivity and specificity for abacavir hypersensitivity in caucasians, prompting the usfda and many other countries to recommend screening for it before prescribing the drug. the hla-b* test for immunologically-mediated abacavir hypersensitivity has very high sensitivity ( %) and specificity ( . %) as well as positive predictive value ( %) and negative predictive value ( %) [ ] . hla-b* is a potentially useful predictive marker for allopurinol-induced sjs/ten or dress, with % positive predictive value and almost % negative predictive value for detecting allopurinol-induced sjs/ten or dress in chinese (table . ). this association was significant in caucasian and other asian populations as well. the recent american college of rheumatology guidelines for the management of gout recommend hla-b* screening for populations with high frequency of the allele [ ] . other recently discovered hla alleles related to drug hypersensitivity of potential usefullness in clinic practice are hla-b* for dapsone hypersensitivity [ ] , hla-a* for carbamazepine-related dress [ ] , and cyp c * for phenytoin hypersensitivity [ ] . the lymphocyte transformation test (ltt) is a widely used in vitro assay for the diagnosis and identification of offending drugs with t cell-mediated drug hypersensitivity [ ] . ltt is based on the activation and proliferation of t cells from pbmc obtained from drug-sensitized patients after stimulation, and incubation with the culprit drug in vitro [ ] . following in vitro stimulation by specific drugs, drug-specific t cells are activated and release several cytokines that promote proliferation of t cells. this in vitro proliferation of specific drug-activated t cells can be detected by the incorporation of h-thymidine during dna synthesis after days of culture. the results of ltt are expressed as the stimulation index (si): the relationship between the h-thymidine uptake in cells (counts per minute (c.p.m.)) with and without the drug antigen [ ] . the general sensitivity of the ltt is - %, varying with different drugs and different phenotypes of delayed-type hypersensitivity reactions; thus, a negative result does not exclude the possibility of drug hypersensitivity. extensive studies on ltt for beta-lactam drugs report even higher sensitivity [ ] [ ] [ ] [ ] [ ] . the specificity of the ltt is - % in different studies [ ] [ ] [ ] ] . ltt for the diagnosis of drug hypersensitivity has limitations. because it is measured by radioisotopes, the sensitivity can be very low and negative results are commonly observed for specific drugs (e.g., allopurinol, lamotrigine) and specific phenotypes (e.g., sjs/ten) [ , ] . several nonradioactive methods have been developed for measuring lymphocyte proliferation or activation in in vitro tests for diagnosis of delayed-type drug hypersensitivity, including the use of carboxyfluorescein succinimidyl ester (cfse) cell staining dye [ , ] , and measuring cytokines or cytotoxic proteins expression, such as inf-γ, il- , il- , il- , il- , granzyme-b, and macrophage migration inhibitory factor [ ] [ ] [ ] [ ] [ ] . flow cytometry-assisted basophil activation test (bat), which measures specific cell makers such as cd or cd c to quantify basophil activation after antigenspecific stimulation, has been widely used in the diagnosis of immediate-type drug hypersensitivity [ ] . bat directly measures basophil responses instead of ige sensitization. it has been applied to the diagnosis of different drugs implicated in immediate-type hypersensitivity, including beta-lactam antibiotics, neuromuscular blocking agents, aspirin, nsaids and radiocontrast media [ ] [ ] [ ] . the sensitivity of bat varies in different types of drugs: that for beta-lactam antibiotics ranged from . to % [ , ] ; that for nsaids ranged from to % [ , ] . recent data have shown that the unique interaction between drug, t-cell receptor and hla molecule is a key factor in the development of immune-mediated adverse reactions to drugs. the discovery of strong association of specific hla alleles with specific drug-induced hypersensitivity (e.g., hla-b* to carbamazepine-sjs/ten, hla-b* to allopurinol-sjs/ten/dress, and hla-b* to abacavir hypersensitivity), and studies of the functional role of hla-b* allele (e.g., hla-b* ) directly interacting with a specific drug (e.g., carbamazepine) and unique t-cell receptor support the hypotheses of the 'pharmacological interaction with immune receptors' (p-i) [ , , ] . in recent years, bioinformatics and computer modeling have been applied to elucidate how drug molecules interact with specific hla in drug hypersensitivity. hla alleles have been associated with liver injury induced by different drugs (such as flucloxacillin). using silico strategies to examine hla haplotype relationships, and bioinformatics tools, alfirevic et al. 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antibiotics usefulness of the basophil activation test (bat) in the diagnosis of life-threatening drug anaphylaxis basophil activation and sulfidoleukotriene production in patients with immediate allergy to betalactam antibiotics and negative skin tests diagnosis of immediate-type beta-lactam allergy in vitro by flow-cytometric basophil activation test and sulfidoleukotriene production: a multicenter study the flow-cytometric determination of basophil activation induced by aspirin and other non-steroidal antiinflammatory drugs (nsaids) is useful for in vitro diagnosis of the nsaid hypersensitivity syndrome basophil activation tests in the diagnosis of drug reactions direct interaction between hla-b and carbamazepine activates t cells in patients with stevens-johnson syndrome in silico analysis of hla associations with drug-induced liver injury: use of a hla-genotyped dna archive from healthy volunteers key: cord- -rz pj a authors: dodds, p. s.; minot, j. r.; arnold, m. v.; alshaabi, t.; adams, j. l.; dewhurst, d. r.; reagan, a. j.; danforth, c. m. title: probability-turbulence divergence: a tunable allotaxonometric instrument for comparing heavy-tailed categorical distributions date: - - journal: nan doi: nan sha: doc_id: cord_uid: rz pj a real-world complex systems often comprise many distinct types of elements as well as many more types of networked interactions between elements. when the relative abundances of types can be measured well, we further observe heavy-tailed categorical distributions for type frequencies. for the comparison of type frequency distributions of two systems or a system with itself at different time points in time -- a facet of allotaxonometry -- a great range of probability divergences are available. here, we introduce and explore `probability-turbulence divergence', a tunable, straightforward, and interpretable instrument for comparing normalizable categorical frequency distributions. we model probability-turbulence divergence (ptd) after rank-turbulence divergence (rtd). while probability-turbulence divergence is more limited in application than rank-turbulence divergence, it is more sensitive to changes in type frequency. we build allotaxonographs to display probability turbulence, incorporating a way to visually accommodate zero probabilities for `exclusive types' which are types that appear in only one system. we explore comparisons of example distributions taken from literature, social media, and ecology. we show how probability-turbulence divergence either explicitly or functionally generalizes many existing kinds of distances and measures, including, as special cases, $l^{(p)}$ norms, the s{o}rensen-dice coefficient (the $f_ $ statistic), and the hellinger distance. we discuss similarities with the generalized entropies of r{'e}nyi and tsallis, and the diversity indices (or hill numbers) from ecology. we close with thoughts on open problems concerning the optimization of the tuning of rank- and probability-turbulence divergence. driven by an interest in developing allotaxonometry [ ] -the detailed comparison of any two complex systems comprising many types of elements-we find ourselves needing to compare zipf distributions: heavytailed categorical distributions of type frequencies [ ] [ ] [ ] [ ] . we take a relaxed definition of what a heavy tail means for a distribution: a slow decay over orders of magnitude in type rank. though not required, power-law decay tails are emblematic signatures of heavy-tailed distributions commonly presented by complex systems [ ] [ ] [ ] [ ] [ ] , both observed and theoretical, and provide important examples to contemplate in our efforts to develop a comparison tool. across fields, efforts to measure and explain how two probability distributions differ have led to the development of a great many probability divergences [ ] [ ] [ ] [ ] . divergences have been constructed for a host of motivations quite apart from our focus here on allotaxonometry, with example families scaffolded around l p -norms, inner products, and information-theoretic structures. as we will discuss, for heavy-tailed distribution comparisons * peter.dodds@uvm.edu which exhibit variable 'probability turbulence' [ , ] , we find these divergences lack appropriate adaptability. here, we introduce a tunable, interpretable instrument that we call probability-turbulence divergence (ptd) along with related allotaxonographs-visualizations which show in detail how two categorical distributions differ according to a given measure. we refer the reader to ref. [ ] for our motivation for creating allotaxonometry and allotaxonographs, the notion of rank turbulence, and a detailed justification for the form of rankturbulence divergence (rtd) we developed there. we establish probability-turbulence divergence using largely the same arguments. we will therefore be concise in our presentation and expand only when the probability version's behavior departs from that of its rank counterpart. in sec. ii, we formally define probability-turbulence divergence. we describe the divergence's general analytic behavior as a function of its single parameter, α, and we determine its form for the two limits of the parameter, α= and α=∞. when α= , in particular, we find an interesting departure from the equivalent tuning for rank-turbulence divergence, and which we will later connect to the sørensen-dice coefficient [ , ] and the f score [ ] . in sec. iii, we then provide realizations of probabilityturbulence divergence as an instrument through example allotaxongraphs. for three disparate examples, we con-typeset by revt e x arxiv: . v [physics.soc-ph] aug sider . frequency of n-gram use in jane's austen's pride and prejudice, . frequency of n-gram use on twitter, and . tree species abundance [ ] . we show how for the kinds of heavy-tailed distributions we are interested in that a probability-turbulence divergence histogram can be constructed to accommodate both a logarithmic scale and the presence of zero probabilities. similar to rankturbulence divergence histograms, these graphs clearly show whether or not a probability-based divergence is a suitable choice for any given comparison. to feature the tunability of probability-turbulence divergence, we present flipbooks as part of our online appendices (compstorylab.org/allotaxonometry/) for allotaxonometry. in sec. iv, we show that probability-turbulence divergence is either a generalization of or may be connected to a number of other kinds of divergences and similarities (e.g., the sørensen-dice coefficient), and then discuss limited functional similarities with the rényi entropy and diversity indices [ ] [ ] [ ] [ ] [ ] . we outline data and allotaxonometry code in sec. v and offer some concluding thoughts in sec. vi. we aim to compare two systems Ω and Ω for both of which we have a list of component types and their probabilities. for simplicity, we will speak of probability, acknowledging that relative frequency, rate of usage, or some other term may be the more appropriate descriptor for a given system. we denote a type by τ and its probability in the two systems as p τ, and p τ, . we represent the probability distributions for the two systems as p and p . we call types that are present in one system only 'exclusive types'. in general, we will use expressions of the form Ω ( ) -exclusive and Ω ( ) -exclusive to indicate to which system a type solely belongs. in general, we are interested in divergences that are some function of a sum of contributions by type. here, we will consider a single parameter family of divergences that are of the simplest form, i.e., a direct sum of contributions: by the rank-ordered set r , ;α , we indicate the union of all types from both systems, sequenced such that the contributions δd p α,τ (p p ) are monotonically decreasing (hence the necessity of an α subscript). we impose this order for general good housekeeping, secondarily allowing us to handle possibilities such as truncated summations due to sampling, or convergence issues for theoretical examples. following on from ref. [ ] , we define probabilityturbulence divergence as: . ( ) where the parameter α may be tuned from to ∞ and n p , ;α is a normalization factor. per ref. [ ] and below in sec. ii b, the roles of the prefactor (α + )/α and the power /(α + ) are to govern the behavior of ptd in the limit α → . by construction and regardless of the choice of normalization factor, we can see from eq. ( ) that probabilityturbulence divergence will equal when both distributions are the same. (we show below that for α = , distinct distributions can also register d p α (p p ) = .) the core of eq. ( ) is the absolute value of the difference of each type τ 's probability raised to the power of α: this α-tuned quantity controls the order of contributions by types to the overall value of ptd. as α → , lower probabilities-corresponding to the rare types-are relatively accentuated. for α → ∞, the higher of the two probabilities will dominate (unless they are equal), meaning the most common types will come to the fore. as for rank-turbulence divergence, we choose n p , ;α so that when the two systems are entirely disjoint-that is, they share no types-then probability-turbulence divergence maximizes at . the normalization is thus specific to the two distributions being compared. we imagine that the types in each system have an extra descriptor specifying belonging to Ω ( ) or Ω ( ) . with no matching types, the probability of a type present in one system is zero in the other, and the sum can be split between the two systems' types: where r and r are the zipf ordered sets of types for each system. we can more compactly express the normalization as: where, as for the definition of probability-turbulence divergence in eq. ( ), the sum for the normalization is again over the ordered set r , ;α . types that appear in both systems will have their contribution [ p τ, ] α/(α+ ) and [ p τ, ] α/(α+ ) counted appropriately. b. limit of α= for probability-turbulence divergence the limit of α= requires some care and will vary from the equivalent limit for rank-turbulence divergence [ ] . first, at the level of individual type contribution, if both p τ, > and p τ, > then if instead a type τ is exclusive to one system, meaning either p τ, = or p τ, = , then the limit diverges as /α, which would seem problematic. we nevertheless will arrive at a well-behaved divergence through the normalization term n p , ; . requiring as we have that the extreme of disjoint systems have a divergence of , we observe that each of the types in the case of disjoint systems would contribute /α. therefore, in the α → limit, we must have: because the normalization also diverges as /α, the divergence will be zero when there are no exclusive types and non-zero when there are exclusive types. we can combine these cases into a single expression: the term δ pτ, , + δ ,pτ, returns if either p τ, = or p τ, = , and otherwise when both p τ, > and p τ, > . (by construction, we cannot have p τ, = p τ, = as each type must be present in either one or both systems. ) we see then that d p (p p ) is the ratio of types that are exclusive to one system relative to the total possible such types, n + n . if and only if all types appear in both systems with whatever variation in probabilities, then d p (p p ) = . the limit of α = therefore exhibits special behavior as for α > , probabilityturbulence divergence only scores for exactly matching distributions. c. type contribution ordering for the limit of α= in terms of contribution to the divergence score, all exclusive types supply a weight of /(n + n ). we can order them by preserving their ordering as α → , which amounts to ordering by descending probability in the system in which they appear. and while types that appear in both systems make no contribution to d p (p p ), we can still order them according to the log ratio of their probabilities, eq. ( ) . the overall ordering of types by divergence contribution for α= is then: ( ) exclusive types by descending probability and then ( ) types appearing in both systems by descending log ratio. d. limit of α=∞ for probability-turbulence divergence the α → ∞ limit is straightforward and in line with that of rank-turbulence divergence [ ] : where the normalization from eq. ( ) has become the dominant contributions to probability-turbulence divergence in the α → ∞ limit therefore come from the most common types in each systems, providing they are not equally abundant. we assert that the successful use of our rankand probability-turbulence divergences is best achieved through consideration of rich graphical representations by what we have called allotaxonographs. in this section, we present and describe three sets of allotaxongraphs comparing probability distributions using probabilityturbulence divergence for: • normalized usage frequencies of -grams in the first and second halves of jane austen's pride and prejudice [ ] for α= / , , and ∞ (figs. , , and ); • normalized usage frequencies of n-grams in all english-identified tweets on / / and / / for n = , , and and for α = / , / , and ∞ (figs. , , and ); • relative abundances of tree species on barro colorado island for five year census concluding in and (fig. ) . the pride and prejudice examples show how ptd may be adjusted to fit well (α= / ) or poorly (α= and ∞). we bin all non-zero probability pairs (log pτ, , log pτ, ) in logarithmic space. colors indicate counts of -grams per cell, and we highlight example -grams along the edges of the histogram. for pairs where one of the probabilities is zero, we add a separate rectangular panel along the bottom of each axis (lighter gray and lighter blue). contour lines indicate where probability-turbulence divergence is constant (the jump to the zero probability region necessitates a break in smoothness). based on the histogram, we choose α= / to engineer an approximate fit to the histogram's periphery. the gray scale for -grams is indexed by their percentage contribution to probability-turbulence divergence, δd p / ,τ , showing a mixture of rare and common -grams. ranked list on the right: we order the most salient -grams according to their overall contribution δd p / ,τ which we mark by bar length. we show the rank pair for each -gram in light gray opposite each -gram. corresponding flipbook: flipbooks s , s , and s in the paper's online appendices (compstorylab.org/allotaxonometry/), show how the instrument changes for the same comparison with α being tuned from to ∞ for -, -, and -grams. see ref. [ ] for a general introduction and motivation for allotaxonometry and allotaxonographs in the context of rank-turbulence divergence. the examples for -grams and -grams can also be seen as demonstrations of possible comparisons of features of complex networks and systems (e.g, -grams in text as directed edges). as for rank-turbulence divergence [ ] but with some key modifications, our allotaxonographs for probabilityturbulence divergence pair two complimentary visualizations: a map-like histogram and a ranked list. in isolation, both the histogram and the ranked list have important but limited descriptive power. the histogram helps us see how well our choice of α performs, information that is entirely lost by the ranking process. and the ranked list would be difficult to intuit from the histogram alone. many aspects of our allotaxonographs are configurable. on gitlab, we provide our universal code for generating allotaxonographs for rank-turbulence divergence, probability-turbulence divergence, and other probability divergences (see sec. v b). in the paper's online appendices (compstorylab.org/allotaxonometry/), we complement all of our allotaxonographs with pdf flipbooks which move systematically through a range of α values. compare with the good fit afforded by α= / for the allotaxonograph in fig. . the contour lines for α= do not conform well to the histogram. at this extreme of the parameter's range, probability-turbulence divergence elevates exclusive types above all types that appear in both systems, and the ranked list on the right comprises only system-exclusive -grams. per sec. ii b and eq. ( ), exclusive types each equally contribute (n +n ) to d p while types appearing in both systems have zero weight. the ordering of -grams is determined by maintaining their contribution order as α approaches . we force the contour lines in the main body of the histogram to remain equally spaced, even as they all represent in the α → limit. see sec. iv for the connection between d p and the sørensen-dice coefficient [ , , ] and the f score [ , ] . see also flipbooks s , s , and s in the paper's online appendices (compstorylab.org/allotaxonometry/). for a primary, familiar example to help us explain our probability-turbulence divergence allotaxonographs, we compare the normalized usage frequency distributions of -gram usage between the first and second halves of pride and prejudice [ ] . we note that we are mostly intent here on showing how allotaxonographs function. we are not attempting to reveal astonishing insights into one of the most well regarded and well studied novels of all time. in fig. , we show an allotaxonograph with α= / which we will contend below provides a good fit. first, the histogram on the left bins all pairs (log p τ, , log p τ, ). the form we see here is typical of comparisons between systems with heavy-tailed zipf distributions. we rotate the axes so as not to privilege one system over the other, as this might lead to a false sense of an independent-dependent variable relationship [ , ] . we indicate counts per cell using the perceptually uniform colormap magma [ ] . because of the logarithmic scale, the cells start to separate for lower values of probability, corresponding to counts of , , , and so on. in general, types that are common in both systems will be located towards the top of histogram, those that are in both will be at the bottom, and those appearing more prevalently in one system will appear further away from the vertical midline. exactly how much this latter category matters is a function of the divergence at hand. the most extreme cells can be readily understood. the bottommost pair of cells represent all -grams that appear once in one half of pride and prejudice and zero the contour lines show constant values of δd p / ,τ . we provide a key for the contour lines in the upper right with the histogram removed. as a guide, the gray-scale for the annotations varies with each -gram's contribution to the overall divergence score, with darker meaning higher contribution. for α= / , 'miss bingley' stands out in particular, while 'irretrievable that' on the left is backgrounded. for all of our allotaxonographs, we place contour lines on each half of the histogram's diamond. these contour lines are evenly spaced not by height but are rather anchored to the bottom axes of the main histogram where they are evenly spaced in logarithmic space. while it may appear that we have omitted annotations internal to the histogram for convenient purposes of visualizing the histogram more cleanly, our annotations are intentional. because individual -grams internal to the histogram will never dominate standard divergences, highlighting them would be badly misleading [ , ] . for our allotaxonographs, the annotations along the bottom of the histogram potentially fall into this trap: 'irretrievable that' and 'were during', each appearing once overall, are just two examples of tens of thousands of such -gram hapax legomena. such types may matter in aggregate but not individually. now, our allotaxonographs for probability-turbulence divergence must depart from those of rank-turbulence divergence because we have to accommodate instances of log p when p = . for ranks, types with counts in one system-exclusive types-are assigned a tied rank for last place, necessarily a finite number. here, on a logarithmic scale our exclusive types would have to be located on one axis at log p = −∞. we end the main histogram's domain for the lower value of log p τ,i such that log p τ,i > . we then add lighter colored regions to the bottom of both sides of the histogram, and locate log p τ,i = along their midlines. the transition is a discrete jump (we do not smoothly interpolate), and we connect the contour lines with a dotted line. as can be seen in fig. , the jump is increasingly clear for contour lines for lower values of δd p / ,τ , nearer the histogram's vertical midline. the last piece for the histogram is the list of balances at the bottom right. these summary quantities are intended to be both informative and diagnostic, and they have important if subtle differences. the first bars show the balance of total -gram counts which for our example of pride and prejudice is / by construction. the second and third balances refer to sizes of lexicons, and these are well balanced too. if we create a lexicon of all -grams for pride and prejudice, about . % of them appear in the first half and . % in the second. if we instead create separate lexicons of -grams for the two halves of pride and prejudice, the third line of the balances records the percentage of -grams that are exclusive to each half. while it could be said that we are ultimately creating a simple -d histogram for a joint probability distribution with heavy tails, to our knowledge, there have been relatively few other attempts to do so [ , ] . as we have described them, we believe our histograms are crafted with a number of special details that make them well suited to their task. the ranked list on the right maps the two dimensions of the histogram onto an ordered single dimension of divergence contributions, largest first. the left-right arrangement is solely done to be consistent with the histogramall contributions are positive. the light gray numbers opposite each -gram (e.g., for 'miss bingley') indicate the -gram's zipf rank in the first and second fig. . allotaxonograph using probability-turbulence divergence to compare normalized -gram usage ranks on two days of english-language twitter, / / and / / . details are the same as for fig. and . we see that the comparison of -gram distributions produces different, broader histogram that that formed by -gram distributions (fig. ) . we choose α= / to provide a balance of -grams across five orders of magnitude for non-zero probability. in contrast to the -gram version, the top -grams are more evenly distributed on both sides of the list. while some -grams are function words combined with the -grams we saw in fig. , meaningful -grams also appear ('tom hanks','toilet paper', 'george floyd', and 'police brutality'). see flipbook at the paper's online appendices (compstorylab.org/allotaxonometry/) for the instrument's variation as a function of α. half of pride and prejudice (and in general, Ω ( ) and Ω ( ) ). we see dominant contributions from character names and references to characters (first half: 'her uncle') functional -grams (first half: 'she had', second half: 'in the'), and non-specific references to places and people (second half: 'the room', 'young ladies', 'young man'); in the ranked list, we add open triangles to types if they are exclusive to one system, corresponding to those appearing in the zero probability expansions of the histogram. for example, 'to brighton' appears only in the first half of pride and prejudice, and 'the parsonage' only in the second. finally, we see that the choice of α= / generates a list with -grams from across the rare-to-common spectrum. the balanced darker shadings of annotations in the histogram add further support. in reducing such a high dimensional categorical space-where each unique type represents a dimension-we have first collapsed the data to a -d histogram, and then to a -d list. being able to find the shape in the histogram to which can apply an instance of probabilityturbulence divergence gives us some suggestive proof in the pudding. we see in both allotaxonographs that the contour lines do not match well with the edges of the histogram, in contrast to those realized by the choice of α= / . we also see how α= favors exclusive -grams (i.e., those that do not appear in either the first or second half of the novel), while α=∞ privileges -grams that are common . allotaxonograph using probability-turbulence divergence to compare -gram usage ranks on two days of english-language twitter, / / and / / . details are the same as for fig. and . the histogram has broadened even further out from the -gram, and now is well suited to probability-turbulence divergence with the extreme of α=∞, d p ∞ . fragmentary and meaningful -grams appear alongside each other, including 'world health organization' and 'black lives matter'. social amplification is also apparent as -grams for highly retweeted tweets dominate the rank list. see flipbook at the paper's online appendices (compstorylab.org/allotaxonometry/) for the instrument's variation as a function of α. in one or both halves. as we showed in sec. ii b, when α= , the divergence contribution for all types that appear in both systems is δd p ,τ = , while for all exclusive types, δd p ,τ = (n +n ) (as reflected in the equal bars in the ranked list). thus, the vertical contour lines in fig. , which again are present because we anchor them at evenly spaced locations along the bottom of the main histogram, all correspond to a divergence of δd p ,τ = . the dashed parts of the visible contour lines then collapse to the bottom zero point, showing how the exclusive types provide the only non-zero contributions to δd p ,τ . finally, in flipbooks s , s , and s in the paper's online appendices (compstorylab.org/allotaxonometry/), we give the reader a view into how our allotaxonometric comparisons of the usage frequencies of -grams, -grams, and -grams in the two halves of pride and prejudice behave as a function of α. all of the flipbooks are especially informative in showing how contour lines and ranked lists change with α. for our second set of allotaxonographs, we compare two key dates of two major events through the lens of english-speaking twitter: / / , the date that covid- became the major story in the united states, and / / , four days after the murder of george floyd in minneapolis, minnesota by police officer derek chauvin. we compare day-scale normalized usage frequency distributions for -, -, and -grams for these two dates in figs. , , and . we choose / / as a key date for the covid- pandemic for several reasons. first and primarily, the world health organization (who) officially declared the covid- outbreak to be a pandemic on / / , a decision that was amplified immediately online but discussion of which most strongly appeared in the news and on twitter on the following day. the date of / / also saw a confluence of three major events that jolted the united states and dramatically elevated the story of the pandemic, all occurring tightly around a minute period between and pm edt ( am to am utc). first, the national basketball association (nba) abruptly suspended its season. the central event was the abandoning of a game just before tipoff between the utah jazz and oklahoma thunder, upon the league learning that rudy gobert, a center for the utah jazz, had tested positive for covid- . other players would test positive in the coming days and weeks, as would staff for teams and members of the media. just a few days earlier, gobert had joked with the media about his perception of institutional overreaction to the coronavirus, by touching microphones at an interview. second, tom hanks announced that both he and his wife rita wilson had tested positive for covid- while hanks was working on a baz lurhmann film in australia. hanks was at the time the most high profile figure known to have contracted covid- . third, president donald trump gave an oval office address, the second of his presidency, "on the coronavirus pandemic." the address marked a strong shift in trump's rhetoric regarding the danger of the covid- outbreak. the main decision announced was the ban of travel from europe to the us for days, which was needed to be clarified later to not also mean a ban on trade. futures on us stock market dropped during the speech. combined, these disparate events were a major part of the covid- pandemic becoming the dominant story for what would become weeks and then months ahead. the murder of george floyd on / / , memorial day in the us, precipitated black lives matter protests and civilian-police confrontations in minneapolis. the protests would grow over the following weeks, and begin to spread around the world. and, at least in the first week, george floyd's murder overtook coronavirus as the dominant story in the us [ ] . with the above context in mind, we can sensibly examine the allotaxonographs of figs. , , and our primary observation is that the three histograms vary considerably as we move through -, -, and grams. the histograms broaden with increasing n, with the -gram histogram losing a scaling form and squaring up in the axes. the rapidly growing combinatoric possibilities of n-grams with increasing n means that we see more and more exclusive n-grams as we look across the three allotaxonographs. for -grams, around % of each date's lexicon are exclusive, for -grams, the percentage increase to around %, and -grams we reach % (see the bottom of the three balance summaries in each allotaxonograph). the maximum counts per cell is for -grams, is for -grams, and for -grams. the cells with the most n-grams are of course the the hapax legeomenathe bottommost two cells in the histogram-those n-grams which appear once on one of the dates and not at all on the other. to obtain good balance for the most dominant ngrams, we select α= / , / , and ∞. different kinds of terms dominate depending on n with 'coronavirus', 'the coronavirus', and 'tested positive for' leading on / / , and 'minneapolis' 'george floyd' and 'of george floyd' at the top on / / . because social amplification is encoded in twitter's data stream through retweets, dominant -grams and especially -grams are liable to belong to the most retweeted messages of the day, and may lead to some variation in the dominant n-grams. (by contrast, we do not have a measure of popularity of individuals phrases or sentences within pride and prejudice with just the bare text.) for example, 'toilet paper' and 'world health organization' appear as dominant -grams and -grams but none of their five distinct -grams are near the top of the ranked list in fig. . on the other hand, some dominant -grams may be used in diverse -grams and -grams and thus may not appear in the ranked lists for -grams and -grams. examples from fig. are 'antifa' and 'breonna'. for all three n-gram comparisons of these two dates on twitter, we provide flipbooks , , and at the paper's online appendices (compstorylab.org/allotaxonometry/). readers may use these to easily explore how the choice of α affects the fit for the contour lines in the histogram and the ordering of which n-grams dominate probability-turbulence divergence. we include one final allotaxonograph from an entirely different field of research, ecology. in fig. we show a probability-turbulence divergence allotaxonograph for tree species abundance in barro colorado island for censuses completed in and . this example also shows how allotaxonographs can be used to inspect how well divergence measures perform for data sets that are much smaller than our examples from literature and twitter. the species that dominates the overall divergence score is one that has diminished in abundance, piper cordulatum [ ] [ ] [ ] [ ] . in ref. [ ] , we compared these distributions with rank-turbulence divergence, and the overall orderings of dominant species are broadly consistent. in flipbook at the paper's online appendices (compstorylab.org/allotaxonometry/), we show how the dominant contributions of species vary as a function of α. allotaxonograph using probability-turbulence divergence to compare tropical forest tree species abundance on panama's barro colorado island (bci) for year censuses completed in and [ ] . the choice of α= / produces a set of dominant species reasonably well balanced across the abundance spectrum. see ref. [ ] for the corresponding rank-turbulence divergence allotaxonographs. see flipbook in the supplementary information for the instrument's variation as a function of α. a. links to existing probability-based divergences probability-turbulence divergence shares some characteristics with other divergences (see refs. [ ] and [ ] for two example compendia). in particular, we find known distances and similarities which correspond with or function similarly to probability-turbulence divergence for α= , / , , and ∞. for α= , probability-turbulence divergence partners the similarity measure sørensen-dice coefficient, s sd (p p ) [ , , ] , which was independently developed in the context of ecology by dice ( ) and sørensen ( ) (see also ref. [ ] ). for two systems, the sørensen-dice coefficient is the number of shared types relative to the mean of the number of types in each system. using our notation, and referring back to eq. ( ), we have: where we are again summing over the union of types r , ; . the quantity − δ pτ, , − δ pτ, , is when a type appears in both systems and otherwise. the sørensen-dice coefficient has arisen in many settings, with different names. for example, in statistics, the sørensen-dice coefficient is the f score of a test's accuracy [ , ] . examples of divergences matching the internal structure of d p / include the hellinger [ ] , mautusita distance [ ] , and squared-chord distance [ ] . in terms of the probability-turbulence divergence's internal structure of |[ p τ, ] α −[ p τ, ] α |, a large selection of divergences match up with the the α= instance. these include l (p) -norm type constructions of the form: while the overall divergence values for these various divergences will differ, the rank orderings of the contributing types will be identical to that of d p . finally, in the α=∞ limit, d p ∞ agrees, up to a normalization factor of , with the motyka distance [ ] . while none of these other divergences provide direct tunability of the type probability-a severe limitation, as we hope our examples have conveyed-there are well established quantities which do. as we observed for rank-turbulence divergence in [ ] , the parameter α's effect is similar to its counterparts in various kinds of generalized entropy [ ] [ ] [ ] and, more directly, the diversity indices (or hill numbers) from ecology [ , ] . rényi entropy, α h, and the associated diversity index, α n , are defined as: where α ≥ . we acknowledge that, at the risk of a minor dislocation from relevant literature, we have had to confront some notation peril here as a standard notation for the diversity index is α d. we have also already used n in our present paper but this choice tracks sensibly: as α → , we retrieve the natural logarithm of the number of distinct types n (species richness in ecology) for rényi entropy, and therefore the diversity index is n = n . as α → ∞, the most abundant type will dominate, with min-entropy the limit: ∞ n = min τ /p τ = /max τ p τ . in the α → limit, we recover shannon's entropy, h, as well as n = e h = e h . there are similar aspects for probability-turbulence divergence and the diversity index in the limits of α= and ∞. for α= , for example, both reduce to quantities involving simple counts of distinct types. nevertheless, we note that we cannot construct probability-turbulence divergence from manipulations of rényi entropy or the diversity index. we can, roughly speaking, only create a difference of sums whereas we need a sum of absolute differences with suitable exponents. pride and prejudice: we sourced a plain text version of jane austen's pride and prejudice from project gutenberg (http://www.gutenberg.org/ebooks/ ). normalized n-gram usage frequency on twitter: we collected around % of all tweets sent on these dates based on coordinated universal time (utc) meaning they covered : : am to : : am eastern daylight time (edt) and : : am to : : am pacific daylight time (pdt). for the eastern and central time zone's in the united states, especially, this shift provides a better, more functional coverage of people's activity. we provide historical access to the top -grams, -grams, and -grams across more than languages as part of our storywrangler for twitter project [ , ] . species abundance on barro colorado island: we accessed the dataset for bci censuses performed roughly every years over years through the online repository described in ref. [ ] . all scripts and documentation reside on gitlab: https://gitlab.com/compstorylab/allotaxonometer. for the present paper, we wrote the scripts to generate the allotaxonographs in matlab (laboratory of the matrix). we produced all figures and flipbooks using matlab version r a. we welcome ports to other languages. as is, the core script is highly configurable and can be used to create a range of allotaxonographs as well as simple unlabeled rank-rank and probabilityprobability histograms. instruments accommodated by the script include rank-turbulence divergence [ ] , probability-turbulence divergence, and generalized symmetric entropy divergence which includes jensen-shannon divergence as a special case. we have defined, analyzed, and demonstrated the use of probability-turbulence divergence as an instrument of allotaxonometry. as the probability-based analog of our rank-turbulence divergence, the instrument is able to perform well when comparing heavy-tailed zipf distributions of type frequencies. we have shown further that probability-turbulence divergence generalizes a range of existing probability-based divergences, either in matching in exact form or equating in how types are ordered by type contribution. while we view rank-turbulence divergence as our most general, interpretable instrument, for systems in which probabilities (or rates) of types occurring are well defined, and the resulting distributions involved are heavy-tailed, probability-turbulence divergence provides a more nuanced instrument. we also favor divergences which compare distributions in as transparent a way as possible. to that end, we have made the core of probability-turbulence divergence a simple difference of powers of probabilities (eq. ( )). by contrast, we view some divergences as being problematic in being overly constructed. we venture that jensen-shannon divergence (jsd), which we ourselves have used elsewhere, is one such instrument. the creation of an artificial mixed distribution is a contrivance we avoid here, and is perhaps indicative of taking information theory too far [ , ] . in our experience, we have also found that the visual information delivered by our allotaxonographs, especially in their coupling of histograms and ranked lists, has been essential to working effectively with divergences of all kinds. one caution we make is that in the examples we have explored in the present paper, we have taken distributions as they are. that is, we have not contended with issues of sub-sampling and missing tail data [ ] . we can say that types appearing with high rate (e.g., common n-grams on twitter) will not be affected by accessing more data, as they are well estimated rates. in our paper on rank-turbulence divergence, we examined how truncation of distributions affects allotaxonographs, and such an approach is always available for any divergence. finally, in our present paper and in ref. 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island, panama phenology of neotropical pepper plants (piperaceae) and their association with their main dispersers, two short-tailed fruit bats, carollia perspicillata and c. castanea (phyllostomidae) hierarchical fruit selection by neotropical leaf-nosed bats (chiroptera: phyllostomidae) stigler's law of eponymy neue begründung der theorie quadratischer formen von unendlichvielen veränderlichen decision rules, based on the distance, for problems of fit, two samples, and estimation ( ), storywrangling.org the bandwagon on the limitations of jensen-shannon divergence and its generalizations: allotaxonographs and critique robust estimation of microbial diversity in theory and in practice text mixing shapes the anatomy of rank-frequency distributions the authors are grateful for the computing resources provided by the vermont advanced computing core which was supported in part by nsf award no. oac- , financial support from the massachusetts mutual life insurance company and google open source under the open-source complex ecosystems and networks (ocean) project. key: cord- -pgyzluwp authors: nan title: programmed cell death date: journal: experientia doi: . /bf sha: doc_id: cord_uid: pgyzluwp nan it is widely held that all developmental cell death is of a single type (apoptosis) and that neuronal death is primarily for adjusting the number of neurons in a population to the size of their target field through competition between equals for target-derived factors. we shall draw on our research and on that of others to criticize these views and replace them by the following. at least three types of neuronal death occur, only one of which resembles apoptosis; a neuron can choose between several self-destruct mechanisms depending on the cause of its death. the purpose of the death is to regulate connectivity, not neuron number. competitors for trophic factors are unequal, and many losers have made axonal targeting errors. a neuron's survival and differentiation depend on multiple anterograde and retrograde signals. activity affects retrograde signals and some but not all anterograde ones. the pattern of activity is more important than the overall amount. in rodents, the period of naturally occuring cell death of motoneurons is followed by a period of supersensitivity to axonal injury. thus, in newborn rodents lesion of the facial nerve leads to a rapid degeneration of the injured motoneurons. we have tested whether overexpression, in rive, of the bcl- proto-oncogene was capable of preventing death of axotomized motoneurons. to address this question we used transgenic mice whose motoneurons overexpress the bcl- protein. one of the two facial nerves of newborn mice was transected on the nd- rd post-natal day. seven days after the lesion, the morphology of the facial nuclei was analyzed. in control mice, and when compared to the intact nucleus, to % of axotomized motoneurons had disappeared. in contrast, in the transgenic animals, the number of motoneurons on the lesioned side remained unchanged when compared to the eontralateral nucleus. furthermore, their axons remained visible up to the distal lesion site. these experiments show that, in rive, motoneurons overexpressing the bcl- protein survive after axotomy, and suggest that, in rive, bcl- protect neurons from experimentally induced cell death and could be a target for treatment of motoneurons degenerative diseases. messmer s., mattenberger l., sager y., blatter-garin m-c., pometta d., kate a., james r.w. drpt de mrdeeine, drpt. de pharmacologie, div. de neurophysiologie clinique, facult de mrdecine, gen~ve. clusterin is a widely expressed glycoprotein, highly conserved across species. numerous functions have been postulated for this protein. the most important are roles in lipid transport, as elusterin is associated with apolipoprotein ai in hdl, complement regulation and tissue remodelling, in particular during cell death and differentiation. using cultures of rat spinal cord neurones ( % neurons and - % non-neuronal cells), we have studied the expression of clusterin and ape e in glutamate-induced neuronal cell death to examine potential roles in lipid management. up-regulation of the two proteins was observed. clusterin and ape e appear in the conditioned medium respectively h and . h after incubation with glutamate. control studies, in the presence of a noncompetitive nmda receptor agonist showed the secretion of clusterin and ape e to be diminished by > %. no up-regulation of either protein was observed in complementary studies with exclusively non-neuronal cell cultures. the cellular origin of the secreted proteins is presently under investigation. programmed cell death and tissue remodelling are consequences of hormonally induced restructuring of the rat ventral prostate after castration and the rat mammary gland after weaning. we used the "differential display"-method (liang and pardee, , science : ) to detect and isolate edna fragments whose corresponding rnas are regulated either coincidentally, or in an organ specific fashion during mammary gland involution and postcastrational prostate regression. partial sequencing of clones revealed high, but not absolute homology of fragments with sequences, previously characterized in different biological contexts. these five encode functions which could be anticipated to be important for cell growth and/or programmed cell death, we are presently investigating the functions of several of these transcripts in cell culture and in rive. antisense oligos are being employed in vivo to determine whether these genes contribute to the phenotype of programmed cell death. b epitopes derived from the envelope gp glycoprotein (ep ) or from the viral superantigen of mmtv have been incorporated into inert or live vaccines. the inert vaccine consists of purified chimeric proteins which contain the b epitopes alone or fused to multimeric promiscuous t helper epitopes from tetanus toxin. mice were immunized subcutaneously with these chimeric proteins. the live vaccine consists of an avirulent strain of salmonella typhimurium which expresses the mmtv epitopes in the form of chimeric proteins fused to the nucleocapsid protein of hepatitis b virus. this vaccine is given to mice in one oral dose. the level, duration and isotype of the immune response generated by each vaccine have been measured and compared. the level of protection has been investigated by systemically challenging immunized mice with the relzovims. a reduced binding of oxytocin (ot) occurs with aging in some, but not all, areas of the rat brain (arsenijevic et al., experientia , , a ) . the candate putamen showed the most impressive loss of ot receptors. two other regions, the hypothalamic ventromedial nucleus (vmh) and the islands of caueja (icj) had also an important deficit of ot binding sites. on the other hand, these two regions were known to be sensitive to sex steroids. in the present work, we treated from month old rats during one month with testosterone propionate ( #g/kg s.c., once every days) dissolved in oil. three rats of the same age injected with oil only served as controls. we labelled ot receptors throughout the brain of old rats using a i-labelled ligand specific for ot receptors. analysis of autoradiograms by an image analyzer revealed that the testosterone treatment increased ot binding sites in the vmh, in the icj, and, to a lesser extent, in the bed nucleus of the stria terminalis, a region also sensitive to sex steroids, by contrast, in the caudate putamen, the disappearance of ot receptors was not compensated. in conclusion, the decrease of ot receptors occurring in vmh and icj with aging can be reversed by administration of gonadal steroids. in contrast, the loss of ot receptors in the striatum appears to depend on another mecanism. vasopressin (avp) receptors are expressed transiently in the facial nucleus during development (tribollet et el., , dev. brain res., , - ) . avp may therefore play a role in the maturation of neuromuscular connexions in the neonate rat, and possibly in the restanration of these connexions after nerve lesion in the adult. in order to investigate the latter proposition, we have sectionned the facial nerve in adult rats and used quantitative autoradiography to look at avp binding sites in the facial nucleus at various postoperative times. we observed a massive and transient increase of avp binding sites on the operated side. the number of facial avp binding sites reaches a maximum about one week after nerve section, remains stable during - weeks, then begin to decrease towards control level. the induction of avp receptors is markedly delayed if the proximal stump of the nerve is ligated. to assess whether other motor nuclei would also react to axotomy by up-regulating the expression of avp receptors, we have sectionned the hypoglossal nerve and the sciatic nerve. in both cases, the binding of avp receptor ligand increases massively in the respective motor nuclei, with a time-course similar to that found in the facial nucleus. altogether, our data suggest that central avp could be involved in the process of nerve regeneration. cytotoxic t-cell mediated apoptosis schaerer,e, karapetian,o.,adrian,m. and tschopp,j. inst.de biochimie, univ.de lausanne, epalinges. an apoptotic cell death mechanism is used by cytolytic t cells (ctl) to lyse appropriate target cells. ctl harbor cytoplasmic storage compartments, containing the lytic protein perforin and serineproteases (granzymes), whose content is released upon target cell interaction. we show that these granules are multivesieular bodies and that degranulation releases these intragranular vesicles (igv) having granzymes, t-cell receptor and yet undefined proteins associated. isolated igvs and perforin induce dna breakdown in target cells within minutes. microscopic analysis demonstrates that igv specifically interact with target cell via the t-cell receptor and that their contents is taken up by the target cell. already min. after interaction, distinct igv proteins are found in the nucleus of the target cell.one of the molecules has been identified to be granzyme a, previously reported to be involved in apoptosis. we propose that lymphocytes transfer apoptosisinducing proteins to the nucleus of the target cells using vesicles as vehicles for delivery. cytotoxic t cells kill their targets by a mechanism involving membranolysis and dna degradation (apoptosis). recently, two sets of proteins have been proposed as dna breakdown-inducing molecules in t cells: granzyme a, b and tia-i. in this study, we cloned and further characterized the tia-i mouse homologue. aa sequence comparison with the human tia- showed an overall identity of %. devoid of a signal peptide, tia is yet localized to cytotoxic granules, probably targeted via a gly-tyr-motif. as tia-i, its mouse homolcgue contains three rnabinding domains. expression of tia during development shows a very strong signal in the brain and weaker signals in thymus, heart and other organs. during embryonic development several structures that contribute to organogenesis form transiently and are later eliminated by apoptosis. this pattern of tia expression could indicate its involvement in apoptosis. prostate involution occurs after castration in rats and is associated with the death by apoptosis of a large fraction of the epithelial cells. we have isolated several genes from a prostate involution bacteriophage lambda library using differential screening methods. among these clones, one d~monstrated an especially strong signal when used as a probe against northern blots of prostate mlhna obtained before, and at different times after castration. this gene is down-regulated after castration by -fold within days. intramuscular injection of a testosterone depot resulted in complete restoration of expression within hours. upon sequencing it became apparent that this clone has a high degree of homology to a known ndah dehydrogenase encoded in mitochondrial dna. the clone failed to hybridize to any transcripts from rat organs other than prostate. we are now in the process of isolating the htm~n hc~olog to this gene for use as a biomarker in study of benign hyperplasia and developing carcinoma. this gene is a possible indicator for testosterone-independent cell populations or of cells lacking ftl~ctional testosterone receptor. during the first three postnatal weeks the rat lung undergoes the last two developmental stages, the phase of alveolarization and the phase of microvascular maturation. the latter involves a decrease of the connective tissue mass in the alveolar septa and a merging of the two capillary layers to a single one. speculating that programmed cell death may play a role during this remodeling, we searched for the presence of apoptotie cells in rat lungs between days and . lung paraffin sections were treated with y-terminal transferase, digoxigenin-dutp, and anti-digoxigeninfluorescein-f(ab)-fragments, and the number of fluorescent nuclei was compared between sections at different days. while the number of apoptotie ceils was low until the end of the second week and at day , we observed an about eight fold increase of fluorescent nuclei towards the end of the third week. we conclude that programmed cell death is involved in the structural maturation of the lung. brunner, a., wallrapp, ch., pollack, i, twardzik, t. and schneuwly, s. lehrstuhl genetik, biozentrum universit~t w~rzburg, mutants in the giant lens (g/l) gene show a strong disturbance in ommatidial development. in the absence of any gene product, additional phetoreceptors, cone cells and pigment cells develop. opposite effects can be seen in flies in which the gene product of the giant lens gene can be ectopically expressed by heat shock. a second very typical phenotype is the disturbance of photoreceptor axon guidance. molecular analysis of gil shows that it encodes a secreted protein of aa containing three evolutionary conserved cystein-motives very similar to egf-like repeats. we propose that gil functions as a secreted signal, most likely a lateral inhibitor for the development of specific cell fates and that gil, either directly or indirectly, is involved in targeting photoreceptor axons into the brain. the decrease in cellularity during scar establishment is mediated through apoptosis desmouliere, a., redard, m., darby, i., and g. gabbiani department of pathology, cmu, rue michel server, gen~ve dudng the healing of an open wound, granulation tissue formation is characterized by replication and accumulation of fibroblastic cells, many of which acquire morphological and biochemical features of smooth muscle cells and have been named myofibroblasts (sch rch et el., histology for pathologists, t ). as the wound evolves into a scar, there is an important decrease in ceuuladty, including disappearance of myofibroblasts. the question adses as to which process is responsible for myofibroblast disappearance. during a previous investigation on the expression of (z-smooth muscle actin in myofibroblasts, we have obsewed that in late phases of wound healing, many of myofibroblasts show signs of apoptosis end suggested that this type of cell death is responsible for the disappearance of myofibroblasts (darby et al., lab. invest. : , ) . we have tested this hypothesis by means of electron microscopy and morphometry and by in situ end-labeling of fragmented dna (wijsman et al., j. histochem. cytochem. : , t ) . our results show that the number of apoptotic cells increases as the wound closes and suggest that this may be the mechanism for the disappearance of myofibroblasts as well as for the evolution of granulation tissue into a scar. (supported by the swiss national science foundation, grant n~ s - r. jaggl, a. marti and b. jehn. universit~t bern, akef, tiefenaustr. , bern at weaning the mammary gland undergoes a reductive remodelling process (involution) which is associated with the cessation of milk protein gene expression and apoptosis of milk-produclng epithelial cells. this process can be reversed by returning the pups to the mother within day. elevated nuclear protein kinase a (pka) activity was observed from one day post-lactation, paralleled by increased c-los, junb, ]und and to a lesser extent c-]un mrna levels. ap- dna binding activity was transiently induced and the ap- complex was shown to consist principally of cfos/jund. oct- dna binding activity and oct- protein were gradually lost from the gland over the first four days of involution, whereas oct- m_rna levels remained unchanged. comparing nuclear extracts from normal mammary glands with nuclear extracts from glands which had been cleared of all epithelial cells three weeks after birth revealed that pka activation, ap- induction and oct- inactivation are all dependent on the presence of the epithelial compartment. the increased fos/jtm expression and the inactivation of oct- may be consequences of the increased pka activity. when involution is reversed, both, pica activity and ap- dna binding activity (and fos andjun mrna levels) are reduced to basal levels. our data suggests a role for pka and ap- on progranlmed cell death of manlnmry epithelial ceils. bcl- ~ does not require membrane attachment for its survival activity c. borner*, i. martinout, c. mattmann*, m. irmler*, e. sch&rrer*, j.-c. martinou-j-, and j. tschopp*. * institute of biochemistry, university of lausanne, epalinges, institute of molecular biology, glaxo inc., plan los ouates. cl- (z is a mitochondrial or perinuclear-associated oncoprotein that prolongs the life span of a variety of cell types by interfering with programmed cell death. how it exerts this activity is unknown but it is believed that membrane attachment is required. to identify critical regions in bcl- o~ for subcellular localization and survival activity, we created by site-directed mutagenesis, various mutations in regions which are most conserved between the different bcl- species. we show here that membrane attachment is not required for the survival activity of bcl- o< a truncation mutant of bcl- (z lacking the last amino acids (t ) including the hydrophobic domain is soluble, yet fully active in blocking apoptosis of sympathetic neurons induced by ngf deprivation or l fibroblasts induced by tnfc~ treatment. we further provide evidence for a putative functional region in bcl- which lies in the conserved domains and upstream of the hydrophobic cooh terminal tail. the breakdown of nuclear dna is considered to be a hallmark of apoptosis. we previously identified the perinuclear membrane localized dnase i as the endonuclease involved in the formation of oligonucleosomal-sized fragments (dna ladder). it is not clear how the nuclease is activated and has access to the dna. we show that in thymocytes induced to undergo apoptosis, lamin breakdown preceded dna laddering. by transfeeting hela cells with a constitutively active cdc mutant, nuclear envelope breakdown and typical apoptotic features (ehromatin condensation) were observed. moreover, co-transfection with cdc mutant and dnase i led to dna degradation. we propose that apoptosis can be induced by wrongly timed and hence abortive mitosis leading to uncontrolled nuclear membrane disintegration. s - s - platelet-derived growth factor (pdgf) is thought to play an active role in fibrosing diseases. bronchiolitis obliterans-organizing pneumonia (boop) is a condition characterized by intraluminal proliferation of connective tissue inside distal air spaces. to evaluate pdgf expression in boop we performed immunohistoehemistry on lung biopsies from patients and controls free of fibrosis. sedal sections were stained with an antibody against either pdgf or the monoeyte/macrophage marker cd , in both groups the pdgf ~ cells were essentially tissue macrophages. using point counting to measure volume fraction (vv) , pdgf-pesitive cells represented . + . % (mean+sd) of the volume occupied by lung tissue in the boop cases, and , + . % in the controls (! < , ). similarily, . + . % of the lung tissue was occupied by cd e~ macrophages in the boop cases, compared to . :~ . % in the controls (p<o. ). a positive linear correlation was found between the v v of pdgf ~ cells and the v v of cd @ cells on the cases (r=g. p< . o ). we conclude that boop is characterized by a recruitment of tissue macrophages and that a significant proportion of them express pdgf. as pogf can also induce the macrophage chemo-attractant mcp- , we speculate that maerophages are involved in a positive feed back regulation, and may play a pivotal role in the pathogenesis of boop. barrier lesions in hydrostatic pulmonary edema d.wu, h.bachofen, u.gyger and e.r. weibel department of anatomy, university of bern, ch , bern pulmonary edema induced in isolated perfused rabbit lungs by moderate elevation of perfusion pressure ( tort ) results in the formation of characteristic bleb-like extensions of the thin cytoplasmic leaflet of alveolar epithelium ( bachofen et al. am rev resp dis. vo . . pp - , ) . in this study, we show by means of electron microscopy and morphometry that the frequency of the blebs correlate with the distribution of alveolar edema fluid. we find that % of the epithelial surface is involved in the formation of blebs. three-dimensional reconstruction showed that blebs had near-spherical shape and that their opening toward the interstitium is irregular. these finding demonstrate a great plasticity of epithelial cell extensions when subjected to moderate pressure stress. at high pressure the cell linings demonstrate clear breaks. two major roles have been defined for no: cellcell communication mediated by the stimulation of cgmp synthesis, and cytotoxicity by direct interaction of the free radical no with cellular target. to investigate these effects in human epithelia, which constitute a major source of no in man, we introduced the murine inos sequence into sv -t immortalized human bronchial epithelial cells (beas- b). inos activity was higher than pmol citrulline/min/ mg prot in the first passages of inos transfected cells, but it decreased by subculturing. no inos activity was detected in cells transfected with control vector. in inos transfected cells, no stimulated a cgmp synthesis and c-los expression which could be inhibited by addition of lnma. thus, in human epithelial cells no is able to significantly alter signal transduction pathway. partial pneumoneetomy, i.e. the resection of lung tissue, is known to initiate compensatory growth in the remaining lobes. under appropriate conditions this results in a complete restoration of lung parenchymal structure and function. unfortunately the molecular mechanisms controlling the onset of this compensatory growth are poorly understood. the aim of our study was to find and eharacterise genes which are up or down regulated during this time. for isolating such genes, we chose the differential display approach (p.liang and b.pardee , science :p ). therein a 'end fragment of a subset of reverse transcribed mrnas is amplified by the polymerase chain reaction. the fragments can be separated on a dna sequencing gel. genes differentially expressed in pneumonectomised and sham-treated rats show different patterns on these gels. the fragments of such genes can be isolated from the gel and cloned into vectors in order to provide tools to characterise their gene. a set of such genes, some up-regulated and others downregulated following pneumonectomy, have been isolated, partially sequenced and characterised. time periods. our new plasma marker consisting of highly concentrated gold nanospheres was infused via a femoral vein catheter into the right atrium of anaesthetised and ventilated rabbits. five or seconds after start of infusion, the blood flow was stopped and the lung was fixed by instillation. silver enhanced paraffin sections revealed labeled and unlabeled blood vessels. electron-microscopic sections showed various plasma gold particle concentration. morphometric analysis of electron micrographs showed an increase of the portion of marked capillaries from shorter to longer circulation times. based on our results, we conclude the existance of inhomogeneous plasma flow velocities within the pulmonary microvascular bed. (supported by snsf grant - . ) in schizosaccharomyces pombe pairing of meiotic chromosomes is not accompanied by the formation of a tripartite synaptonemal complex (sc), a structure which connects the homologs in most other eukaryotes. meiotic nuclei revealed novel structures ("linear elements") that might be related to the axial elements ($c precursors) of other organisms and function in meiotic chromosome pairing, recombination, and segregation (baler et al. ( ) jcb : ). to get further insight into the behavlour of meiotic chromosomes, we performed fluorescence in situ hybridizations on spread nuclei with probes from different chromosomal regions. interestingly, all centromeres and telomeres become clustered during meiotic prophase. the centl:omeres are paired already before meiosis, whereas the telomeres initiate pairing specifically during meiotic prophase, followed by pairing of interstitial regions. painting of whole chromosomes revealed that the homelogs occupy together a specific territory in the nucleus. an expression vector containing the vai gene read by rna polymerase ill, injected into xenopus zygotes, yields high levels of rna in virtually all embryonic cells. anti sense fina is produced from a segment of the xhoxla gene inserted in opposite orientation into the vai gene. immunological staining of whole mount embryos shows that target mrna level and xhoxla protein expression of xhoxla protein is diminished in about half of the antisense treated individuals. accordingly, nearly half of the embryos develop typical malformations in regions where the xitoxla gene is normally expressed. somites el the anterior trunk are heavily disorganized and mesodermal denvalives surrounding the intestinal tract are reduced or missing. antisense inhibition, through production of rna in situ from expression vectors, thus represents a useful tool to study the functional role of zygotic genes in xenopus development. cloning and characterization of the mouse homolog to s.cerevisiae sep encoding a dna strand exchange and homologous pairing protein vladimir bashkirov and wolf-dietrich heyer. institute of general microbiology, university of bern, baltzer-strasse , bern. homologous pairing and dna strand exchange are the central steps postulated in the current models of genetic recombination. to date no mammalian gene encoding a homologous pairing protein has been cloned. to get insight into the mechanism of recombination in higher eukaryotes we were interested to clone such genes. protein sequence comparison of the s.cerevisiae homologous pairing protein, sepi, and its homolog from s.pombe, p ex~ revealed several conserved stretches of amino acids in their nh -terminal regions. using fully degenerate primers and mouse testes edna as a template for the polymerase-chain-reaction a product with the expected length has been amplified. this bp dna was shown to encode a putative polypeptide with high degree of homology to both proteins from the two yeasts. the mouse pcr-product was used as a hybridization probe for screening a gtlo mouse testis cdna library. after several rounds of successive screening a total of about kb of mouse edna (msep ) was cloned and sequenced. the putative translation product revealed high homology throughout the entire sequence to its s.pombe and s.cerevisiae counterparts, % and % of amino acid identity, respectively. the msep sequence was shown to be unique in the genome and the chromosomal gone is likely to contain many introns. we want to establish a functional full length edna clone of msepi and characterize the gone and protein product. we have earlier developed a cell free system to study recombinational repair of dna double strand breaks and deletions. the method allowed us to purify and characterize a high molecular weight multiprotein complex (rc- ), which catalyzes dna recombination. a dna polymerase and dna ligase as well as other enzymatic activities copurify with rc- .we have commenced an investigation of homologous dna recombination in nuclear extracts which were prepared fzom normal and mutated mouse thymocytes. the scid mutation in mice causes an aberrant v(d)j joining reaction, an elevated sensitivity to ionizing radiation and a defect in recombinational repair. moreover, the normal mouse thymocytes were sorted into the cd /cd double negative (dn), double positive (dp), and single positive (sp) subclasses and their nuclear extracts, and extracts from rag- -/-mice, were tested. only the scid mice and the cd /cd sp thymocyte extracts were inactive in the recombinational repair reaction. this indicates a development stage specific activity and a scid specific defect of recombinational repair in thymocytes. restoration of the recombination activity was observed in thymocyte extracts derived from scid, which express a wansgene for the t-cell receptor i~ chain. a protein could be purified from normal thymus cells to near homogeneity which specifically rescues the recombination activity in scid extracts. this protein, srsp ~cid recombination stimulatory ~otein), migrates as a single band of app. kda in sds polyacrylamide gel electrophoresis. the ade -m mutation in the ade gene of the fission yeast schizosaecharomyces pombe gives rise to a hot spot for meiotic homologous recombination. to address whether transcription plays a role in m activity, the effect of the deletion of the ade promoter in combination with either the m and m mutations on recombination frequency was studied at the tetrad level. deletion in eis of the promoter abolishes m hot spot activity relative to the m control deletion strain. it is possible that deletion of the promoter has eliminated a sequence necessary for m hot spot activity, rather than lack of transcription being responsible for the loss of m activity. the role of transcription in m hot spot activity is currently being examined. we are analysing meiosis in a temperature sensitive top mutant. complementary temperature-shift experiments showed that topoisomerase ii is required for both meiotic divisions; early meiotic events are not impaired. in a meiotic time course, dapi staining revealed that the cells are blocked before the first meiotic division. interestingly, in meiotic spreads we found that the spindle pole body (spb) cycle is not affected by the block: the final arrest point showed nucleus with completely separated spbs. we want to analyse the number of spbs and spindle formation with immunofluorescence. we are planning to analyse the course of meiosis in a top rec double mutant (rec is recombination deficient) to see if topoisomerase ii activity is necessary for separation of recombined chromosomes. analysis of meiosis in a top rec double mutant (rec is recombination deficient and does not form linear elements) could give us a clue about the function of the linear elements. genetic recombination is able to induce cancer and to mediate its further progression. in cells from cancer predisposed individuals, mitotic recombination can lead to loss of heterozygous tumor suppressor genes as is e.g. observed in the hereditary form of retinoblastoma. one mechanism for gene loss is inter-or intrachromosomal recombination involving short duplicated sequences. our aim is to identify xenobiotics that are able to induce mitotic recombination and to elucidate the involved molecular mechanisms. for that purpose we have constructed transgenic d. melanogaster ( ). rn.). cell lines. cells were stably transfeeted with an extrachromosomal dna element providing a putative recombination substrate+ "l%e constructed element contained two ta'uncated fragments of the neomycin resistance gene (nee r) interrupted by a hygremycin resistance marker (hygr). the hyg r insert was flanked on both sides by identical bp fragments of nee r, different d. m. promoters shown to be constitutively active in various d. m. tissues were inserted ' to both e. coil genes. restoration of a functional nee r gone may occur by deletion of the hyg r after recombination between the homologous regions. stably transfected schneider line cells were obtained and preliminary results concerning chemical induction of genetic rearrangements will be shown. fleck, ., sch~r, p. and kohli, j., institute of general microbiology, baltzerstr. , bern to detect mismatch-binding proteins of s. pombe we performed band shift assays with radiolabeled oligonucleotides containing a defined mismatch. we found two distinct mismatch-binding activities. one shows high affinity to most of the mismatches, but is absent for c/c. this protein might belong to the general mismatch-repair system, which is thought to be related to the bacterial muts dependent pathway. the second activity preferentially binds to those mismatches containing a cytosine and therefore represents a novel type of mismatch-binding protein. baur m., sch~r p. and kohli j., institute of general microbiology, baltzerstrasse , ch- bern homologs of the salmonella typhimurium (mutl, s and and streptococcus pneumoniae (hexa, b) genes involved in mismatch repair have been identified in several distantly related organisms. so far no mismatch repair gone is known in schizosaccharomyces pombe. in order to get more insight into the ~chanisals of mismatch repair in s. pombe we started the cloning of a mutl homolog. degenerated oligonucleotide primers based on conserved regions of mutl homologs were used in the polymerase chain reaction (fcr) to amplify a corresponding dna fragment from s. pombe. a dna fragment of about bp was amplified whose deduced amino acid sequence shared a high degree of homology with paiql of saccharomyces cerevisiae and mutl, hexb. with the help of this dna fragment the gone was isolated from a genomic dna library by colony hybridization. sequence analysis of this mlhl gene (mlh = mut~ homolog) shows an orf of the expected size with three putative introns. while the deduced amino acid sequence of the ' region shares strong homology with the procaryotic genes mutl, hexb and the eucaryotic gene pmsi, the amino acid sequenoe of the ' region shows homology only to pmsi and hexb. the central region of the protein seems to be conserved only weakly. parisi s., and kohli j., institute of general microbiology, university of bern, ch- bern rec and rec are supposed to be genes with major functions in meiotic recombination since mutations in these genes reduce the frequency of meiotic intragenic recembination ~ fold. these genes have been isolated and sequenced but no enzymological activity could be assigned to the protein. a first step toward enzymological and biochemical characterization of rec and rec is to make beth protein products available in sufficient quantities and in reasonable pure form. for this purpose both genes will be cloned in a s. /x~nbe over-expression system using the strong and inducible nmtl promoter.in parallel both genes will also be expressed in e. coli as gst fusion proteins in order to produce polyclonal antibodies in rats and rabbits. these antibodies can then be used for protein purification in s. pombe as well as for immunofluorescence experiments. induction of expression in e. coli produces a band of the expected size of kda for rec and of kda for rec . to remove the gst carrier, the rec and rec fusion proteins can successfully be cleaved by thrombin or factor xa respectively. production of antibodies is in progress. d bbeling, u., nobi, r,, berchtold, m.w. and kuenzle, c.c. institut fdr veterinarbiochemie, universit t zurich, wintertharerstrasse , normally, only pre b-and pre t-cells can rearrange their immunoglobulin genes by v(d)j recombination, but when other cell types are transfected with the rag-i and rag- genes they also become able to perform v(d)j recombination. by transiently transfeeting nih t fibroblasts with both intact rag genes we found da recombination rate of . %. no recombination was detected, when we transfected the intact rag- gene with a mutant rag- gene, which lacks c-terminal amino acids, or the intact rag- gene with a rag-i gene containing a deletion in the topoisomerase iz homology region. these experiments show that these two regions of the rag genes are essential for v(d)j recombination. when the l fibroblast cell line, which has been rendered recombination competent by stable transfection with the rag genes was overtransfected with both wild type rag genes, we found in contrary to an expected increase of v(d)j recombination a reduction by a factor of - . . this decrease was not observed with the mutant rag genes.this result indicates, that there exists an optimal intracellular rag concentration for efficient v(d)j recombination. m. fischer, a. sailer, h. blieler, t. riilicke*, a. aguzzi +, p. autenried and c. weissmann; nstitut fiir molekularbiologie l universitfit ziirich, h nggerberg, ziirich, *biologisches zentrallabor and + lnstitut fiir neuropathologie , universitdtsspital ziirich, zfirich, switzerland the infectious agent of transmissible spongiform encephalopathies such as creutzfeldt-jakob disease in man or scruple in sheep is thought to be prpsc, a posttranslationally modified form of the normal host protein prpc we have shown that mice devoid of prpc (prn-polo) are completely resistant to scrapie. here we report that also heterozygous prn-p o/+ mice with reduced levels of prp c show enhanced resistance to scrapie, as manifested by a long delay in the onset and slow progression of clinical disease. conversely, prn-po/o animals overexpressing prp transgeues exhibit shortened incubation times and accelerated progression of the disease. propagation of the scruple agent is completely abolished in prnpolo animals. high titers of infectivity, about logld units/g of brain, are detected in prn-po/+ mice weeks after inoculation (the earliest time point measured), weeks or more before the animals die of scrapie. in contrast, wildtype animals survive no more than weeks after reaching similar titers of infectivity. a practical implication of our findings is that moderate inhibition of prpc synthesis could mitigate disease progression in spongiform encephalopathies. the ruvb protein has been shown, biochemically and genetically, to be involved in the process of homologous recombination in e. coli. one of its functions is to promote branch migration within holliday junctions formed during the process of recombination. to investigate the mechanism by which ruvb promotes branch migration we have used conventional electronmicroscopy, cryo-electron microscopy, scanning transmission electronmicroscopy, and image analysis to study the interaction of ruvb protein with dna. we observed that ruvb forms multimeric rings on dsdna which encircle the dna. we also observed that such rings tend to localize at the holliday junctions. we propose that ruvb rings can actively move along dna using the energy of atp hydrolysis.this movement continues upon encountering a holliday junction, thus forcing branch migration along the dna. gschwind m. and huber g., pharma division, preclinical research, in some families with early onset aizheimer's disease mutations on the -app gene have been identified which cosegregate with the disease. so far all these pathogenic mutations have been identified on exons and in humans framing the sequence of the -amyloid itself.a genomic clone was isolated from mouse strain c black/ containing the coding regions of the last three exons ( ) ( ) ( ) according to the human sequence. sequencing of all three exons including the large ' non coding region showed a % homology to the previously described balb/c mouse cdna. exon-intron boundaries were determined at the same positions as in human and rabbit genome and the flanking regions in the introns are also very similar, not only is the homology between the human and mouse f$-app very high ( . %) but also the genomic organisation of their genes is nearly identical. therefore homologous recombination can be applied to introduce molecular -app changes and to study their functional consequences on the protein level. we systematically investigated the ability of reca protein to push the dna strand exchange reaction through heterologous regions. we observed that reca can push the strand exchange through substantial heteroiogy (up to bp) when the heterologous insert was placed on the so called proximal end of the linear duplex. in the case of the medial beterology the strand exchange reaction could overcome heterologies of up to about bp. heterology at the distal end was most difficult for reca to resolve, and already bp long insert was completely blocking the progress of the strand exchange. these results, together with earlier electron microscopy studies, allow to propose a molecular mechanism by which reca can exchange strands between partially heterohigous dna molecules. institut for veterin~r-virologie, universit~t bern, ch- bern bovine viral diarrhae virus is a positive-stranded rna virus of the pestivirus group. two biotypes, cytopathic and non-cytopathic in cell culture, can be distinguished. all cytopathic strains characterized so far carry insertions in their genomes. they probably result from a strand-switching activity of the viral rna polymerase during viral replication. to study the switching capacity of the viral enzyme, rna isolated from cells infected with two different strains of bovine viral diarrhea virus were analysed for homologous recombination in an rt-pcr assay. performing the assay we found that fragmented rna present during reverse transcription, the reverse transcriptase enzyme itself and viral rna present in the pcr reaction result in the production of artificial recombination during the assay. it was not possible to detect a homologous recombination activity of the viral polymerase above the background recombination frequency of the rt-pcr assay. our results strongly question the use of the rt-pcr assay for the study of recombination of rna viruses. transpositional rearrangements mediated by insertion sequence (is) elements represent an important source of genetic variation within populations . we analyze dna rearrangements at the level of the complete genome by rflp, using is elements as probes for hybridisation to southern blots samples taken at different time points from independent cuttures of e coli b grown by serial transfer for ' generations and stored at - ~ are used to study structure and evolution of genetic diversity within populations. the seven is elements contribute differently to genetic variation and lead to a succession of mutants affecting the genetic structure of the population. we now search for correlations between rflp patterns and the increase of relative fitness over time observed previously naas t., o~ m, fitch w.m and arber w, ( ) the complete dna sequence of the locusta migratoria mitochondrial genome has been derived from four cloned mtdna fragments. the sequence is . kb in length, contains peptide coding sequences, and genes for trnas and two rrnas. the organisation of the genome shows a strong resemblance to drosophila, the gene order and orientation being the same except for the positions two trnas. this contrasts with the only other non-dipteran insect mtdna to have been sequenced, apls mellifera, which shows differences in the positions of ii trnas. this finding is discussed in relation to the at content of the different genomes. the sequences of the three mtdna genomes are also compared directly and the results related to existing knowledge concerning the evolution of insects. udem, new jersey medical school, newark, nj - , usa nonsegmented negative strand rna viruses are so far not amenable to reverse genetics. the genomes must associate with nucleocapsid (n), phosphoprotein and polymerase proteins to form a functional rnp. as a step towards reverse genetics of measles virus (mv), plasmid vectors were constructed allowing synthesis of rnas corresponding precisely to a mv genome in which all coding and intercistronic regions are replaced by the chloram-phenicol acetyl transferase (cat) coding region, transfection of these negative polarity transcripts into mv infected hela or cell lines gave rise to cat activity which could be serially transferred and massively amplified together with progeny helper virus in cell cultures. both expected mv/cat chimeric mrna and replication product were identified in the progeny. preliminary experiments indicate that only rnas in which the total number of nucleotides is a multiple of six replicate and transcribe efficiently, consistent with the data on natural and modified copyback di rna of sendal virus (calain and roux, j. virol. ( ) ). precise end to end encapsidation of rna, each n protein contacting six nucleotides, might be required. the embryonic tissue specificity of the human cytomegalovirus immediate-early (hcmv-ie) promoter/enhancer (- to + ) was examined by l~-galactosidase staining of transgenic mouse embryos carrying hcmv-ie regulated lacz genes. embryos of three transgemc lines were analyzed between . - . dpc. for all lines, hcmv-ie transcription was primarily confined to subsets of neural and vascular cells. however, extents of transgene expression along the embryonic primary axis were slightly and greatly restricted in two of the lines relative to the third line at all stages analyzed. these differences most likely represent integration site-dependent chromatin constraints that affect levels of hcmv-ie-directed lxanscripfion. together, the three lines can be taken to define a graded potential for hcmv-ie transcription along the anteriorposterior axis of the embryo with greatest permissivity in mid-axial structures which include the dorsal neural tube at the level of the hindbraln and upper cervical spinal cord, the inner ear and vestibular acoustic ganglia, branchial arteries, aortic sac, and lung buds. the cell typespecificity and axially graded permissivity for hcmv-ie transcription in the mouse embryo provide a basis for understanding the pathology and relative frequencies of different types of birth defects caused by congenital hcmv infection in humans. stauffer, y., fford, e. and beard, p., swiss institute for experimental cancer research (isrec), ch-i epalinges, switzerland. hpvi is associated with genital carcinoma. like other human papillomaviruses, hpvi cannot be easily propagated in cell culture, because the viral growth cycle is dependent on the differentiation of its host cell, the keratinocyte. moreover, very little viral capsid protein is found in vivo in infected tissues. the hpvi l and l genes code for the major and minor capsid proteins, respectively. to obtain the viral capsid proteins we made constructs with the l and l genes for expression in e. coli (the pqe system) and in recombinant vaccinie virus (a modification of the phgs-i system) infected cells. the l and l proteins expressed in e. cell contained a histidine tag encoded by the vector. this allowed us to purify the proteins on a sickelaffinity column in order to raise antibodies. these will permit us to detect the presence of capsid proteins expressed from recombinant vaccinia viruses. we expect the l and l proteins expressed together from the vaccinia-based vector to selfassemble to form virus-like particles. to papain-like proteinases. the role of these putative catalytic residues in the activity of the proteinase was studied by site-directed mutagenesis. a minimal proteinase has also been constructed by n-and c-terminal deletion to determine the boundaries of the proteolytic domain, sequence analysis of the c-terminal cleavage product should indicate the site of cleavage. cell entry, uncoat~ng and nuclear transport of adenovirus z . u. f. greber and a. helenius. yale university school of medicine, new haven, ct, u.s.a. to enter cells, viruses must achieve three major goals: transfer their genome across the plasma membrane into the cytosol, target the genome to the replication site and decondense it for replication. adenovirus, an icosahedral nonenveloped particle with a double stranded genomic dna and ii to structural proteins~ enters into human kb cells by a sequential disassembly program during receptor-mediated endocytosi~, acid-dependent penetration into the cytosol, and targeting to the nucleus. during internalization, the fibers, primarily responsible for virus binding to the cell surface, are detached. in early endosomes, the capsid-stabilizing proteins ilia and viii are released. fenton base, a vertexassociated protein implicated in penetration across the endosomal membrane, is removed in endosomes, if penetration is blocked with lysosomotroplc reagents. in the absence of inhibitors, a large fraction of penton base stays associated with the cytoplasmic capsid. the viral dna is disconnected from the shell after proteolysis of the internal protein vi~ a capsid and dna binding component. removal of protein ix, a capsid binding factor, further destabilizes the coat, and the remaining 'stripped-down' virus particles associate with nuclear pore complexes to release their dna-genomes into the nucleoplasm. this stepwise dissociation program is thought to provide the high efficiencies of the entry process needed to successfully deliver the viral dna across multiple barriers to the cell nucleus, the site of replication. ch. schmidt and w. arber; biozentrum der universit~t basel, abt. mikrobiologie, klingelbergstr. , ch- basel bacteriophage p , carried as a single copy plasrnid in e. col~, is widely known for its horizontal gene transfer ability (transduction) in enterobscteria. as a temperate phage, it can lysogenize its host and thereby provides it with accessory functions (restriction of foreign dna by ecop , functions expressed by transposons carried on the p genome or integrative suppression). these properties reflect both symbiotic and evolutionary functions of pi. p regulates expression from its genes by two different mechanisms: trans(:ription of early regulatory genes is repressed by the phage encoded repressors c and bof, whereas transcription of its morphogenetic genes from p specific late promoters is induced by the early expressed gpl . by site-directed mutagenesis the late promoter ps, composed of the - inverted repeat aagttactt and the - box, was functionally characterized. p and its derivatives compare well with several other late promoters of pt with regard to their sequence dependent strengths: the strong promoters ps, pdar and lpr b all share the perfect inverted repeat around position - , whereas the weaker promoters lpr , lpr a, lpr b and lpr contain mismatches in their inverted repeats or the inverted repeat is positioned mere upstream relative to the transcription initiation site. homologies. however, the phages formed two immunity groups. the results may suggest a common genetic trait providing a selective advantage to lysogenic aa. by using a dna substrate with defined gap size we found that human immunodeficiency virus type reverse transcriptase (hiv~rt) was able to perform strand displacement dna synthesis. this activity was not affected first by calf thymus proliferating cell nuclear antigen and replication factor c and second by escherichia coli single-stranded dna binding protein~ which together allow dna polymerase ~ to perform strand displacement dna syrlthesis (podust , v. and h~bscher, u. ( ) nucl. acids res. , . '-azido- ', '-dideoxy-thymidine tripbosphate inhibited displacement completely indicating that dna synthesis is required for this reaction. the hiv-rt p polypeptide alone could perform limited strand displacement dna synthesis, whereas the hiv-rt p polydeptide was completely inactive, likely due to its inability to replicate extensively on a mi dna template, on the other hand the hiv-rt psl polypeptide e~hanced the strand displacement activity of the hiv-rt d subunit at a molar ratio of : mainly by chasing short products into longer ones. furthermore kinetic experiments after complementation of hiv=rt p with kiv-rt psl indicated that hiv-rt psl can restore rate and extent of strand displacement activity by hiv-rt p compared to the hiv-rt heterodimer d /d , suggesting a function of the kda polypeptide, the mouse mammary tumor virus proviral dna contains an open reading frame in the ' long terminal repeat which can code for a kda polypeptide with a putative transmembrane sequence and five n-linked glycosylation sites. this gene is known to code for a superantigen which deletes a specific subset el co + t-lymphocytes in vivo, the superantigen encoded by the exogenous mouse mammary tumor virus of the gr strain acts specifically on v[ bearing t-cells. we produced recombinant vaccinia viruses to express either the complete or a truncated orf protein after infection of primate cells in culture. the complete err gene in mammalian cells leads to the production of a kda protein which is specifically detected with an anti-orf-peptide antiserum. the kda protein can be labeled with d-[ - h]mannose and its synthesis is inhibited by tunicamycin, an n-linked glycosylation inhibitor, indicating that it is a glycoprotein. the truncated orf protein beginning at the second atg of the open reading frame is also modified, but the c-terminal half of orf, starting at the fifth atg (orf ), has the expected size of the non modified polypeptide. pulse-chase experiments indicate that the kda orf g}ycoprotein has a short half-life of about . - hours in this system whereas the orf truncated protein is even less stable; its half-life is about minutes. the cellular localization of the orf protein and the role it could play in vivo are currently under investigation. conclusion was based on a restriction fragment length polymorphism (rflp) with probes of is-elements. the mutants displayed different growth behavior. the evolutionary stability of of these mutants was further tested by competing a mixture of them for ten days. polymorphism was maintained during this competition but two fitter mutants took over % of the population. implications of these results will be discussed in terms of generation and selection of genetic variants for the adaptation of microbial population. mouse mammary tumor virus (mmtv) is produced in the mammary gland of infected females and transmitted by the milk to suckling new-boms. the passage of mmtv from the gut to the mammary gland is still poorly understood, the nature of the tissue tropism of mmtv has not been elucidated, a single receptor present on mammary epithelial and lymphoid cells might serve for the entry of the virus. a cell-type specific entry mechanism could in part be responsible for this phenomenon. alternatively different surlace molecules might be involved in its uptake. we have studied the cell susceptibility to mmtv using a sensitive method of detection of newly acquired exogenous proviral dna sequences, prior to viral integration and transcription. thereby we confirm that mmtv infects mouse, rat and monkey cell lines of different tissue origin in vitro demonstrating the lack of a strict host range specificity in tissue culture as previously described. we have also observed the infection by mmtv of various human cell lines. furthermore various infection susceptibilities are observed for cell lines of a same species. r. cattaneo, p. spielhofer, k. kaelin, m.a. billeter mo/eku/arbio/ogie /, universit~t zorich, hsnggerberg, zorich subacute sclerosing panencephalitis (sspe), a rare, lethal disease is caused by clonally selected defective measles viruses (dmvs) characteristically mutated. this emerged from sequencing of five mv genes from sspe cases and by functional tests of the envelope glycoproteins hemagglutinin (h) and fusion protein if). these expressed h and f variants migrate poorly to the cell surface but maintain some cell fusion activity whereas the envelope associated matrix protein (m) appears dispensable. thus, the spread of dmv genomes through the brain seems to occur by membrane fusion at local cellular contacts, essentially hidden from the massive immune responses. in more than half of sspe cases, the propagated dmvs contain m genes in which up to % of u residues are replaced by cs. such "biased hypermutations" are likely due to the simultaneous conversion of many a residues to inosine in double stranded rna regions accidentally formed, mediated by an ubiquitously occurring adenosine deaminase. similar events at less spectacular degrees have now been observed in functional genes of several negative strand rna viruses. thus, this enzyme might be an important evolutionary driving force for mononegavirates. pull, i., wieland, s., nieder~st, b., keist, r., walter, e., and blum, h.e. department of medicine, university hospital z'tirich infection by hbv-positive organ donors remains a serious problem in transplant surgery. here we present a case of a -year-old woman who received a heart transplant from a hepatitis b surface antigen (hbsag) positive donor. the patient was negative for all hbv markers prior to transplantation. two months after transplantation she became hbsag positive and died from subacute liver failure about seven months later. cloning and sequencing of hbv dna revealed several hbv mutants in the serum of the recipient, each with a bp in-frame deletion in the pre-s region. transfection studies in huh- cells with the predominant pre-s deletion mutant showed a higher replication competence compared to wild-type hbv. sequence analysis of pcr products from serum of the donor showed mainly wild-type hbv dna in the pre-s region without the pre-s deletion. these data demonstrate that mutant viruses accumulate in immunesuppressed patients. specific mutants may play a critical role in the severe or fatal clinical course of hbv-infection in transplanted patients. in non-neuronal cells herpes simplex virus (hsv) establishes a productive lyric infection starting with transcription of its immediate early (ie) genes by corecruitment of a viral protein (vpi ) and a cellular factor (oct-l) onto characteristic oetamer-like sequence motifs ftaatgarat) in the promotors of the ie genes. in neurons, however, hsv establishes latent infections whereby transcription of the ie genes is prevented and the lyric program aborted. our efforts are focused towards determining which neuronal factors are involved in the regulation of hsv ie gene transcription and whether they may be involved in hsv latency or reactivation in neurons. in electrophoretic mobility shift assays (emsa) using mouse brain nuclear extracts we found that neuronal oct factors (noct- , noet- and noct- ) bind to taatgarat sequences from the viral icp and icp promoters. an additional brain protein also binds to these sequences but not to a consensus octamer sequence (atgcaaat) as do neuronal oct factors. emsa assays with mutated taatgarat probes show that the taat sequence is crucial for this binding and we have tentatively named this brain protein taat- . we are currently testing the noct factors in in vivo and in vitro transcription assays to study their involvement in hsv ie promoter regulation. biochemical and functional analysis of a vaccinia virus recombinant containing two copies of the p k gene. schmutz, c., and wittek, r. institut de biologie animate, univei-sit~ de lausanne, ch- lausanne. the most abundant protein of the vaccinia virus envelope is the palmitated k protein. this protein is required for envelopment and subsequent release of virus from infected cells. the amount of extracellular enveloped virus (eev) produced varies considerably depending on the virus strain and host cell line but remains in any case low (< % of the virus production). if p k is a limiting factor for envelopment, its overexpression might be a taeans to increase the production of eev. for this purpose a vaccinia virus recombinant containing a second copy of the p k gene was enginee,'ed. western blot analysis clearly showed increased levels of this protein. titration assays revealed slightly lower yields of both intracellular naked virus (inv) and extracellular enveloped virus (eeu in ceus infected with recombinant virus. unexpectedly, with recombinant virus, the ratio of eev versus inv was significantly lower when compared to wild-type virus infection, despite the higher level of p k expression. we are currently testing whether this is a consequence of a lower production, or a reduced infectivity of eev particles in cells infected with recombinant virus. hanspeter stalder, christian hertig and ernst peterhans institut f~r veterin~r-virologie, universit-st bern, ch- bern bovine viral diarrhoea virus (bvdv) causes acute transient infection in animals exposed to virus postnataly and persistent infection in animals infected in utero in the early phase of gestation. the latter is associated with specific immunotolerance to the infecting viral strain. animals infected in utero may be born normal and remain healthy despite the persisting infection with a noncytopathic biotype of bvdv. mutation to the cytopathic biotype, or superinfection with an antigenically similar cytopathic biotype, results in lethal mucosal disease. we have pcr amplified and sequenced parts of the region coding for the surface glycoprotein e and p , a nonstructural protein associated with enzymatic activities. over a time of one year, virus obtained from an immunotolerant persistently infected animal remained identical in its nucleotide sequence in the analyzed regions. contrasting with this remarkable evolutionary stasis over some - virus generations is the heterogeneity of viral isolates obtained from different persistently infected animals. we propose that the muitilineage distribution of bvdv is the result of antigenic drift in the population of acutely infected animals, combined with evolutionary stasis in immunotolerant animals. in to % of naturally infected goats, the sevedty of disease in infected animals correlates with the antibody titers to the viral envelope protein (enw) and especially to the gp transmembrane podion (tm). in order to analyze linear b epitopes of env, we produced a random library of caev env polypeptides fused to ~-galactosidase and expressed in e. coli. the complete env gene obtained from an infectious clone of caev-co was subcloned in a puc plasmid and dnase digested, random fragments of bp were inseded in ~,gtl dna and expressed in e. coil y . as the first step of b epitope mapping and in order to identify immunodominant group epitopes we used high titer sera from naturally infected swiss goats to screen the library. six immunoreactive clones were obtained and subsequently sequenced. all six mapped to the tm region of env. a fine mapping of these immunoreactive regions was performed using pepscan technology and elisa with synthetic peptides. four epitopes could be identified including the eysteine loop corresponding to an immunodominant epitope in other lentiviruses. these peptides were used in vitro to test the reactivity of sera from naturally and experimentally caev-infected goats and in rive to modulate the immune response of goats to tm. the minute virus of mice (mvm) has a kb linear single stranded dna genome. at both termini, short selfcomplementary sequences are folded into stable hairpin structures. the ' (right) hairpin is a single stem structure with only three unpaired bases within the stem. these unpaired form a bubble structure. we examined the requirement in mvm lytic infection for the bubble structure in the ' terminal hairpin of mvmp. mvmp rf dna containing the entire ' extremity and extending to the hha i site downstream of the axis of symmetry of the ' extremity was cloned into pgem-szf(+). this construct contains more than half of the ' palindrome, however it lacks the sequence information required to form a bubble. murine fibroblast a cells were transfected with this dna and the ' terminal sequence of resulting virus was analysed. a bubble was seen to be generated, however the sequence of nucleotides contributing to the bubble was of n n-viral, plasmid origin. the nucleotides in question were removed from the original plasmid construct and the experiment repeated. virus was obtained as before. sequence analysis of the ' end indicated that both flip and flop forms were present. the corresponding ' hairpin deduced from these two forms contains no unpaired bases at the position where the bubble is normally located in wild type mvmp. the immediate early lie) gene promoters of herpes simplex virus type- share a similar sequence which in some cases overlaps with a binding site for octamer binding proteins. this sequence confers responsiveness to a viral transaetivator, vp . on these promoters, vp combines with multiple cellular proteins to form an activating complex. we set up a reconstituted in vitro system consisting of hela nuclear extract and recombinant oct- /pou domain protein (without the activation domain of oct- ) and vp where we showed that the activation of ie genes is not mediated by oct-i itself. rather, oct- serves to recruit vp that has no intrinsic dna binding capacity. vp , however, possesses a stong acidic c terminal activation domain of - amino acids which is indispensable for the in vivo function of the protein. as this and other acidic activation domains possess only a relatively weak activating capability when we test them in in vitro transcription assays we are interested in investigating the role of this acidic activation domain in vitro i.e. under condition of cell free extracts and on naked template dna. we are using recombinant oct- /pou domain protein and recombinant vp that has been truncated at the c terminus. additionally, we are also testing different truncations of vp in vitro that have been produced in cos cells. furthermore, we are also trying to see whether these different truncations already affect the assembly of the actvating multiprotein complex. we have studied the kinetics of synthesis of transcripts initiated from the vaecinia virus . kd gene early promoter. unexpectedly, after a first burst of rna synthesis early in infection, the promoter showed a second peak of activity in the late phase.reactivation of transcription was neither dependent on the presence of the sequences upstream of the early promoter, nor on the location of the promoter in the genome. reactivation seems to be dependent on virus assembly since it is prevented by rifampicin, that specifically inhibits an early step of viral morpho-g~nesis. analysis of several other early genes showed that reactivation is not unique to the . kd early promoter. analyzing, by in situ hybridization using digoxigenin-labelled riboprobes, the expression of the viral genome relative to that of an array of cytokines including tnf-(z; ifn-% il-i~, il- , il- and gm-csf. initial experiments suggest that viral genome expression is restricted in the inflammatory infiltrates whereas certain cytokines, particularly tnf-oq are strongly expressed in the synovial membranes. overall, the results point to a prominent role of macrophage activation in the maintenance of chronic inflammation and possibly also in the development of arthritic lesions. the core protein of the hepatitis b virus (hbv) interacts with the viral rna pregenome and the reverse-transcriptase.jdna-polymerase to form a core particle which allows for replication of the viral genome. deletion analysis of the core protein revealed, that the arg-rich carboxyterminal domain is required for rna encapsidation and productive viral dna synthesis. we demonstrate, that the duck hepatitis b virus (dhbv) core protein can be derided into two domains comprising amino acids - (n-ter) and - (c-ter), respectively. the co-expression of these two core subdomains leads to the formation of replication competent core particles. furthermore, we demonstrate that the carboxy-terminal domain of dhbv core (c-ter) can be functionally replaced by the corresponding domain of the human hbv. the complete hbv core protein, however, does not form replication competent core particles with the dhbv pregenome and rt. these results imply that the carboxy-terminal part of the hbv core proteins define an exchangeable, functionally conserved domain. influenza ha mediates the adhesion and penetration of host cell membranes. acylation of three conserved cysteine residues in the membrane spanning region of several ha subtypes has been shown. the biological significance of this hydrophobic modification remains to be elucidated. a possible role for the membrane fusing activity has been controversially discussed in the past. removal of covalently bound fatty acid from protein by incubation with hydroxylamine is a commonly applied method. as we shall show for a number of different envelope viruses with acylated glycoproteins, this treatment leads to quite different consequences in a functional seiase depending on the virus used. as an alternative approach to study the biological significance of palmitoylation we expressed wild-type influenza ha and an hamutant lacking the fatty acid binding sites using the baculovirns system. both wild type and fa--mutant ha, after expression in insect cells bind to red blood cells, induce hemolysis, and fuse efficiently with fluoreseently labeled erythrocyte ghosts. hydroxylumine treatment of both recombinant wt-and mutant-ha leads to a loss of hemolysis and of fnsion activity. at least in the ease of fa--i-ia its inhibitory action must be related to some other effect than fatty acid cleavage. these results indicate that hydroxylamine treatment may not always be suitable for the generation of fatty acid free glycoproteius or virus particles for functional studies. gesemann and j.g. nicholls, biocenter univ. basel, basel, switzerland depolarization of leech neurons leads to growth cone collapse and neurite retraction. this response to depolarization is influenced by the substrata on which the cells are growing and is mediated by the influx of calcium (s. grumbacher-reinert and nicholls, , j. exp. biol. : - ; neely, , i. neurosci. : - . the morphologieal changes induced by depolarization of leech neurons growing on leech extracellular matrix extract is accompanied by a loss of microfilaments in the growth cones. leech neurons growing on a different substrate, the plant lectin coneanavalin a (coma), did not retract their neudtes or lose rnierofilaments, but continued to grow after depolarization. we attribute this to the reduced calcium influx during depolarization of neurons on cona (ross et al., , pnas : - ) . increasing the intraeellular calcium concentration by incubating neurons with the calcium-ionophore a , led to cessation of motility and disruption of microfilaments but not microtubutes in growth cones on coma. to investigate the mechanism by which calcium affects the organization of microfilaments we stained neurons with antibodies against gelsolin, a protein that severs microfilaments when it is activated with calcium. we have observed that these antibodies colocalize with microfilaments in leech growth cones, we are now analyzing the nature of this antigen that cross-reacts with the anti-gelsolin antibodies with the goal of establishing its potential role in the calcium-induced disruption of microfilaments in leech neuronal growth cones. this work was supported by a grant from the swiss nationalfond no. . to j.g. nicholls. activation of metabotropic glutamate receptors (mglurs) was studied in voltage-clamped ca cells in rat hippocampal slice cultures using the whole-cell pateh-clamp configuration. in saline containing mm k +, s, r-acpd ( ~tm), a mglur agonist induced an inward current associated with an increase in membrane conductance. the reversal potential, assessed with a voltage ramp protocol from - to - mv and calculated by linear regression, was - . • . mv, and was shifted to - . • . mv when the concentration of external monovalent cations was halved. these results indicate that the conductance underlying this current is selective for cations (mainly k + and na+). the steady-state i/v curve for the s, r-acpd-induced inward current showed a slight inward rectification. in most of the cells, this inward current was followed (or overlapped) by an outward current concomitant with a decrease in a k + conductance (ere v = - . ~ . mv in [k+]o = mm and shifted to - . :t: . mv in [i +]o = ram). this k + current was voltage-independent in the membrane potential range of- to - mv. similar results were obtained with methacholine, a cholinergic muscarinic agonist. the non-specific cationic current, seen only in the presence of elevated extracellular k + concentration, could play an important role during intense neuronal activity. the c-myc protein has been implicated in the regulation of cell growth and differentiation, c-myc specifically interacts with max which in turn forms heterodimers with mad and mxil and homodimera with itself. we present evidence that this network o! proteins is regulated both at the level of relative protein concentration as well as by post-translational mechanisms. in the hematopoietic cell lines u- , ml- and hl- o e-myc mrna and protein are rapidly downregulated after induction o| differentiation whereas max expression is not significantly altered. in contrast the expression of mad is induced with properties similar to immediate early genes, taxi is induced to a lesser degree and with slower kinetics, in an effort to study the regulation of o-myc we have mapped all the major in vivo phosphorylation sites in both c-mye and max. two sites in the n-terminal transactivation domain are important for the regulation of the transforming potential el c-myc both in established cell lines as well as in primary rat embryo tibroblasts. however, no difference in the transactivating potential of corresponding mutants was observed. phosphorylatien of max at sites close to the basic region increases both on-and off-rates of either myc-max hetero-as well as max homodimers to dna. brain research institute, university of z~rich, ch- z~rich, switzerland gaba b and adenosine receptors act via pertussis toxinsensitive g-proteins of the gi/g o class to mediate longlasting inhibition in the cns. this class of g-protein is negatively coupled to the enzyme adenylyl eyclase. our aim was to determine whether the inhibition of adenylyl nyclase mediated by these receptors has functional consequences for electrical signaling in hippocampal neurons. the calciumdependent k + current, i~, underlying adaptation of cell firing, was used as an electrophysiological bioassay for adenylyl cyclase activity, as it is very sensitive to intracellular levels of camp. accordingly, the ~-adrenergic agoniat iaoproterenol ( - nm), that activates g,-linked receptors, reduced the amplitude of i~ by . • . % in voltage-clamped ca pyramidal cells in ttx (n= ). reduction of imm by isoproterenol was curtailed to . • . %, (p < . ) in the presence of baclofen (i ~m), acting at gaba b receptors, or adenosine ( nm). thus, the inhibition of adenylyl cyclase activity due to activation of gaba a and adenosine receptors can modulate responses to other neuretransmitters by an interaction occurring at the level of intracellular signal transduction. furthermore, stimulation of these receptors will tend to enhance i~, suppressing repetitive cell firing. this represents a further mechanism which will result in prolonged inhibition of hippocampal neurons. a study of the possible modulation by nitric oxide (no) of endogenous dopamine (da) release was performed in bovine retina in vitro. isolated half retinas were incubated in kkg at ~ for successive -rain incubations, and da was measured in the incubation medium by hplc with ec detection. when retinas were incubated in the presence of the no-generating compound hydroxylamine ( ttm), a decrease in either basal or k +-( ram) stimulated da release was observed during the nd -rain period. tested under similar conditions, dibutyryl cgmp ( mm) was ineffective. the no synthase inhibitor l-ng-nitro-arginine methyl ester (l-name, gm) increased basal da release during the st and nd incubation, whereas it did not affects the k+-stimulated da release. in addition, its effect were potentiated in calcium-free medium. finally, we have also shown in cell-free experiments that retinal no synthase activity was totally calcium-dependent and blocked by l-name. taken together, these results suggest that endogenous no regulates da release via a cgmp independent mechanism. division of endocrinology, university hospital, ch- geneva while the stimulation of aldosterone biosynthesis by angiotensin ii (angii) in bovine glomerulosa cells clearly depends upon a sustained influx of ca +, the pathway for ca § entry is not yet accurately defined. at least two mechanisms seem to be involved: ) the opening of voltage-operated ca z § channels, and ) the stimulation of a "capacitative" ca + entry regulated by intraocllular ca § stores; however the relative contribution of these pathways to the stimulation of steroidogenesis needs to be determined. two pharmacological tools were used: nieardipine, a blocker of to and l-type ca + channels, and thapsigargin, which releases ca ~+ and therefore induces a capacitative ca ~+ influx. nicardipine ( lam) completely blocked the cytosolic ca + response to k +, which exclusively activates voltage-operated ca . channels, but only partially ( %) the response to angli. in the presence of nicardipine, angll was unable to stimulate a ca + influx aiter depletion of ca § stores with thapsigargin, demonstrating that angli principally stimulates a capacitative ca + entry pathway. in addition, nicardipine completely blocked the steroidogenic response to k +, but only % of the response to angll. thapsigargin-induced aldosterone formation, as the response to angll, was markedly potentiated by low concentrations of k +. in conclusion, this study shows that in bovine glomerulosa cells, angli activates a sustained ca + influx, principally through a capacitativc pathway, leading to aldostcrone formation. this finding could partially explain the poor efficiency of dihydropyridine ca § antagonists for preventing aldosterone secretion. harald holder and john m. lucocq*. institute of anatomy, university of berne, ch berne *depanment of anatomy and physiology, university of dundee, uk dundee dd hn. subjection of hela cells to ~ for minutes leads to a complex activation pattern of map-kinases as revealed with renaturadon gels. exponentially growing hela cells show activallon of a myelin basic protein (mbp) phospborylating kinase migrating with an apparent molecular mass of around kda upon heat shock induction. in contrast, in cells arrested in go by serum starvation for h, essentially three different proteins phosphorylafing mbp are activated subsequent to heat shock treatment. these kinases migrate with apparent molecular masses of around kda, kda and kda. western blot analysis with antibodies recognizing the human boraologues of erkl and erk revealed that considerable pools of erkl and erk show retarded elec/rophnretic migration behavioar indicating that they are phosphorylated and probably activated in heat subjected and growth-arrested hela cells. down-regulation of protein kinase c (pkc) by a prolonged treatment with -o-tetradecanoyl-phothol- -acetate (tpa) did neither change the major activation pattern observed upon separation of cell lysates on renaturation gels nor that one observed on western blots decorated with anti-erkl or anti-erk antibodies. these results suggest that heat shock induced activation of at least erkl, erk and the kda mbp-kinase is not mediated via activation of pkc. this work was supported by swiss nsf grant no. - . . effects of dominant negative glucocorticoid receptor mutants in cell cultures and transgenic animals stefano brenz verca, stefan schneider, sandro rusconi, institut fiir molekularbiologie h der universitat zf~rich, winterthurerstr. , switzerland the glueocorticoid receptor (gr) is involved in a large number of developmental and biochemical processes. recently, we obtained a trans-dominant negative gr mutant (tdn) -called gr[aia] -by a frame shift of the cag-repeat of the rat gr (see poster by hug et al.) . the gr[ala]-mutant shows normal llgand-binding and dna-binding but cannot stimulate transcription (lanz et al., steroids; in press) . modulatable versions of the tdn-mutant have also been created by substituting the hbd of the grmutant with the hbd of sex-hormone receptors. these constructs have only been tested so far in transient cotransfection assays. therefore, we are currently trying to express them permanently in cell lines (rat hepatoma cell line fto- b) in order to verify whether they can affect the transcription level of a known endogenous gr-dependent target-gene, like the tyrosine aminotransferase (tat) gene or a permanently transformed mtv-iacz reporter. so far no rapid test-system has been described, that allows the quantitative analysis of such effects in transient expression assays. we are about to set up such a rapid and generally applicable transient test-system. in the future we plan to express dominant negative gr mutants in a tissuespecific manner in transgenic mice. it will be particularly interesting to investigate their effects on the immune system or on liver functions. these studies investigated growth cone responses m brief local encounters with surface molecules implicated in neuronal pathfinding using chick drg neurons. we tested two interrelated hypotheses: ) discrete points of information (guideposts) override sustained surface information and dominate growth cone behavior and ) guidance molecules provide such information by activating second messengers instead of simply increase adhesivity. the potential guidance molecule lamialn was covalentiy coupled to polystyrene beads in order to mimic guidepost cells. encounters with laminin model guideposts significantly altered the behavior and morphology of growth cones advancing on fibronestin or polylysine. growth cones steered towards theses beads in a saltatory medea once a long lasting contact with a single filopodium had been established. subsequent to a pause at the bead, growth cones advanced with a significantly increased growth rate for the next llmin. the average increase in rate stimulated by bead contact was times the basal rate on polylysine and . times that on fibronectin. positioning of multiple model guideposts along a path and within filopodial reach maintained this bead stimulated rate of outgrowth and directed growth cone advance. the laminin induced pattern of events was totally blocked in the presence of either h or sphingosine, two pkc inhibitors, or neomycin, a plc inhibitor. neither of these inhibitors affected neurite outgrowth on the substrata alone. our results demonstrate the necessity of filopodial contacts, the importance of spatially restricted stimuli and the activation of transducing pathways as key mechanisms in neuronal pathfinding. mechanisms of glucocorticoid receptor desactlvation by homopolymeric alanines martin hug, manuela h&fferer and sandro rusconi, institut fiir molekularbiologie der universitat uz zart'ch, winterthurerstrasse , ziirich, switzerland the rat glucocorticoid receptor (gr) edna contains in its y-portion a (cag-repeat that encodes a siring of gin residues interrupted by arg. a mutant with the frame-shifted repeat which codes now for poly-ala residues (called gr[aia]) is completely trans-inactive, is more cytoplasmitally located in absence of ligund and exhibits dominant-negative properties in presence of ligand (lanz et al., steroids, in press) . we generated gr recombinants with increasing numbers of cag-triplets in three possible reading frames (oligo-gln, -ser or -ala). the gr-oligo[ala]~-~. mutants showed an activity comparable to wild type gr, whereas the groligo[ala]>_ were inactive. the severity of the effect of oligo a.la su'ings seems to be dependent on the position of the repeat along the primary sequence (e.g. progressive decrease of the effects by moving toward more earboxy-position). we have also examined the effects of the oligo[ala] , , , , on gal chimeric factors and found that the inhibition by ala repeats is probably factor-specific. our results made us speculate that possible differences in post-translational modifications may be at the root of the negative effects by the ala repeats. we believe that these effects are due to transient association of the nascent gr[aia] mutant proteins with intraceuular membranes. these hypotheses are currently tested. the activation of steroidogenesls in calcium-clamped bovine glomerulosa cells and its modulation by angiotensin ii and camp, python cp, laban op, rossier mf, vallotton mb and capponi am division of endocrinology, university hospital, geneva, switzedand. the ca +-messenger system plays a crucial role in the regulation of steroid secretion in adrenal zona glomerulosa cells, as it is known to mediate the action of angiotensin ii and potassium ion. in the present study, we used intact isolated glomerulosa cells in which the cytosolic free ca + concentration ([ca +]c) was clamped at various levels with the ca +-ionophore, ionomycin, in order to locate the sites of action of ca +, to determine their sensitivity towards ca + and the modulation by other physiological factors. by measuring simultaneously steroid synthesis and [ca +]c, we show that ca +-clamping ( - nm) induced a concentration-dependent increase in the production of both pregnenolone ( ___ % of the basal level) and aldosterone ( + % of the basal level, ec = nm). by contrast, ca + did not influence the conversion of t-deoxycorticosterone into aldosterone at concentrations up to rim, although at higher concentrations we observed a modest but significant inhibition. in addition, ca +-clamping did not affect the formation of pregnenolone from a freely diffusible analogue of cholesterol, hydroxycholesterol, indicating that ca + acts at a step upstream of cholesterol side-chain cleavage. both angiotensin ii and a membrane-permeant surrogate of camp, dibutyryl cyclic amp, potentiated the steroidogenic response to increases in [ca +]c . in summary, these studies reveal an exquisite sensitivity of the glomerulosa cell steroidogenic pathway toward very small physiological changes in [ca +]c. radtke, rainer heuchel, oleg georgiev, gerlinde stark#, michel aguet# & walter schaffner institut fiir molekularbiologie , universilat ziirlch, z' rich # lnstitut rir molehdarbiologie i, universitiit ziirich, ztirich we have previously described and cloned a factor (mtf- ) that binds specifically to metal-responsive dna sequence elements in the enhancer/promoter region of metallothionein genes. mtf- is a protein of . kda that contains six zinc fingers and multiple domains for transcriptional activation. here we report the disruption of both alleles of the mtf- gene in mouse embryonic stem ceils by homologous recombination. the resulting null mutant cell line fails to produce detectable mounts of mtf- . moreover, due to the loss of mtf- the endogenous metalinthionein-i gene is silent, showing that mtf-i is required for both basal and metal-induced transcription. accordingly, reporter genes containing either synthetic or natural metal-reslxmsive promoters were not transcribed after transfection into the null mutant ceils. cotramsfection of the mtf-i expression vector rescued both basal and heavy metal-induced transcription. these results demonstrate that mtf- is essential for normal metallothionein gene regulation. rat pheochromocytoma cells (pc ) were differentiated with ngf for - days, a time sufficient for the formation of growth cones and elongation of neurites. addition of the phosphatase inhibitor calyculin a (cl-a) ( . . . nm) led to a concentrationdependent collapse of growth cones and retraction of the neurites within minutes. after the retraction, the cell bodies showed the appearance of a grape-like domain opposite to the cell nucleus. they recovered to normal shape within about - minutes if the inhibitor was washed out. the neurite retraction was further analysed using both low-light level fluorescence video-microscopy and fura- single cell microfluorimetry. the onset of retraction started in the central part of the growth cone prior to a complete collapse of filopodia. the basal intracellular free calcium concentration ([ca +] i) remained constant during neurite retraction. as a measure for the viability of cl-a-treated cells, agonist-isduced responses of [ca +] i were analysed. ca + entry across voltage-activated ca -~ channels was inhibited by % after minute treatment with cl-a, while the atp-induced ca + entry via ca *-permeable cation channels was not significantly reduced. however, the onset of the atp-induced rise in [ca +]i started at the grape-like domain. the speed of the ca + wave across the cell body was slower than in control cells. the addition of . gm okadaie acid, a different type of phosphatase inhibitor, caused no cell shape change or neurite retraction within hours. taken the specificity of the two phesphatase inhibiters into account, the observed effects seem to be induced by inhibition of a ppl-class phosphatase. myosin in nematocytes of hydra michel nakano & robert p. stidwill dept. zoology, university of ztirich, switzerland the functions of nonmuscle myosins during cell locomotion are not clear. several models have been presented reaching from very active participation of the molecules in different parts of cells to only minor or no roles at all. there is increasing evidence that the functions of myosin ii (capable of forming bipolar filaments) are quite distinct from the ones of myosin i and generally that the different members of the growing family of myosin-like proteins may not all have identieal functions. we have attempted to demonstrate the occurrence and determine pattern of myosins in tissue of hydra and especially in migrating nematoeytes mainly for two reasons. first, nematocytes are fairly rapidly moving cells which can be studied in vitro and, second, the question is of interest from a evolutionary point of view. we have utilized several antibodies against vertebrate and invertebrate myosins for immunofluoreseenee (confocal laser scanning microscopy) and western blotting studies. in addition to these results findings on the screening of a ~.zap c-dna library are presented. the jaki protein tyrosine kinase is a member of a family of intracellular kinases characterized by having two kinase catalytic domains: a bona fide tyrosine kinase domain and an amino terminally positioned kinase-related domain. recently it has been shown that jaki and the other members of this family (jak , tyk ) are intimately involved in cytokine and growth factor signalling. the presence of two putative nuclear localizing sequences in the amino terminus of the protein prompted us to examine the subcellular localization of this protein. immunohistochemical and biochemical analysis revealed that a part of jaki was localized to the nucleolus. metabolic labelling showed that (a) the jaki protein partitioned rapidly into the nucleus and (b) the nuclear jaki became smaller with time. the size difference was not due to either co-translational glycosylation or phospherylation. these results suggest that part of the jaki protein is rapidly translocated to the nucleoli where it becomes processed. revealed that this clone is expressed predominantly in spleen, mammary gland and thymus as two transcripts, a major species of approximately kb and a minor species of approximately kb. nucleotide sequence analysis revealed a % homology to the porcine syk gene. the syk gene is expressed as a major . kb transcript and has been reported to be lymphoid specific. due to the discrepancy of transcript size and number, we suggest that our cdna clone represents a novel member of this family of ptks. we are currently isolating a full length sequence from a mouse spleen cdna library. in addition, we want to characterize which cell(s) in the mammary gland are responsible for the observed expression. expression and function of g-protein isoforms were studied in differenflaring ( c ) and nondifferentiaflng (( ) subclones of red- rauscher murine erythroleukemia cells. erythroid differentiaflon induced by the combined action of erythropoietin (epo) and dmso was associated with a selective loss (> %) of immunoreactive gai . simultaneously, a iruncated form of g~ accumulated in the cytosol, while the membrane concentrations of gai , gets and gaq/ remained unchanged. nondifferenfiating ( cells contained sigrtificanfly higher levels of most get subunit isoforms that remained unchanged upon epo/dmso treamaent. changes in adenylyl cyclase activity and in intracellular ca ion levels ([ca]i) resulting from activation of g-protein-coupled receptors were monitored for differentiation-dependent effects on transmembrane signaling. adp-mediated changes of [ca]i in native and differenflaflng c cells were consistent with p t receptor coupling to gai . thrombin receptors seemed to couple preferentially to gai in g cells and to gaq in c cells. our results document substantial alterations in g-protein-mediated signaling during erythroid differentiation. to investigate the involvement of protein tyrosine kinases (ptks) in the growth control of the mammary gland, we have used pcr based cloning to identify ptks expressed during normal mammary gland development. this approach led to the isolation of three members of the eph-related family of receptor ptks: myk-;, a novel member expressed predominantly in lung, heart and mammary gland; m-eck, the murine homologue of the human eck gene and mek , the murine homologue of the chicken cek . all three ptks were expressed in a distinct and narrow window of mammary gland development: puberty and estrus cycle. no expression was found either in late pregnancy or in the end differentiated state of lactation. vet-expression was found in the undifferentiated and invasive tumors of transgenic mice expressing the h-ras oncogene. in contrast, no elevated expression of all three genes was detected in the well differentiated, non-invasive mammary tumors characteristic of c-myc expressing transgenic mice. these results suggest that members of this family of ptks are involved in the control of proliferation of the mammary gland but are incompatible with its differentiation. np-tcii is a lymphocyte specific transcription factor (lattion a.-l. et el., mol. cell. biol., , : - a nucleotide receptor that mediates phosphoinositide and phosphatidylcholine hydrolysis by phospholipases c and d, respectively. here we report that atp and utp potently stimulate mesangial cell proliferation. both nucleotides stimulate phosphorylation and activation of map kinase and the up-stream map kinase kinase. activation of map kinase by both nucleotides is dose-dependently attenuated by the p receptor antagonist suramin. stimulation of protein kinase c (pkc) by phorbol ester increased map kinase phosphorylation and activation, and downregulation of pkc partially inhibited atp-and utp-induced map-kinase activation. inhibitors of pkc which display a selectivity for the ca +-dependent isoenzymes (c~, [ , ) , as compared to the ca +-independent isoenzymes ( , e, ~, rl) such as staurosporine and the specific pkc inhibitor cgp did not inhibit nucleotide-stimulated map kinase phosphorylation, thus implicating the involvement of a ca +-independent pkc isoform. in conclusion, these data suggest, that atp and utp trigger the activation of the map kinase signalling cascade in mesangial cells and this may be responsible for the potent mitogenic acitivity of both nucleotides. subcellular ion-gradients in ca signaling p. lipp & e. niggli, department of physiology, univexsity of bern recent experimental evidence has indicated an important role for submicroscopic concentration gradients during ca-signaling in various cell types. in heart muscle cells influx of ca via l-type ca channels triggers ca release from the sarcoplasmic reticulum (sr) and links electrical excitation to mechanical contraction (ec-coupling). we used a ratiometric confocal method (fluo- and fura-red) to follow the intraceuular ca-concentration while ha-and ca-currents (inn, ica) were elicited in the whole cell mode of the patch-clamp technique. both, icand in, were able to trigger ca release from the sr. kinetic differences between ini and ic -induced ca-transients, li-substitution experiments and the existence of a residual inn-induced ca transient in the absence of sr function suggested that these ca signals were mediated by the ha-ca exchange running in the ca influx mode. control experiments showed that ins-induced ca transients did not result from spurious activation l-type ca channels. the significant increase of the na concentration required to activate the ha-ca exchange can only occur provided cytosolic diffusion of na is restricted in the vicinity of the exchangers. this limited diffusion results in significant ha-gradients in the subsarcolemmal space. in conclusion, i~iinduced ca influx may represent a safety factor for ec-coupling while ic, ensures sustained ca-release and is the source for refilling the sr with ca. supported by snf. the regenerative ca-induced ca release (cicr) mechanism is an important amplifier in cardiac signal transducilon. the cicr contributes to the ca transient responsible for mechanical activity, but also generates ca waves propagating within the cytosol. we investigated subcellular ca signals in neonatal rat and adult guinea-pig ventricular myocytes using ratiometric confocal microscopy with a mixture of two fluorescent ca indicators. individual resting myocytes exhibited spontaneous ca release events from the sarcoplasmic reticulurn (sr) that were attributable to three distinct patterns: i) local ca release events without propagation; ll) planar ca waves propagating across the cell; iii) spiral ca waves spinning around a nucleus or shifdng along a subcellular region without entering it. transitions between these patterns were frequently observed, suggesting that a particular release pattern is not the property of an individual cell but reflects the functional state of the sr. the existence of focal release and refractory zones within a single cell indicates that the sr is not uniform on the subcellular level. the sr seems to he a network of elements with distinct functional properties. a suhcellular variability in the positive feedback of the cicr mechanism can account for the observed features of ca signaling. the degree of positive feedback exhibited by a single sr element may depend on its ca content. to characterise the intracellular signalling and regulatory properties of the cloned parathyroid hormone (pth) receptor we have stably transfected two renal epithelial cell lines with cdna's encoding the pth receptor (provided by dr g. segre). the proximal tubular, porcine (llc pk [clone ]), and the distal tubular, madine darby canine (mdck) cell lines were transfected since the culture of either cell line on permeant filter supports leads to the development of polarised epithelial cell layers that mimic the epithelial organisation in vivo. transfection of the cloned pth receptor into each of these cell lines allowed us to examine, independently, the functional properties of the receptors expressed at the apical and/or the basolateral cell surface. while the basolateral application of pth produces a large increase in camp production in both cell lines, the relative efficacy of an apical application of pth differs between the cell types. the transfected llc pk cells exhibit a greater response to apical pth compared to the transfected mdck cells. pth-mediated regulation of intrinsic phosphate transport was also examined in polarised, transfected mdck cells. neither apical or basolateral application of pth affects intrinsic apical or basolateral ha-dependent or phosphate transport activities. similar experiments are being performed with the transfected llc pk cells. a. and preiss, a. biozentrum der universit&t basel, ch basel hairless, a recessive larval lethal mutation, causes dominant defects in sensory organs of adult drosophila flies.manifold genetic interactions indicate that hairless antagonizes neurogenic gene function suggesting an involvment also in neurogenesis. in order to gain insight into its function we have cloned the hairless gene which encodes an extremely basic, very serine rich protein of ii kd calculated molecular weight. no significant homologies to proteins of known function could be identified. therefore, we are concentrating by various means on a structure -function analysis of the hairless protein. firstly, we are testing a series of hairless deletion constructs for rescue capacity and generation of anti-hairless phenotypes after overexpression compared with the wild type gene. secondly, we are analysing hairless protein distribution throughout development, also at the subcellular level. heat induced hairless protein runs at ~ kd, possibly due to phosphorylation which might be intrinsic to hairless function, a hypothesis we are currently scrutinizing. thy- and cd surface glycoproteins are both capable of delivering proliferative signals to t cells upon cross-linking with appropriate antibodies. the tyrosine phosphatase cd is a key regulator of t cell proliferation as it controls the activity of src family kinases p /~k and p fy n. associations of thy- with cd , and of cd with cd have been reported after chemical cross-linking of intact cells and it is likely that such associations constitute part of the structural basis for the t-cell stimulating capacity of thy- and cd accessory molecules. we report that thy- , cd and cd can be specifically crosslinked at the cell-surface and isolated as multimolecular complexes containing a kda protein that is recognized by an anti-bip (grp ) antibody. in vitro kinase assays show the selective association of p ick and p fy n with thy- and cd contained in multimolecular complexes. the multimolecular structure we describe could representa transducing unit incorporating surface receptors and regulators of tyrosine phosphorylation that are stabilized through interaction with the bip protein in the plasma membrane. structure-function-analysis of hairless, a gene involved in drosophila nervous system development maier, d., functional coupling of ppars and trs ijpenberg, a., wahli, w., desvergne, b., institut de biologie animale, lausanne we are interested in a possible functional coupling of thyroid hormone (tr) and peroxisome proliferator-activated receptors (pfar). to this end, we performed cotransfection experiments in nih t cells using cat reporter constructs containing the promoter of either the t responsive malic enzyme (me) gene, or the ppar regulated acyl coa oxidase (aco) gene. the me reporter construct showed very moderate response to the activated mouse ppar (mppar), whereas combination of try and unactivated mfpar potentialized the t induction. a putative ppar response element was identified within this promoter and will be further investigated. the aco reporter constructs were not responsive to trs. in constrast, a tr and t dependent inhibition of the fpar mediated activation was observed. the mechanism of functional coupling of these receptors, which may possibly involve the -cisretinoic acid receptor rxr, will be further investigated by molecular analysis. unliganded steroid receptors form a complex with hsp via their hormone binding domain (hbd). the receptor activation involves a hormone-induced release of hsp . we genetically addressed the role of hsp in budding yeast by analyzing three kinds of hsp (the essential yeast homologue of hsp ) mutants. these mutations affect either the quantity of the protein (low versus normal levels), its integrity (deletion analysis) or its nature (hsp homologues from other species). hsp mutant are examined for their ability to complement a hsp deletion mutant. moreover, they are tested for functional interaction with a hbd. interestingly, a viable deletion of a charged region, suspected to be involved in hsp -hbd interaction, only slightly interferes with hormonal activation of both estrogen or glucocorticoid receptors. furthermore, a short deletion in the last third of hsp leads to the production of a dominant negative mutant which prevents ceil growth at elevated temperature. the ability of various hsp homologues to palliate or not the absence of the yeast protein allows us to define, using chimeras, the regions of the protein which are necessary and sufficient to support cell life. zhang,h.,reynaud, s. and spohr,g. d~partement de biologie cellulaire, sciences iii, , quai ernest-ansermet, ch- gen&ve id cdnas expressed during early xenopus development have been isolated and sequenced. the encoded protein is homologous to the proteins described in higher vertebrates. northern blot analysis reveals that transcription starts soon after mid blastula and decreases to some extent after tailbud-stage. lower levels of transcription are detected also in adult frog tissues such as liver and heart. microinjection into oocytes of in vitro-synthesized myodor/and id-mrna, along with a cat reporter gene whose expression is under the controll of an a -actin promoter supplemented with four e-boxes shows that myo d function is repressed by id. to investigate the importance of negative regulatory sequences we have isolated and characterised the id promoter. as a strategy for identifying cis-regulatory elements, chimeric genes consisting of different ' elements of the promoter were attached to the cat coding sequences and microinjected into embryos. we are studying the tissue distribution of the rat peroxisome proliferators activated receptor (ppar~), a member of the nuclear hormone receptors superfamily, during development and in adults. high levels of ppar~ mrnas are detected in adult liver, kidney and heart. muscle, stomach, and intestine show moderate amounts of the ppar~, whereas brain, testis and lung present a very low expression. during postnatal development, we observe a continuous increase of the ppar~ expression in the kidney, in contrast to a steady level in the liver. anjard, c., groux, b ., gamboni, s. and reymond, c.d., institut d'histologie, unil, rue du bugnon , lausanne. the camp dependent protein kinase (capk) from dictyostelium is composed of a single catalytic (c) and a single regulatory (r) subunit. the c subunit we characterised, pkac is about twice the size of its mammalian counterpart. we showed that it possesses all properties of a catalytic subunit. it associates with the r subunit in absence of camp, it copurifies with capk activity and an increased activity is observed in cells over-expressing pkac. furthermore, we dissected its role during development and cell differentiation. overexpressing pkac under cell type specific promoters allowed to show the requirement for its activity during spore differentiation. pkac seems to play a more complex role in prestalk cells. we are now dissecting the functional regions of the pkac protein, making use of the known tertiary structure of the c subunits of mammalian enzymes. dna binding properties and stimulation of gene transcription of baculovirus expressed xppar~. hihi, a.k, mermod, n., medin, j., ozato, k. and wahli, w., inst. de biol. animale, uni lausanne, ch- lausanne. lab. of mol. growth reg., nih, bethesda, md, usa. the peroxisome proliferators activated receptors (ppars) are members of the steroid/thyroid nuclear receptor superfamily. so far, they have been found in amphibians, rodents and humans. in xenopus laevis, subtypes (xppara,~.y} have been isolated. in order to study the mode of activity of the ~ form, the xppar~ gene product was overexpressed using a baculovirus expression system. the nuclear protein obtained has the correct molecular weight of kd. gel shift assays showed that nuclear extracts containing the recombinant protein are able to specifically bind the 'ppar response element' which is a direct repeat of the core aggtca motif. this dna binding activity is potentiated by another nuclear receptor, the mouse rxr~ and is inhibited by phosphatase, suggesting a role for phosphorylation in ppar binding to dna. a functional approach, using an in vitro transcription system, was chosen to define ppar and rxr action on transcription stimulation, as well as the role of their respective activators. arachidonic acid ( . - ram) induces various patterns of ca +i signal in bronchial smooth muscle cells, as revealed by single cell dynamic video imaging of fura- loaded cells. most frequently, ca +i oscillations were observed, although other patterns (a single ca +i transient; a single peak followed by a sustained elevated level, or a sustained ca *i elevation solely) were occasionally seen. the amplitude of ca *i oscillations was related to the concentration of arachidonic acid. the frequency histogramm was noticeably shifted to higher frequencies by nordihydroguaiaretic acid (ndga, . rtm, a lipoxygenase inhibitor), whereas it was markedly shifted to lower frequencies by indomethacin ( pm, a cyclooxygenase inhibitor) and by , , , -eicosatetraynoic acid (etya, ~tm, a lipoxygenase and cyclooxygenase inhibitor). these observations suggest that: ( ) arachidonic acid per se elicits ca + i oscillations in these ceils; ( ) cyelooxygenase activity products modulate the frequency of these oscillations. promoter analyses of a growth factor regulated gene t. triib, m. kalousek, r. kessler, and r. klemenz dept. of pathology, university hospital, ziirich stimulation of quiescent cells to enter the cell cycle results in altered gene expression. some of the first genes to be induced (immediate early (i.e.) genes) encode transcription factors which are thought to pass on the mitotic signal to the delayed early (d.e.) genes. we have analysed the promoter elements required for growth factor mediated induction of the d.e. mouse gene t which encodes a secreted glycoprotein of the immunoglobulin superfamily. a bp region located between . and . kb upstream of the transcription start site is sufficient for growth factor mediated inducibility. it contains an ap- binding site which is absolutely required for t gene expression. it is surrounded by e-boxes. at least of them are essential for efficient gene induction. thus the i.e. proteins encoded by the fos and jun gene family which form the ap- complex can activate the d.e. gene t in collaboration with helixloop-helix transcription factors binding to the e-boxes. the increase of radiolabelled inositol phosphates ([ h]ips) production in agonist-stimulated human airway smooth muscle cells (asmc) was determined by hplc. in order to observe the role of calcium, pkc and pla on plc activity during agonist stimulation, the effects of pharmacological agents (able to alter calcium, pkc and pla ) were tested on ip production elicited by a sec stimulation with histamine. the inhibitor of pkc staurosporine increased, while pma (an activator) decreased the histamine-stimulated production of [ hi , , -ip and of its degradation products. an inhibition was also observed with the two pla inhibitors, quinacrine and p-bromophenacyl bromide. in calciumfree buffer, no difference in the ip increase was shown, but an inhibition was observed when thapsigargin, an inhibitor of the ca +, mg +-atpasc of the sarcoplasmic reticulum, was added in the same conditions. the calcium ionophore ionomycin increased the lp production in presence of external calcium, whereas it completely blocked the stimulation in calcium-free buffer. the results suggest that plc activity, in human asmc, is modulated by a positive feedback of calcium and by a negative feedback of pkc and pla . regulation of hormone-stimulated calcium signals m. boolman, afrc, laboratory of molecular signalling, dept. zoology, cambridge, uk stimulation of cells with hormones that activate inositol , , -trisphosphate (insp~) formation, leads to an increase in the intracellular ca . concentration ([ca +]~). these hormone-stimulated [ca § signals have a complex temporal and spatial regulation, with the [ca *]~ increase often taking the form of a series of repetitive spikes or oscillations, arising from a steady basal level. the spatial counterpart of a [ca +]~ spike is a wave, where [ca § is first elevated in a localised region of a cell, and then spreads throughout the cell in a regenerative manner. the [ca~*]~ spike frequency is modulated by several environmental factors including hormone concentration, extracellular calcium and thiol reagents. current evidence suggests that a cell can interpret these complex [ca +]~ changes to regulate a diverse range of cellular activities. although many of the biochemical steps between hormonereceptor activation and the [ca § response are known, the precise mechanism generating these complex [ca +]~ signals is unclear. in order to understand this mechanism more clearly, we have investigated the regulation of insp~-mediated ca ~ § release from intracellular stores in intact cells. our current data suggests that under certain conditions, the insp -sensitive intracellular stores behave as functionally discrete units, and that during a ca § spike these stores become functionally coupled by the diffusion of ca z+ from one store to the next, triggering a regenerative ca + release. phenylarsenoxide as a tool for identifying proteins involved in the secretory process wiedemann. c., schaefer, t.,*gitler, c., burger, m. m. friedrich-miescher institut, p.o.box , ch- basel "department of membrane research and biophysics, weizmann institute of science, rehovot , israel phenylarsenoxide (pao) at btm blocks exocytosis in chromaffm cells of the bovine adrenal medulla. this inhibition can be reversed by small dithiois, such as dithiothrcitol or , ~limercaptopropanol. because trivalent arsenicals interact specifically with dithiol containing proteins, we conclude that dithiol-proteins do play an important role in regulated secretion in chromalym cells. to identify dithiol-proteins putatively involved in the secretory process, we compare pao-binding proteins from chromaffm and pci cells and from fibroblasts as well as from sulx:ellular fractions by -dimensional gelelectrophoresis. the proteins are identified by radioactive pao bound to the dithiol in a complex that witltslands sds-page. affinity chromatography on pao-scpharose with various protein fractions is used to purify the dithiol-proteins of interest. enzymatic activity, e.g. kinase or phosphatase activity, can be measured in the eluates of such a column to give further hints at the possible function of dithiol-proteins. rac-pks (~elated to pka_ and pkcc protein kinnses) represent a serine/threonine kinase family whose catalytic domain is closely related to those of protein kinases a and c. they contain a ph (pleckstrin homology) domain n-terminal to the catalytic domain. in addition, rac-pk~ was shown to be a proto-oncogene. in order to investigate the role of rac-pk in cellular signallimg we undertook genetic and biochemical analysis of the drosophila homolog (drac-pk). the drac-pk gene encodes multiple classes of transcripts. antisera raised against the drac-pk recombinant protein specifically recognize distinct molecular weight forms ( , and kda). all forms are expressed throughout development and are both maternally and zygotically regulated. the drac-pk gene is localized at b -i region of the third chromosome, betwee~ the pannie~ and the stubble genes. universit de lausanne, rue du bugnon , lausanne when spinal meninges homogenates were incubated with reduced glutathione (gsh), a cofactor of prostaglandin (pg) biosynthesis, [ c] arachidonate (aa) was bioconverted into a major and a minor product which comigrated on tlc with pge and pgd respectively. in absence of gsh: ) pgd could not be detected; ) the level of pge was dramatically reduced but surprisingly counterbalanced by accumulation of an unusual [i c] aa metabolite which exhibits particular traits of migration on tlc and of retention time on hplc. this aa metabolite: ) does not correspond to a degradation product of pge ; ) crossreacts with pge antibody; ) fits the conditions required for a hydroperoxy pge (chemical degradation into pge by hydroperoxyde reducing reagents such as snc or gsh-hemin). it is therefore inferred that depletion of gsh favours accumulation of hydroperoxyprostaglandins which would participate to oxidative injury. the ecdysteroid receptor (ecr) and ultraspiracle (usp) from both, chironomus tentans and drosoplu'la melanogaster, were expressed in e. coil as fusion proteins with glutathione-s-trsusferase. the identity of the expressed recombinant proteins was confirmed by western blot analysis. the drosophila ecr binds to its cognate hormone response element as a heterodimer with usp as a partner. we now want to compare the capabilities of the two receptor partners from both insect species in regard to dna binding and heterodimerization by means of gel mobility shift assays. the use of naturally occurring andartificial hormone response elements will allow to define a hormone response element consensus sequence for ecr-usp heterodimers and, if existing, for ecr and usp homodimers, respectively. for other members of this receptor family it was shown that the dna binding domain and adjacent sequences alone sufficiently defines response element specificity for both homo-and heterodimers. the dna binding domains of ecr and usp from the two species show a high similarity ( % and % identity, respectively). the corresponding sequences were selected for overexpression in e. coli. in combination with immtmoprecipimtion of receptor-dna complexes and pcr amplification steps, the expressed proteins will be used for the detection of naturally occurring hormone response elements within genomic dna. university of fribourg, ch- fribourg. various agonist-induced cell responses in neutrophils and fibroblasts, such as chemotaxis and cytoskeletal rearrangements, have been shown to parallel the synthesis of ptdins( , , )p . but the importance of this rise is not clear. we show here that wortmannin blocks pdgf-induced production of ptdins( , , )p in human foreskin fibroblasts with an ic of about nm. a similar inhibition was observed in in vitro assays (ics =lnm) with pi -kinase ii~nunoprecipitated by antibodies directed against the kd subunit (p ). on the other hand, wortmannin did not affect pdgf-mediated phosphorylation of p , indicating the correct interaction of p with the pdgf-~ receptor. the pl /p complex of the pi -kinase remained intact in the presence of ~m concentrations of wortmannln. these results are consistent with a direct, specific inhibition of pi kinase by wortmannin at concentrations relevant for its previously reported effects on cellular responses. when stimulated with pdgf, human foreskin fibroblasts form circular membrane ruffles rich in filamentous actin. the fact that wortmannin inhibits these pdgf-mediated aotin rearrangements suggests the need of pi -kinase activity as a signal for this cell response. caicineurin is a calcium/calmodulin-regulated protein phosphatase which is comprised of a catalytic subunit a and regulatory subunit b. although calcineurin was discovered in brain, the calmodulin stimulated protein phosphatase which has caicineurin like structure has been described in other tissues. the protein structure show that the calcineurin b is very conserved in different tissues and species, using anti-calcineurin b monocicnal antiserum, the tissue extracts from rat have been explored. immunoreactivity was obverved in all tissues, although its intensity was different. the highest intensity was found in brain. spleen, heart and thymus had an intermediate level intensity. the results were supported by the quantitative measurement of calcineurin. the caicineurin concentration was to fold higher in brain than that in other tissues. furthermore, the distribution of the calcineurin activity in rat tissue was studied using dephosphorylaticn assay of calctneurin. the highest caicineurin activity was found in brain too, but was only to fold more than that in other tissues. the specific activity of calcineurin was different in tissues. these results indicate the caicineudn activity might be regulated in tissue specific fashion. burst and cytoskeleton arcaro a. and wymann m.p., institute of biochemistry, university of fribourg, ch- fribourg chemoattractants like n-formyl-met-leu-phe (fmlp) stimulate in neutrophils a rapid and transient increase in the levels of ptdins( , , )p (pip ) which is due to the activation of ptdlns( )-kinase. pip has been proposed to be a second messenger molecule, but its cellular targets are presently unknown.here we report that pretreatment of human neutrophils with wortmannin inhibits the fmlp-etimulated production of pip in a dose-dependent manner (ic = nm). furthermore, ptdins( )-kinase activity immunoprecipitated from resting cells is totally abolished by i nm wortmannin (ic = nm). these results show a potent and direct effect of wortmannin on ptdins( )-kinase. wortmannin also inhibits the respiratory burst of neutrophils at nanomolar concentrations, which suggests that pip is involved in the signalling pathway controlling activation of the nadphoxidase.when pretreated with wortmannin and stimulated with fmlp, neutrophils display oscillatory changes in factin content that correlate with oscillations in cell ~hape. this, and the inhibition of ptdins( )-kinase, suggest a modulatory role for pip in cytoskeletal rearrangements.we are currently investigating the effects of pip on the functions of gelsolinl a protein that is proposed to mediate actin polymerization and can be regulated by polyphosphoinositides and ca +. matthias gstaiger, walter schaffner and christopher hovens institut f'tir molekularbiologie ill der universitat z'tirich, ch ziirich the octamer motif atgcaaat plays a central role in mediating the activity of a number of both lymphoid specific and ubiquitous promoters and in the immunoglubulin heavy chain intronic enhancer. the activity of thils motif in lymphoid cells is presumably mediated by the lymphoid cellspecific factor oct- a. when ectopically expressed in non-lymphoid ceils, oct- a is not sufficient to mediate enhancer activity of a multimerized bp enhancer core element derived from the immunoglobulin heavy chain intronie enhancer, a segment, which is however highly active in b-cells. this and other findings support the supposition that additional b-cell specific factor(s) can account for the specificity and extent of the octdependent transcription observed in lymphoid cells. to further our understanding of the molecular mechanisms involved in mediating lymphoid-specific expression, we have employed the yeast two hybrid system to identify human proteins capable of physically associating with human oet- a. to this end, a oct- a expressing yeast strain has been constructed which bears two integrated reporter genes, his and lacz under the control of the octamer motif. transformation of this strain with a cdna library has allowed us to isolate clones interacting with oct- a. we are currently analysing these candidates to determine the veracity of these interactions in higher enkaryotic cell background. in general, untransformed nuclear hormone receptors are predominantly confined to either the nucleus or the cytoplasm of a cell. receptors of the latter class are translocated to the nucleus upon interaction with ligand. our studies of the use of the ecdysteroid receptor complex from insects, consisting of a heterodimer formed between ecr and usp, as a tool for target gene activation in vertebrate cells have revealed a novel mechanism of nuclear translocation of ecr that does not depend on hormone action. after transient transfection of respective expression plasmids into hela or cv- cells, the subeellular distribution of the individual receptor types was analyzed by indirect immanofluorescence staining. while ecr alone was detected in both cellular compartments, nucleus and cytoplasm, usp was predominantly found in the nucleus of either host cell. cooxpression of both ecr and usp resulted in an efficient transloeation of ecr into the nucleus. usp appears to trap ecr in the nucleus, presumably by the formation of a heterodimeric complex. thus, heterodimer formation of ecr and usp not only appears to be required for high affinity binding to cognate hormone response dements (and subsequently for transactivation of an adjacent target gene) and for binding to ecdysteroid but in addition for an enhanced translocafion of ecr into the nucleus. activation of rodent macrophages with cytokines or bacteria is accompanied by the induction of a ca +-independent nitric oxide (no) synthase which plays a key role in antimicrobial and antitumoral activity. firm evidence for expression of a similar enzyme in other mammals or in man has been lacking. we now show that bovine bone marrow-derived macrophages produce nitrite in an l-arginine-dependent manner upon stimulation with heat-killed grampositive or gram-negative bacteria. homologous interferon- and tumor necrosis factor-~ induced little no -production, but primed macrophages for enhanced n -production induced by salmonella dublin or lps. this is one of the first demonstrations of no -production by nonrodent macrophages and encourages a search for an involvement of reactive nitrogen species in the killing of parasites or tumor cells by macrophages from mammals other than rodents. lectins are used in many analytical methods to study the carbohydrate structure of glycoproteins. we report here a technique which combines the high resolution of two-dimensional gel electrophoresis ( -dpage) to separate plasma proteins, the specificity of lectins to bind carbohydrates and the sensitivity of chemiluminescence. this method is compared with that of a commonly used colorimetrio reaction. since the reference plasma protein map obtained by -d page is available (i), the technique described here allows a quick and specific identification of plasma glycoproteins. therefore any ma j or glycosylation modification which may happen in some diseases can be detected. (i) electrophoresis ; : - . this work was supported in part by fcar (quebec, canada). in our laboratory we use genetic and biochemical approaches to study translation initiation, in particular the initiation factor eif- a in yeast. this factor from higher eukaryofic cells is known as an rna dependent atpase and functions as a rna helicase together with another initiation factor, elf- b. it belongs to a family of putative rna helicases, the d-e-a-d proteins. to find new genes involved in translation initiation, suppressors of a temperature sensitive eif- a mutant were isolated. one of the suppressors (stm ) is a single copy gene which encodes a protein resembling the human translation initiation factor eif- b. disruption of the gene is not lethal to the cell, but affects growth. polysome analysis of strains with a deleted stm gene have shown that this new gene is also involved in translation initiation. it carries six repeated sequences of amino acids of unknown function and has -like the human eif- b -a rna binding domain. the second suppressor (stm ) is also a novel gene. it encodes a large protein which shows no homology to other proteins present in the data libraries. it contains high portion of charged amino acids and is rich in glutamines and serines. its function in translation initiation is currently under investigation. tobacco translationini~ationfactore~ aisencoded by a large anddiverse genefamily karl brander, therese mandel, isabelle lutziger, george owttrim and cris kuhlemeier, institute of plant physiology, university of berne, altenbergrain , ch- berne using heterologous probes we isolated cdnas coding for translation initiation factor eif- a from tobacco, eif- a is encoded by a large multigene family of which at least nine members are expressed in leaves and most if not all other organs of the tobacco plant. while screening genomic libraries we found several additional genes. two of them code for genes highly homologous with eif- a throughout the coding region, but with changes in the gkt, ptrela and dead-box motifs. a third gene is expressed exclusively in the developing male gametophyte. this eif- a-related gene may have a role in the well-documented regulation of translation in pollen. in order to study the function of these genes we have begun altering their expression in transgenic tobacco plants. seauence reauirements for a non-aug protein initiaf~jon non-aug initiation codons are still infrequent and were initially observed in animal viruses, but a number of cellular examples have now also been reported. several years ago we reported the use of an acg in the p/c mrna of sendal virus (sen) to initiate the non-structurar c' protein. we recently described a c' protein in the human parainfluenza virus type (hpiv ), which is initiated from a gug. in both cases, the c' protein is an n-terminally ebngated form of the c protein which is initiated at the second aug, in the + reading frame relative to the first aug. this aug is used to initiate the p protein, an essential component of the viral rna polymerase. unlike the acg in sen, the gug in hplvt is clearly not a weak start codon since c' is the most abundant protein expressed from this mrna both in rive and in vitro. it appears that not any upstream gug in an optimal context will function as a start codon. for instance, the p gene of hpiv cannot express a c' protein (the orf is closed by stop codons) but it does contain a gug in an optimal context upstream of the p protein aug, which could encode a p' protein. nevertheless, we have been unable to demonstrate that this gug functions as a start cedon. with the use of chimeric mrnas between hpivl and , we have shown that positions + and + (the first g of the gug triplet being + ) are the major determinants of the efficiency of gug as an initiaton codon for protein synthesis. we are currently investigating the possibility that these nucleotides may increase the initiation rate of weak non-augs such as the acg of sen. biological activity of the heterodimeric subunit srp / of the signal recognition particle is retained in fusion proteins fabrice bovia & katharina strub, d partement de biologie cellulaire, universit de gen~ve, ch- gen~ve the targeting of secretory proteins to the membrane of the endoplasmie reticulum is mediated by a cytoplasmic ribonucleoparticle, the signal recognition particle. it is composed of proteins and f rna molecule (srp rna). the smallest proteins srp and srp are required for the translational control function of srp. it effects a specific arrest in the biosynthesis of er-targeted proteins. these polypeptides bind to the srp rna exclusively as a heterodimer (srp / ). we have constructed single-chain variants of this dimer using a short peptide linker of aa to connect the c-terminus of the kd subunit to the n-terminus of the kd subunit ( - ) and vice versa ( - ). we found that both proteins bind srp rna with a similar efficiency as the wild type protein. glutaraldehyde cross-linking experiments and sedimentation analysis indicate that the fusion proteins fold into a similar structure as srp / and bind srp rna as a monomer. furthermore, they can functionally replace srp / in the elongation arrest and translocation assay. since the fusion proteins are circular permutations of each other, we can conclude from this results that n-and c-termini of both proteins have no essential role in folding, rna-binding and in mediating their biological activity. furthermore, the possibility to express an oligomeric protein as a single polypeptide facilitates the analysis of its functions as well as its structure. a s - studer, r. and hunziker, p. e., iochemisches institut der universit~t z rich, ch- z rich metallothioneins (mts) are small cys-rich proteins which are inducible by a variety of agents such as bivalent transition-metal ions, tumor promoters, cytokines and hormones. the biological role of mts is still unclear. initially postulated to serve in the sequestration of the toxic heavy metals such as cadmium and mercury, they are now believed to play a major role in the cellular metabolism of essential metals such as zinc and copper. to explore this function further we have now established a method to quantify basal mt production in several human cell lines. thus, cellular isomt contents were monitored by h.pj.c, and the rate of synthesis was followed by the incorporation of ~ss-cysteine. the results show that maximum mt synthesis and accumulation occurs when the cells enter the exponential growth phase. with increasing celldensity the mt content decreases progressively until confluence is reached. in correlation with preliminary measurements our results suggest a close relationship between the mt content, the cell growth rate and the intracellular abundance of zinc. vincent bernard, adrian etter, heinz tobler and fritz mtiller. institute of zoology, university of fribourg, fribourg (switzerland). the nematode ascaris lumbricoides undergoes chromatin diminution which causes a loss of dna in all somatic ceils. we have identified a ribosomal protein (rp) gene (coding for alep = ascaris lumbrieoides eliminated protein ) which is located within the germqine specific material. alep shows strong homologies with the ribosomal protein s (rps ). the transcription of its gene takes place only in the germ-line. d-gel electrophoresis on germ-line and somatic purified ribosome fractions shows that the alep protein (rps ) is present only in the germ-line ribosome. does the alep germ-line specific protein have a homologue in the somatic ribosomes? we have cloned a homologue of alep from ascaris somatic tissue named rps ', we are currently studying the expression pattern of this rps '. these results indicate the existence of a developmentally regulated ribosome heterogeneity between the germ-line and somatic ceils. what is the function of rps ? since ascaris is not suitable for genetic analyses, we isolated rps from the nematode caenorhabditis elegans. the c. elegans rps cdna shows only % homology at the nucleic acid level. rps is located on the c. elegans chromosome iv. the identification of a rps mutant in c. elegans will indicate its function. favre, d, l, pavlovic, j and michel, m.r. t institute of medical microbiology, university of beme, ch- berne. institute for immunology and virology, ch- ziirich we have successfully purified the recombinant c (recc) protein from spodoptera frngiperda (sf ) insect cells which were infected with acnpv-c (a). the recc protein retained the biological activities of native c protein obtained from wild type sfv (b). our results suggest that the c protein is responsible for the cellular shut-off of host protein synthesis by inhibiting the initiation of translation. we are currently investigating the molecular mechanisms involved in this shut-off. tif is a new eukaryotic initiation factor which shows % identity with the sequence of mammalian translation initiation factor eif- b. the tif gene is not essential for growth; however, its disruption results in a slow growth and coldsensitive phenotype. in vitro translation of total yeast rna in an extract from a tif gene-disrupted strain is reduced as compared to a wild-type extract. the translational defect is more pronounced at lower temperatures and can be corrected by the addition of purified yeast tif ,or mammalian eif- b. in vivo translation of ~galactosidase reporter mrnas with varying degree of rna secondary structure in the ' leader region in a tif gene-disrupted strain show preferential inhibition of translation of mrna with more stable secondary structure. chloroplast protein synthesis requires the import of elongation factors ef-tu (tuf-gene) and ef-g (fusgene). it seems that the expression of both genes is light regulated. we recently cloned and sequenced a chloroplast specific nuclear fus gene in soybean (biochim.biophys acta : - , . further analysis revealed that a second very similar (sequence, anatomy) nuclear fus- gene exists which, however, shows strong sequence diversion in the 'trailer region. several cdna clones were partially sequenced but sofar only transcripts from the fusi gene were retrieved. we currently study the promoter regions of either gene to test possible differential expression of fus genes. comparative mapping and sequencing studies of the two genes in the "-region indicate that a major dna insertion occurred distal to the coding part of both fus genes what may also influence gene expression. the rna-binding domains in the kd and kd subunits of the signal recognition part~clehavea putative~-h~licalstructure. nazarena buiand katharina strub dpt de biologie cellulalre, science ill, universit gen~ve the signal recognition particle (sr ), a cytoplasmic ribonucleoprotein is important for targeting nascent secretory proteins to the endoplasmic reticulum. srp and srpl are constituents of srpandare, together with the alu sequences of srp rna, required for the elongation arrest activity of the particle. srp and srpi form a heterodimer in the absence of the rna and bind the rnaexclusively as a heterodimer. the primary sequence of srp and srpi revealed that both proteins are highly basic and lack structural similarities to other characterized rna-binding proteins. we have therefore been interested in characterizing the molecular nature of rna-protein interactions. the analysis of n-and c-terminal deletion mutants of the proteins have shown that both proteins contribute to the formation of an rnabinding pocket, the rna-binding and dimerization domains in both proteins, are found in regions that have a predicted ~~helical structure. in sp~pi , this u-helical region is located betwen aa and i at the c-terminus; it contains the rna binding and dimerization domains adjacent to each other. our results support the model that the hydrophobic face of the putative u-helix is implicatedinprotein-protein interactions and the positively charged face in rna-protein interaction. in sp~pg, the two rna-binding domains found n-and c~ terminally, also have a predicted amphipatic s-helical structure. it has been proposed that the decline in protein synthesis observed in aging organisms may result from a decrease in the protein synthesis elongation factor ef-la. john shepherd's finding that transgenic drosophila melanogaster carrying an additional copy of the ef-la gene have an extended lifespan further indicated that the ef-la gene may play an important role in determining longevity (shepherd et al., ) . in order to test this hypothesis, we have quantitated ef-la mrna, ef-la protein, as well as catalytic ef-la activity in john's flies. young and aging ef-hx transgenic (ef) and control lines (( ) were examined. because the the additional ef-lc~ copy is under the control of the hsp promoter, flies were aged either at ~ or at ~ the results show that transgenic ef flies do not express more ef-lct than the control flies, neither at the rna nor at the protein level. the question arises, whether the lransgene can be induced at all. this was tested by doing rnase protection assays. transgenlc message can be detected only in ef flies heat shocked at ~ but not in ef flies grown at ~ nor in control flies at ~ from this experiment we conclude that the transgene is indeed inducible, but is not expressed in detectable amounts at ~ we conclude that the difference in the lifespan does not result from the overexpression of the ef-la transgene. phenobarbital (pb) induces the expression of liver enzymes involved in the metabolism of drugs and other foreign compounds (xenobiotics). these enzymes include several cytochromes p , nadph:p reductase, aldeyde dehydrogenase, epoxide hydrolase, udp-glucoronyltransferases and glutathione-s-transferases. many other proteins not yet recognized may however be induced by pb. our first approach therefore is aimed at identifying gene products which respond to pb treatment. as model systems we are using either chicken embryos (induction in ovo) or primary cultures of chicken embryo hepatocytes (induction in culture) (althaus et al., jbc pp , . to differentially display mrna of induced vs non-induced cells we are using a rt-pcr technique with sets of random primers. electrophoretic separation of amplification products allows to identify mrna species which are induced (or decreased) following treatment. data obtained from these experiments and sequence information revealed from extracted pcr products indicate that pb treatment results in the transcriptional avtivation (or repression) of a variety of so far unknown genes. antithrombin -binding heparan sulfates from ovarian granulosa cells hosseini g., de agostini, a., clinique de st rilit , hcug, gen~ve. at the time of ovulation, several serine proteases of the plasminogen activator and coagulation cascades are activated in the ovary. these proteolytic activities are tightly controlled in time and space, and the heparin-activated serpins plaminogen-aetivator-inhibitor- , protease nexin- and antithrombin (at) are present in the follicular environment. we have observed that granulosa cells produce a subset of heparan sulfate proteoglycans that bind and activate at. these at-binding heparan sulfates (ahspgs) endow the vascular endothelium with antithrombotic properties. we are now examining the role of ahspgs in the extravascular compartment formed by the ovarian follicle. using i-at binding assays, we have detected ahspgs on granulosa cell surfaces and culture media, in amounts comparable to those found for endothelial ceils. after h of stimulation by the gonadotropin fsh ( - ng/ml) granulosa cells increased the amounts of ahspgs they released in culture media by - -fold, while keeping their cell surfaceassociated ahspgs constant. analysis of s-labelled hspgs revealed that heparan sulfates constitute about % of the surface-associated and % of the soluble granulosa cell glycosamineglyeans. finally, using i-at autoradiography on rat ovary cryosections, we have localized ahspgs on the granulosa cell layers of large antral follicles, while ahspgs could not be detected in smaller follicles. these data suggest that granulosa cell ahspgs could be critically located to modulate proteolytic activities in the changing environment of the growing follicle. division, cibabasel, switzerland. the development of chelators which can mobilise excess storage iron (fe) and permit its excretion is necessary in the treatment of fe overloaded diseases. although oral administration of such a pharmaceutical is highly desirable, no orally active fe chelator is so far in regular clinical use. searches for orally active and safe fe chelators require appropriate animal models of fe overload in order to evaluate the potential of these drugs. an animal model is particularly useful for testing the capacity of new fe chelating ce~oounds in enhancing the mobilisation of clinically relevant storage iron. iron loading in animals was achieved by dietary supplementation with carbonyl fe, fe fumarate and , , -trimethylhexanoyl ferrocene (tmh-ferrocene). liver fe concentrations were increased - times above normal levels. tmh-ferrocene was the most efficient compound to induce stable liver fe overload. the orally given con'pounds induced a predominantly hepatocellular iron distribution and was found distributed among reticuloendothelial cells only at a later stage. this pattern of liver fe storage is similar to that observed in the early stages of primazy ha ~ochromatosis and late stage of transfusional hae~ochromatosis. eeeently, we and ethers have cloned and characterized novel transcription factors called peroxisome proliferator-actirated receptors (ppak). they belong to the superfamily of nuclear hormone receptors as the steroid, thyroid and retinoid hormone receptors. ppars are activated, beside xenobiotic peroxisome proliferators, by natural fatty acids and induce, together with the receptor for -cis retinoic acld (rxr), the transcription of genes involved in the ~-and ~-oxidation of fatty acids. ppar and rxr bind as heterodimers to the ppar response element (ppre), consisting of a direct repeat of aggtca-iike sequences with one intervening nucleotide. now, we show by gel retardation analysis and transient transfection assays that ppar/rxr heteredimers also bind to and transactivate gene transcription through estrogen response elements (ere). the ere is a palindrome with aggtca half-sites separated by three nucleotides. the selectivity and specificity of the transactivation through an ere was demonstrated by the testing of various related palindromic and inverted palindromic repeats of aggtca sequences, which were all inactive. the possible activation of estrogen responsive genes by fatty acids via ppars in vivo is discussed especially with regard to a debated role of fatty acids in breast cancer. the purpose of this study was to evaluate whether intermittent doxorubicin (dox) treatment in vitro selects for multidrug resistance in rpmi myeloma cells and, if so, to determine the underlying mechanism(s). cells were exposed to constant doses of dox for days alternating with growth in dox-free medium for days. after > months of treatment, the cells developed an atypical mdr phenotype with -to -fold resistance to topo ii poisons. cross-resistance to vincristine and taxotere was < -fold, sensitivity to cisplatin and melphalan remained normal. efflux of mdr drugs was incresed with no effect of verapamit; subcellular dox distribution was normal. expression of topo ii a was found to be reduced by around and - % at the rna and protein level, respectively. minimum mdrt rna overexpression was detected when using rt-pcr; p-glycoprotein was not detectable. mrp rna expression was increased by - % relative to the wild type cells without detectable p . this data suggests that atypical mdr might play a role in acquired dox resistance in human myeloma. mucosal surfaces cover a vast area in the human body. they satisfy its need to communicate with its environment, e.g. in terms of nutrient uptake and sexual reproduction. however, at the same time they offer a vulnerable gate for invasion by pathogenic microorganisms. for immunoprotection of these regions, large amounts of iga antibodies are produced in the bloodstream and transported across the epithelial barrier to the lumen by means of polymeric ig receptor. there the receptor is cleaved, and its extracellular portion remains associated as secretory component (sc) to the secretory iga complex. in the context of a project aiming to mass production of secretory iga as a passive vaccine, we evaluated a series of eukaryotic expression systems for production of sc. the cdna encoding polymeric ig receptor was engineered in a way that only its extracellular domains corresponding to sc were expressed in yeast, in insect cells infected with recombinant baculovirus, and in mammalian cells infected with recombinant vaccinia virus. furthermore, a c-terminal poly-histidine tag was introduced for simple purification of the products. we will show optimization of expression levels for each of the systems as well as a quantitative and qualitative comparison of the products. recombinant vaccinia viruses (rvv) that express gone products from other organisms has become a popular and widely used laboratory expression system. our current interest is directed toward the production of secretory component (sc), a subunit of secretory iga antibody complex involved in mucosal immunity. toward this goal, we constructed rvv governing sc transcription under the control of two viral-based and the t bacteriophage promoters. expression of the protein was assessed in several mammalian cell lines, such as human tk-, human hela (grown in suspension or as a monolayer) and monkey cv-i. optimization of infection parameters and culture conditions were also performed. institut de biologic animale de runiversit~ de lausanne protection of mucosal surfaces is mediated by secretory iga ant/bodies (alga) constituted of dimeric iga and secretory component (sc) . toward the aim of reconstituting slga complexes in vitro and using them for passive oral immunization, sc has been produced in yeast, insect and mammalian expression systems. the gone encoding sc has been engineered so that a) the c-terminus of the protein carries a xhis tag and b) the newly synthesized polypeptide is released in the culture medium. culture conditions have been established to permit recovery of sc in serum-free medium. purification procedures based on nickel chelate and classical chromatographies have been optimized to yield sc under native conditions. finally, in vitro reassociation with purified dimeric igas obtained from hybddomas has been used to assess biological activity of recombinant sc. using a magnet-assisted subtraction-hybridisation technique (mast), cdna clones were isolated that are expressed in nqrm~l l~nq %issue but n__d_i expressed in the corresoondina nsclc tissue, ii of the edna clones are highly homologous to edna sequences for the following proteins: pulmonary surfactant proteins sp-a and sp-b; receptor for advanced glycosylated endproducts of proteins (rage); calmodulin-like protein; natural killer gone (nkg ); matrix gla protein (mgp); glutamine synthetase; ubiquitin; vascular smooth muscle alpha-actin; cytoskeletal beta-actin; vimentin. the remaining edna clones may represent parts of still unknown genes. the mast edna clones were derived from a patient who developed squamous adenocarcinoma. northern blot analysis of normal/tumour rna from three other nsclc patients showed that the lack of gone expression was independent of nsclc type and stage. enzymatic methylation of cytosines in mammalian dna is believed to play a fundamental role in basic gene regulation. methylation in the vertebrate genome is restricted to cpg dinucleotides at the c- position of cytosine. in vitro studies have shown that methylation can drastically influence the dna structure. cpg islands remain unmethylated in normal cells, whereas several immortalised, transformed cell lines contain partially methylated cpg islands. transcription of genes can be inhibited by methylation of cpg's. this modification contributes e.g. to the stable repression of genes on the inactivated x chromosome of females. the collagen vi genes show typical cpg islands in their promoter region and they were shown to be down regulated at the transcdptional level in src or myc transformed cells. given these facts we dee~ded to investigate the effects of methylation in the collagen vi gwene. e could demonstrate that the promoter activity of (x (vi) chicken collagen gone was strongly diminished by methylation in vitro. furthermore dna methylation completely prevented binding of a novel transcription factor to the promoter, whereas binding of sp was not affected. to show evidence of methylation in vivo, we are currently iocalising the exact positions of methylated cpg's in normal and transformed cells by genomic sequencing. alterations of putative tumour suppressor genes in human non-small cell lung carcinoma (nsclc). r. shipman and c.u. ludwig, molecular oncology, lab (zlf), basel, ch- chromosomal subregion llp displayed non-random allelic loss in nsclcs. llp contains the genetic loci for catalase (cat), the wilms' tumour gene (wti) and the follicle-stimulating hormone ~-chain (fsh~). % of the nsclcs were deleted at cat suggesting that loss of a gene(s) near cat may be involved in the progression of nsclc. yac clones containing the cat locus are being prepared for use in a direct selection procedure for cdnas encoded at this region. although % of the nsclcs were deleted at wti, no mutations were detected in the remaining wti allele. wti expression was subsequently demonstrated in fetal lung, fetal hematopoietic tissue, normal adult lung and nsclc. allelic deletion at p , immuno-histochemical and sequence analysis of the p gene were also examined in our nsclcs. these analyses support the contention that aberrant expression of the p gene is a pivotal event in the progression of nsclc. $ - recently, a number of advances have been made which enable the experimentalist to study the effects of low levels of ionizing radiation. the results suggest a threshold to radiation damage exists and that above a critical level, recovery mechanisms in the cell are induced and the biological response falls. thus low levels of radiation cause greater than expected levels of biological response. this has been demonstrated in studies of mutations, cell survival and cancer induction. because the low dose-rates and the low doses per fraction result in the total doses being small, the magnitude of the biological effects induced is insufficient to warrant alarm. identification of genes associated with the revertion of the malignant phenotype of a rhabdomyosarcoma cell line. michele genini, sabina solinas toldo*, ruedi fries* and beat w. schafer, dept. of pediatrics, university of ziirich, steinwiesstr. , ziirich, and *institut for nutztierwissenschaften, eth ztifich, ztirich. the human rhabdomyosarcoma cell line rd is tumorigenic and differentiates very poorly despite the expression of the myogenic factors myf and myf . therefore it can be regarded as natural mutant. to identify genes which are involved in suppression of tumorigenicity or enhance differentiation we applied two different approaches. since the development of human embryonal rhabdomyosarcoma has been associated with abnormalities of chromosome band p , in the first approach we transferred a normal human chromosome into rd ceils via microcell fusion. the microcell hybrids did not show a higher degree of differentiation. suppression of tumorigenicity was observed in one clone but is probably independent of chromosome , as it was shown by chromosome specific painting. ' in the second approach we applied a subtractive hybridization procedure between primary myoblasts and rd cells to find genes specific for the normal phenotype. these genes will be tested in vivo for induction of differentiation and/or tumorsuppression. deficient synthesis of the gpi anchor is the molecular basis of idiopathic paroxysmal nocturnal hemoglobinuria ("pnh") and a similar phenotype can accompany aplastie anemia cpnh-iike"). the population expressing cd , a gpi-anehored membrane glycoprotein, was quantified by flow cytometry in samples from patient p.g. (pnh) and patient m.m. (pnh-like) and amounted to % (p.g.) and % (m.m.), respectively. t lymphocytes from both patients were isolated, expanded and cloned. from both patients, clones expressing the cd marker (cd +) and cd deficient clones (cd -) were obtained. takeda et al. (cell , , - ) showed that in some (cd -) bcell clones, a deletion of the p g-a gene product is responsible for the expression of the pnh-phenotype. pcr analysis of t cell mrna from extracts of both cd + and cd -clones of patient m.m. (pnh-like) using pig-a specific primers revealed a band pattern from which can he concluded that no deletion is present in the pig-a gene product. this result suggests that cd -t lymphocytes of the pnh-like patient express a normal pig-a gene product; a disorder in regulation of pig-a expression or an unrelated biosynthetic defect may explain the cd phenotype in these t cells. further work is aimed at defining the differences in biosynthetic defects of pnh and pnh-like cd -t cell clones. supported by grant - - of the snsf to egb. w@ have recently employed an experimental approach to turn the function of "nuclear messengers" such as c-fos (and c-jun) on and off at will in polarized me/nmary epithelial cells. to this end we used constructs encoding the c-fos (and c-jun) genes fused to the hormone-binding domain of the human estrogen receptor, designated c-foser (and c-juner), we could show that short-term activation ( mins.) of c-foser by estradiole (e ) led to the disruption of epithelial cell polarity within hours, as characterized by the expression of apical and basolateral marker proteins. during the next - days, however, the cells fully regained their polarized phenotype. conversely, long-term activation ( - hours) caused the irreversible loss of epithelial cell polarity, leading ultimately to an epithelial-fibroblastoid cell transition. these cells underwent dramatic changes in gene expression including the complete loss or reduction of epithelial markers such as e-cadherin/uvomorulin, zo-i and cytokeratins, and the de novo expression of mesenchymal proteins like vimentin, type i collagen and fibronectin. expression of the v-ha-ras oncogene (acting upstream of c-fos) did not markedly influence epithelial cell organization when the cells were grown on plastic. however, when cultured within type i collagen gels in the presence of serum, or when injected into the mammary glands of nude mice, rapid cell growth and a clear epithelial-fibreblastoid cell transition was observed. growth properties determine the organ colonization ability of a metastatic murine melanoma cell line. the ability of metastatic tumor cells to specifically colonize distant organ sites strongly depends on the interaction of the metastatic cells with the local organ environment. we have selected a b routine melanoma cell fine (b -ls ) which has an increased ability to colonize the liver, and compared its behavior with either its parental (b -f ) or inng-specific (b -f ) cell line. we have found that adhesion in vitro and organ lodgement in vivo were not different for ls and f . in contrast, b -ls grew at slower rates than the other lines both in vitro and in vivo after subcutaneous or intra-foot-pad injection. analysis of tyrosine-phosphorylated proteins indicated a higher phosphorylation activity in b -ls cells, which might suggest an altered regulation of some kinase(s) in this cell line. intercellular contact with hepatocytes in vitro, or the liver in vivo could restore an efficient growth of b -ls , enabling thus these cells to colonize this organ much better than others. hepatocytes or liver plasma membrane (p.m.) extracts conserved the growth stimulatory activity towards b -ls ceils. chromatographic fractionations of these extracts showed that a major growth sfimulatory protein in this fiver p.m. fraction turns out to be ttansferrin, which in the liver appears to exist in at least two different mature forms. protein import into mitochondria requires atp in two locations: outside the organelle, and in the matrix space. depletion of matrix atp arrests translocation of precursors across the inner membrane, but does not prevent precursors from crossing the outer membrane. as a result, proteins that reach the inner membrane or intermembrane space without passing through the matrix are often sorted normally in atp-depleted mitochondria. external atp is used to maintain precursors in a translocationcompetent state. however, some precursors can fold and still remain translocation-competent, and import of these precursors is independent of external atp. with the folded cytochrome b precursor, it is matrix atp rather than external atp that drives translocation across the outer membrane. thus matrix atp generates a pulling force that can drive both the translocation and the unfolding of precursor proteins. mitochondrial hsp probably functions as the atp-dependent import motor. arsenijcvic d, dulloo ag & girardicr l, dpt of physiology, cmu. geneva, ch. the relationship between body weight, daily energy expenditure (assessed by indirect calorimetry), and immune status (determined by lymphocyte proliferation tests) was investigated in swiss webster mice maintaining a stable body weight several weeks after infection with toxoplasma gondii(me ). following an initial period of weight loss (infection-induced cachexia), the surviving mice could be differentiated into two main groups: (i) those showing a partial regain in body weight (the infected gainers) and eventually stabilizing al a mean body weight % below a non-infected control group, and (if) those showing no weight regain (the infected non-gainers) and eventually stabilizing at % below control levels. immune function was found to be markedly suppressed in the infected non-gainers, whereas it was normal (and similar to control levels) in the group of infecled gainers. daily energy expenditure (and food intake), after normalisation for differences in body weight, was found to be the same in both infected gainers and non-gainers, but lower by % when compared to controls (p< . ); thereby indicating that the infected groups were able to increase their overall efficiency of food utilization in response to the low food inlake. in conclusion, this study conducted during a weight stable phase of chronic infection shows a clear-cut association between the inability to regain body weight and impaired immune function. in contrast, no relationship was found between metabolic rate and body weight nor between metabolic tale and immune function, but the chronically infected mice exhibited the same phenomenon of enhanced metabolic efficiency characteristic of chronic underfeeding per se. identification and functional analysis of chaperonin , the groes homolog of yeast mitochondria. biozentrum, university of basel, klingelbergstrasse , ch- basel, switzerland. phone ++ - - , fax ++ - - . the mitochondrial chaperonin system consists of chaperonin (cpn ; also termed hsp ), which is homologous to e. cull gruel, and chaperonin (cpnlo), which is homologous to e. coil groes. we have used a functional assay to identify cpnl of yeast mitochondria. when dimeric rubisco is denatured and allowed to bind to yeast cpn , subsequent refolding of the enzyme is strictly dependent upon yeast cpnl . in the presence of mgatp, yeast cpn and yeast epnl form a stable complex that can be isolated by gel filtration. the atpase activity of hsp is not inhibited by complex formation with cpnl . a different result is obtained with the e, coil system, where groes is able to completely inhibit the atpase activity of gruel ( ). to test the in vivo role of cpnl , we have cloned, sequenced and disrupted the corresponding nuclear gene cpnio. this gene encodes a protein of , da that is imported into the mitochondrial matrix without detectable cleavage. haploid cells lacking a functional copy of cpnio fail to grow at temperatures between ~ c and ~ c ( ). ( ) rospert, s. et al. ( ) proc. natl. acad. sci. , in press. ( ) rospert, s. et al. ( ) febs lett., in press. characterization of yeast mitochondrial heat shock protein , l. bolliger, b. s. glick and g. schatz, biocenter, university of basel, basel. mitochondrial hsp (mhsp ) is thought to use the energy of atp hydrolysis to pull precursor proteins across the mitochondrial membranes. to facilitate the purification of yeast mhsp , we used the cloned gene (a gift of dr. n. morishima) to modify mhsp with a six-histidine tag at its c-terminus. this construct supports growth of yeast cells lacking wild-type mhsp . we developed a purification procedure that allows us to recover this tagged protein with a purity of about %. experiments are in progress to characterize the nucleotidedependent interaction of purified mhsp with unfolded proteins. in addition, we are looking for partner proteins that may modulate the action of mhspt . we have copurified a protein of about kd together with mhsp . the kd protein could be released from mhsp by atp or adp. when tryptic fragments of this protein were subjected to microsequencing, one fragment showed strong homology to the grpe protein of e.coli. the transmembrane electrical potential of mitochondria (awmit) can be assessed in single hepatocytes by using epifluorescence microscopy. for this end the rhodamine fluorescence of a mitochondria-rich (fr) and a mitochondria-poor region (fp) are measured. the ratio of these signals depends on the awmit according to a modified nernst equation (reber b .f.x., somogyi r. & stucki j.w. ( ) , bba, , - ) . to calibrate this method the cell membrane was permeabifized for monovalent cations with nystatin and gramicidin. then the cytosolie k + was replaced by na + from a k+-free bath solution. subsequent addition of valinomycin made the mitochondrial membrane permeable for k + ions. by the addition of different k + concentrations to the bath, the awmit could be clamped to defined values. the relation between the ratio fr/fp and the awmit could be well fitted to our modified nernst equation. however, the calibration curve flattens out at potentials higher than about inv. therefore the detection limit of changes of a~mit within the physiological range is about - mv. maximal stimulation of the na+/k + atpase by addition of nystatin to the na + containing bath medium caused a drop of awmit of about inv. subsequent addition of ouabain resulted in an almost complete reversal of this drop. this shows that changes in atp consumption can be assessed via changes of audmit. schneiter ph., di vetta v., j quier e. and tappy l. institut de physiologie de l'universitg bugnon , lausanne. the metabolic effects of an exercise performed in the fasting or fed state were studied in subjects over a hour period in a respiratory chamber. a mixed meal containing c labeled carbohydrates was ingested at : am and an exercise session (walking at km/h rain, slope %) was performed at : am (fasting) or am (postprandial). net carbohydrate (net cho ox) and lipid oxidation (indirect calorimetry) and oxidation of exogenous carbohydrates (exo cho ox, breath co ) were measured. glycogen breakdown was calculated as net cho ox -cho exo ox, and glycogen synthesis as cho in -cho exo ox. energy expenditure over hours was similar but net cho ox was % lower, lipid ox % higher and exo cho ox % lower when exercise was performed in fasting vs fed state; moreover, glycogen synthesis was increased by % (p< . ), while glycogen breakdown was increased by % (p= . ). in conclusion, a min exercise in the fasting vs fed state a) increases utilization of endogenous lipids, b) enhances muscle glycogen turnover over a hour period. hormonal regulation of e-abl tyroslne kinase by fusion to a steroid binding domain t. mattioni, o. chini hooft van huijsduijnen, p.k. jackson and d. picard d partement de bielogie cellulaire, universit de geneve, ch- geneve the activities of a wide variety of proteins can be subjected to hormonal control by fusion to the hormone binding domain of steroid receptors. we show that this strategy can also be applied to a tyrosine kinase. in particular, transforming derivatives of c-abl become hormone-inducible oncogenes by fusion to a steroid binding domain. it is noteworthy that the hormone-inducible transformation of nih t cells is completely reversible upon removal of the specific ugand. reversion experiments reveal another facet of abl: its ability to inhibit cell proliferation. hours after hormone depletion, cells are not only morphologically reverted, but the cell cycle appears to be blocked in g . a cytostatic effect of abl overexpression has been reported by several groups but, until today, it could only be examined by comparing different abl derivatives or the same derivatives in different cellular contexts. in contrast, our hormone-regulable system has the advantage that the two distinct functions of abl (transformation versus growth inhibition) can be studied using the same derivative expresed in the same cell line. we are using the hormone inducible system to investigate the mechanisms regulating the transtorming and the cytostatic effects of abl in a variety of cell lines. jiewu yang and herbert zuber institute for molecularbiology and biophysic, eth, z'tirich enzymes from thermophilic organisms show higher thermostability and temperature optima than those of mesophilic organisms. it has been shown that these properties are based on a specific structure of the protein. the primary structure of thermophilic (b. stearothermophilus) and mesophilic(b, megaterium) triosephosphate isomerase(tim) have been determined by cloning and sequencing of the tim genes. comparison of the primary structures of the thermophilic and rnesophilic enzyme showed specific amino acid substitutions, particularly in the c~-helices of the tim barrel, which could play a critical role with respect to thermostability. we have consmacted mutants by exchanging (x-helices between tim of b. stearothermophilus and b. megaterium. helix h , h and h of tim from b. megaterium, corresponding to amino acid residues - , - . and - respectively, have been exchanged. the properties (thermostability and temperature optimum) of the hybrid-mutants were compared with the wild type enzymes. in a comparative investigation of structural and functional parameters related to the oxidative substrate metabolism in skeletal muscle cells of dogs and goats, a close relationship was found between intracellular lipid stores and mitochondria. as revealed in serial sections with subsequent em analysis, each lipid droplet was in direct contact to one or several mitochondria. quantitatively, % of the lipid droplet surface in goats and % in dogs were in direct contact to outer mitochondrial membranes or -in other terms - % of outer mitochondrial membranes in goats and % in dogs were in direct contact to lipid deposits. these findings were in good agreement with the physiological data showing a times higher oxidation rate of fatty acids drawn from intracellular stores for dogs than for goats. human muscle adapts to altered load with changes in the expression of enzymes involved in energy metabolism. endurance exercise in humans is known to increase the oxidative capacity of skeletal muscles (hoppeler, int. j. sports med. ( ), , ) . at the ultrastructurai level, a higher mitochondriai content contributes to the higher oxidative capacity of skeletal muscles. little is known about the molecular mechanisms that lead to such adaptations in humans. the expression of mitochondriai components involves complex regulation of both mitochondriai and nuclear encoded genes. we have developed a pcr approach to quantify dna and rna from human skeletal muscle cryostat sections and addressed the question, whether the structural adaptations in endurance trained athletes are accompanied by concomitant changes in respective rna steady state levels. preliminary data indicate a higher concentration of coxiv and coxl ( . and . times, respectively) in trained subjects. no differences were observed for mtdna, despite a two fold higher mitochonddal content. human skeletal muscle contains three main fiber types which are based on the myosin heavy chain composition of the individual fiber, the isoforms of other contractile proteins within a given fiber type can vary leading to a continuum of different phenotypes. we are studying the fiber type specific expression of the mrnas of myosin alkali light clmins (mlc) using in situ hybridization, especially the rarer slow form mlc lsa. differences were found between shoulder muscles and leg muscles: m. deltoideus yielded stronger signals for mlc lsa and mlc lsb in the least glycolytic type i fibers (ia), weak but detectable signals for mlc lsb and mlc lf/f mrna in more glycolytic type i fibers (ib) and strong signals for mlc lf/ f in type ii fibers. in m. vast. lat. mlc lsb was strongly expressed in type ia as well as ib fibers, mlc lsa mrna was only found in some ia fibers. mlc isa has been shown to be expressed at a particular time point during muscle development and regeneration. in a biopsy of a becker dystrophy patient, we found very strong signals in some small fibers displaying the morphology of regeneralmg fibers. thus this probe could be useful to dete~t regenerative processes in pathological muscle samples. the aim of this study was to validate the complementarity of two new non-invasive methods for the assessment of autonomic regulations. reactions to a moderale exercise ( w on a cycloergometer) were compared in cardiac transplant recipients (ctr) who modulate their heart rate (hr) by means of circulating calecholarnines, their heart being donervated, and in normal subjects where such exercise level doesn't stimulate the sympathetic system. the methods used were: ) the spectral analysis of hr variability where the high frequency component (hf, above . t z, related to the respiratory frequency) is a marker of the vagal activity, the low frequency component (lf; at about . hz) depends on both sympathetic and parasympathetic activities and the lf/rf ratio indicates changes in the sympatbo-vagal balance; and ) a pure sympathetic index obtained from the amplitude of the t-wave of the ecg (twa) which is decreased by adrenergic stimulation of the myocardium. our results indicate that in both groups hr was increased by the exercise. in ctr, a net decrease in twa (- _+ %, n= ) was measured. contrasting with this, in normal subjects, no change in twa (+ _+ %, n= ) but a decrease in beth hf (- _+ %, n= ) and lf (- _+ %, n= ) without change in lf/hf ratio (- +_. %, n= ) were observed. these results indicate that ctr subjects increase their hr by an adrenergic stimulation, whereas the normal subjects regulate their hr for such moderate exercise only by redudng their vagal tone. there was a significant relationship (r= . , p< . ) between ps and the ree. the slope of the regression line indicated a net cost of ps of . kcal/g. we conclude that obesity in children is associated with an absolute increase in whole-body protein turnover consistent with the increased ffm and ree. the "glucose paradox" in the anoxic and reoxygenated embryonic myocardium raddatz, e., tran, l., rochat, a.-c., kucera, p. and de ribaupierre, y. institut de physiologie de l'universitd, ch- lausanne. the hearts of -day-old chick embryos explanted in vitro react rapidly, reversibly and reproducibly to anoxia and reoxygenation. this work deals with the effects of exogenous glucose on the mechanical activity of the hearts submitted to strictly controlled normoxia-anoxia-normoxia transitions. the anoxic periods lasted from to seconds. instantaneous heart rate, amplitude of contraction, velocities of contraction and relaxation of the ventricle wall were determined optically. in presence of and mmol/ of glucose ) the arrest of cardiac activity under anoxia was delayed by and %, respectively, ) the duration of the postanoxic cardioplegia was reduced and ) recovery was faster. paradoxally, these concentrations of glucose induced arrhythmias both during anoxia and reoxygenation. the incidences of such arrhythmias increased with the duration of anoxia. the absence of glucose or blockade of glycolysis suppressed arrhythmias. image analysis showed that the observed myocardial dysfunctions originated in the atrium and were sometimes associated with disturbances of propagation. the microinjeetion of okadaie acid into incompetent oocytas leads to the release of the prophase block with entry into m-phase. these g /m-like alterations include chromatin condensation, germinal vesicle breakdown (gvbd), microtubules reorganization and formation of an abnormal (often multipolar) spindle of meiosis i, with chromosomes not aligned on the metaphase plate. the polar body is never extruded. moreover okadaic acid microiujection induces the emergence of phosphorylated cytolasmic foci as detected by the monoclonal antibody mpm- , known to react with mitotic phosphoproteins associated with centrosomes. the kinetics of gvbd following mieroinjection is extremdy retarded ( - hrs) when compared to spontaneous maturation ( ran-lhr) and depends on the oocyte diameter. interestingly, when inc~ompetent oocytes are cultured during hrs and then microinjeeted with okadaic acid meiosis resumes within a short delay ( -rhrs). mpm- epitopes are present on ecntrosomes only after okadaic acid injection. following gvbd one of the step depending on protein synthesis, the expression of tissue type plasminogen activator, is triggered. indeed oa-injected incompetent oocytes produce small amounts of this activator. altogether these results argue for an effect of okadaic acid favoring entry into mphase including microtubule organization, centrosomes phosphorylatiou and the recruitment of one of the masked mrnas (tpa). st~rillt~, ncb~, c~- l g~. ~t rs-inse~, monf~llier, france. meiotic reinitiation of the mouse oocyte is caracterized by a slow entry into metaphas~ i, b ~jinnin with germinal ve~ir break@own (gvbd) and ending with spindle formation. it is accompanied by a cascade of protein kinases and phosphatases increasir~g protein phosphorylation. the activation of histone hi kinase (maturation promoting factor, mpf) and of the hapk have been compared during spontaneous or okedalc acid (oa)-induced rhetoric reinitiation. in spontaneously maturing oocytes, hist ne hi kinase activity increases before gvbd ( x) and is a~sociated with the disappearance of the upper (tyrosine phosphorylated) migrating form of p cdc , following gvbd, histone hi klnase activity culminates ( x) in metaphase i. activation by phesphorylation of p mapk occurs after gvfld. in contrast, no increase of histone hi kinase is detec~cable in oocytes induced to reinitiate meiosis by a transient exposure %o oa, neither before gvbo nor during the following hours of culture. the upper migrating form of p cdc is present for houra. the activation of p mapk b~lins before gvbd. furthermore, when oa is applied on coclrces microinjected with p sucl, neither iocrease of histone hi kinase activity nor p cdc dephosphorylation are observed although gvbd is induced; p mapk is activated. altogether these results suggest that gvbd may or may not be associated with a detectable activation of histene hi kinase, depending on the experimental conditions. activation of ps cdc and p mapk are separable events. we propose that, in the mouse c~yte, oa might be able to aetivaf an alternative pathw~ leading to gvbd that is cdc -independent and that involves p mapk activation ensuing mpf-independent phosphorylations. s - urich, k., basel, switzerland transformation of cells by polyomavirus is mediated by middle-t antigen. this viral protein is able to form complexes with src family tyrosine kinases (e.g. c-src), phosphatidylinositol- -kinase (pi -k) and phosphatase a (pp a). c-src associated with middle-t is constitutively dephosphorylated at tyrosione , a site negatively regulating the activity of this enzyme. this results in high tyrosine kinase activity in interphase and mitotic polyoma-transformed cells. middle-t is transiently hyperphosphorylated during mitosis as reflected by an increase in the apparent lvl, on sds acrylamide gels. two putative phosphorylation sites for a cyclin-dependent kinase present in middle-t, threonine and threonine , are also phosphorylated by purified p ~a~ in vitro. we constructed mutant middle-t genes where individual phosphorylation sites were converted to alanine residues. mutants lacking threonine were still able to associate with all cellular enzymes described above yet defective for gell transformation. interestingly, the defect of this mutation could be compensated by additional changes in the middle-t protein sequence introduced further downstream in a domain suspected to play a role in the targeting of this protein to intracellular membranes. $ - schmidt, s., fa~khauser, c., and viesturs, s. isrec, epalin~es, switzerland little is understood of the mechanisms which regulate cytokinesis, or how it is integrated with mitosis. we have cloned a number of genes from the fission yeast s. po~be which are required for the initiation of septation and are characterising them. defects in the cdc and cdcll genes result in continued nuclear division without cytokinesis, while cdcl mutants undergo multiple rounds of septum formation without cell cleavage. our analysis suggests that phosphorylation will play an important role in the regulation of cytokinesis and its integration with mitosis. a functional cdcl product is required for maintenance of p r activity in mitosis and the primary sequence of the cdc protein indicates that it encodes a protein kinase. data concerning the role of the cdc , cdcll and cdcl genes will be presented. rfc was originally identified in human cell extracts as one of the cellular factors essential for the replication of sv origin-containing dna in vitro. it is a five subunit protein with one large subunit of -i kda and four small subunits of - kda. rfc binds specifically to primertemplate structures at the '-end of the primer. together with proliferating cell nuclear antigen (pcna) it serves as a primer recognition factor for dna polymerase ~ and ~. to show the invobement of rfc in cellular dna replication we are currently cloning s. cerevisiae rfc. the amino acid sequences of a number of tryptic peptides have been obtained. this information was used to amplify parts of the s. cerevisiae genes by per and to screen a genomic library with these probes. three genes were cloned so far. the large subunit, rfc , codes for a kda polypeptide that is % identical to the large subunit of human rfc (lirfc). the sequences for two of the small subunits were also obtained. rfc , coding for a kda polypeptide, is % identical to the hrfc kda subunit. rfc , coding for a kda polypeptide, is % identical to the hrfc kda subunit. there is also considerable similarity between the different subtmits. rfcj, rfc and rfc , as well as the hrfc subunits, have consensus sequences for a atp/gtp binding site. all three genes are essential in s. cerevisiae. p is a major tumor suppressor gene which spefically interferes with the onset of e phase. we have therefore cxa~ined the possibility that p modulates the normal functions of enzymes and proteins directly involved in dna replication. we have shown that human wtp protein binds physically to calf thymus single stranded dna binding protein a, rp-a, we have also found that p blocks dna helicases in a typical strand displacement assay. this may reflect the fact that p is able to reanneal complementary dna single strands. since both wt and some mutant forms of p share this function, they are unlikely to be related to the p tumor suppressor activity. to our surprise, among the helicases which were tested we determined that the p inhibition could be released by rp-a in the case of cellular but not in the case of viral dna helicases. additionally, we found that p can partially inhibit dna polymerase ~ and s holoenzymes on singly primed mi dna. this inhibition, however, was only evident when e. coli ssb was substituted for rp-a. our data thus show that p exerts an inhibitory effect on several enzymes involved in dna replication and dna repair. the p -rp-a interaction may function to protect replication enzymes from inhibition by p under certain physiological conditions. catalytic subunit showed that pol ~ is the most conserved replicative pol (cullmarm g., hindges r., berehtold m.w. and htibscher u., gene, in press). we synthesized pcr primers and cloned three fragments spanning the whole catalytic subunit. the resulting pcr products, called n-term, middle and c-term have a size of , and bp, respectively. the primer for the n-term and middle fragments contained a histidin tag in order to purify the recombinant proteins by using a ni-nta column. these fragments were cloned into the expression vector pgemex . because of the natural termination codon in the c-term fragment, the histidin tag was placed directly in the vector in front of the fusion protein, generating a new vector, prhh s. all three fragments were expressed in e.coli. the fusion proteins could be purified in a single step and were used to raise polyclonal antibodies. finally, the entire pol sequence was joined together and could be expressed. data on the characterisation of the antibodies and the proteins will be shown. hsp from calf thymus can influence dna polymerase under heat shock conditions. we have generated antibodies against dnak protein, the hap homolog from escherichia coil, and used them for detecting of hsp protein during its purification from calf thymus tissue. the apparently purified protein has a molecular weight of kda, and has been recognized by antibodies against dnak and eucaryotic hsp / , it possesses a very week atpase activity, which can be stimulated by different poly-and oligopeptide substrates. results will be presented showing the influence of hsp on dna polymerase r from calf thymus under heat shock conditions. identification of a vertebrate cdc mutant which is unable to complete the g /s transition. nicole sr and viestars simanis. chemin boveresses , epalinges,.isrec. s. pombe strains have been constructed which should permit the generic and molecular analysis of the events which occur in late gi when cells become committed to the mitotic cell division cycle and the initiation of dna synthesis. a number of mutants of the chicken cdc gene were eonsuueted during the course of analysis of phosphorylation sites some of which lead to the interesting phenotype of cold sensitivity when expressed in a cdc null background, when the only source of p cde is the chicken homologue of the gene, expressed from a muldcopy plasmid. in order to create a genetically tractable strain, the wild-type chicken cdc gone and one mutant into the s. pombe genome to create a strain in which cell cycle progression is dependent upon the chicken cdc gene. analysis of this strain indicates gnat the cells block predominantly before replication of dna. in order to determine whether the cells were arrested before or after the execution of the start control their ability to conjugate at the arrest point was assessed. consistent with a block before the traverse of start and contmim~ent to s-phase, cells were able to conjugate with high efficiency. further support for the view that this mutant is defective only for the g -s transition is provided by the observation that it is able to complement a cdc a l mutation in trans. this mutant is defective only for ( function and not traverse of start. tl~e double mutant is viable at the restrictive temperature of either of tile parents alld is no longer cold sensitive. further work will concentrate on cloning genes involved in the traverse of gi into s phase in fission yeast. (masson and kreis, , j. cell biol. : ) where it is localized on a subset of microtubulea. when caco- cells are grown to confluency and become polarized, e-map- expression levels increase concomitant with a higher microtubule stability. transfection of fibroblastic cells (veto), which do not contain any detectable e-map- , with cdna encoding this protein, results in stabilization of microtubules to nocodazole treatment. these data suggest that e-map- may be involved in the stabilization and reorganization of microtubules in polarizing epithelial cells. since e-map- is a stabilizing protein, its interaction with microtubulen should presumably be modified at the onset of mitosis to allow disassembly of the interphase microtubule network and formation of the mitotic spindle. indeed, immunolabeling for e-map-t varies during mitosis. in early prophase, no e-map- can be detected on the microtubules of the forming mitotic spindle. the protein is observed on the spindle microtubules of metaphase cells and staining becomes bright on the new interphase microtubules of telophase cells. analysis of the protein in cells blocked in mitosis by low concentrations of nocodazole indicates that e-map- is hyperphosphorylated. this hyperphosphorylated form of e-map- is no longer able to co-sediment with microtubules in in vitro microtubule-binding assays. phosphopeptide map and phosphoamino acid analysis show the existence of novel phosphorylation sites in e-map- during mitosis compared to the protein during interphase, we are currently characterizing the kinases involved in the modulation of e-map- activity. little is known about the specific functions of calcium-binding proteins in epithelial cells ~md in particular in tumoral cells of epithelial origin. calretinin (cr) and parvalbumin (pv) are supposed to be involved in cell proliferation. we observed the endogenous expression of cr in several cell lines from human colon adenoearcinomas, and we obtained stably transfected cells for pv in a human ovarian adenocarcinoma cell line. we studied the cell cycle in correlation with the endogenous or ectopic expression of cr and pv, respeetiveiy. g and mitotic celts are strongly labelled with the cr-antiserum , while pv immunoreactivity is dominant in interphasic, but not in mitotic cells. in cr expressing cells, the cell cycle seems to be normal and the rate of proliferation to be proportional to the percentage of positive cells. the cell cycle is inhibited at mitosis after the addition of cr antisense oligo nucleotides to the culture medium. the cell cycle is blocked in gi/s and g in the cells eetopically expressing pv. the results support the hypothesis that these two calcium.binding proteins, cr and pv, could intervene at different moments and probably with different mechanisms on the mechanism of the cell cycle in the tumoral cells studied. production of polyclonal antibodies against calretinin cr- k gander, j.-ch., gotzos, v., cello, m.r. and schwaller, . institute of histology and gen. embryol., university; -fribourg a mrna for an alternatively spliced form of calretinin, ealretinin- k (cr- k) was detected in widr cells (a cell line from a human adenocarcinoma). we therefore decided to produce antibodies specific for this protein. it is directed against the amino-acids localized at the c-terminus of the truncated protein which are unique for this protein and not present in full-length calretinin. two different approaches have been chosen to obtain a specific antiserum. we have produced a chimeric protein containing the cterminal region of cr- k and the (h) (nanp) polypeptide as carrier. the fusion protein was expressed in e. coil and purified on a nickel chelate column. as a second method, a synthetic peptide (corresponding to the last amino acids of cr- k) was crosslinked with the carrier protein klh (keyhole limpet hemocyanin). the antigens were used to immunize two rabbits. after the first boost, the sera were tested. we have observed that both antisera recognize besides the protein used for immunization also cr- k (expressed in e. coil) in a western blot specifically. no other protein bands of either e.coli extracts or from eytosolic extracts of widr cells crossreact with the antibodies. both antisera detected the protein cr- k in immunohistochemieal stainings of widr cells confirming the presence of cr- k in widr cells. the tctp (p ) is a growth related tumour protein which is expressed under translational control. homologous acidic proteins have been found in higher plants and in saccharomyces cerevisiae (ykl ) . the striking conservation of this polypeptide between these very divergent organisms suggests some important but still, unknown cellular function. to address this question, a null mutation was constructed in yeast, by deleting almost all the ykl structural gone. the deleted strain shows no detectable phenotype. therefore, we conclude that, in yeast, this gene is dispensable. the protein ykl has been overproduced in e. coil and injected to rabbits to raise antibodies. we are now characterizing the ykl expression at rna and protein level. ellenrieder,c., forrer,p., kosovsky,j., rischle, m., nueller, r. and jaussi, r., institut f~r medizinische radiobiologie, paul scherrer institut & universitgt zh, ch- villigen radiation therapy of tumors could be improved by influencing cellular radiation sensitivity. yeast strains with defective cell cycle checkpoint genes (e.g. radl, rad , radg) show increased radiation sensitivity. cross-hybridization of a probe from the tad gene on human mrna northern blots yielded a single prominent signal. experiments to clone the corresponding kb cdna are underway. we have cloned a hamster cdna encoding the homologue of human cdk . probably, this cdna encodes the p protein from hamster whose phosphorylation responds to radiation checkpoint signals and who does not bind to p sucl beads. treatment of cells with cdk antisense oligonucleotides is hoped to modulate the radiation sensitivity. proliferation of smooth muscle cells (smcs) is a major event in vascular development and atheromatous plaque formation. in order to characterize smc replicative potential, newborn rats have been injected with h-thymidine ( h-tdr) during their first week of life when most of smcs were proliferating; then, h-tdr labelling was evaluated in adult rats. depending on the number of mitosis that smcs had accomplished after the first week of life, four different smc subpopulations could be defined indicating that rat aortic smcs are heterogeneous in their replicative activity. -bromo- '-deoxyuridine (brdu) incorporation after balloon induced endothelial denudation of rat aorta in adult rats showed that smcs entering into the cell cycle were mainly devoid of h-tdr labelling, this category could derive from two distinct smc subpopulations: smcs which were arrested in their proliferation before or just after birth or smcs which had actively replicated during development. thus, one (or two) subpopulation(s) of rat aortic smcs, characterized by a particular replicative activity during development, is (are) selectively activated after balloon induced endothelial denudation. the situation in vivo of dermal fibroblasts embedded within the connective tissue can be emulated in vitro by growing the cells in collagen gels. we have investigated how fibroblasts cultivated within a matrix react upon wounding of this dermal equivalent. human skin-derived fibroblasts (kd-cells) growing within attached collagen matrices were monitored over a period of days. fluorescently labeled cytoskeletal elements (tubulin, vimentin, acfin) and fibronectin served as phenotypica/ markers. the d-arrangement of fibroblast microtubules, intermediate filaments and microfilaments, as well as of fibronectin was visualized by clsm and optical sectioning. cells at the wound margin, adjacent to the wound, and in non-wounded controls, up to a depth of about gm were analyzed. upon wounding, i.e. upon excision of a mm "punch biopsy" from the center of the collagen matrix, fibroblasts in the wound region oriented towards the matrix defect. layer-like structures of increased cell density evolved over time at the wound margin. the tissue defect was covered by fibroblasts, reminescent of the situation in vivo. global yeast genome expression analyzed by -d page after tctp homolog gene (ykl ) disruption. sanchez a translationally controlled tumor protein(tctp) was identified and microsequenced from a human liver sample. previous publications described the presence of a gene related to tctp in plants, mouse erythroleukemia and ascetic tumor, human mammary carcinoma and more recently in the yeast. tctp has no known function but its high degree of homology from plants to human underlines its probable crucial role in cell function. in order to study multifactorial chan~es in protein expression as a function of the ykl gene disruption m the yeast, we used high resolution two-dimensional gel electrophoresis combined with an efficient computer system (melanie). two groups of gels (ykl +(n= ) and ykl -(n=l )) were analyzed, compared and classified using correspondence analysis. there was no significant difference between the two populations of gels according to either qualitative and quantitative spots expression except for two spots which completely disappeared. the first one corresponded to ykl gsne product. the second one corresponded to a kda peptide with an isoelectric point of . . this ykl associated peptide still needs to be characterized and linked to the yeast genome. demyelinating and neurodegenerative diseases are associated with altered cellular responses including processes mediated by receptor protein tyrosine kinases. using a pcr cdna cloning approach we have identified a novel member of the eck/elk/eph genefamily in myelinating serum-free brain cell cultures. alternatively spliced cdnas encoding complete open reading frames for putative transmembrane proteins have been isolated from both rat and human brain. a recombinant protein derived from the rat cdna was demonstrated to have protein tyrosine kinase activity. an affinity purified antiserum to this protein was used to identify a glycoprotein of kd molecular weight in brain cultures, and developping and adult brain tissue. the protein expression is most prominent during embryonal development and during the first three weeks of postnatal life. by in-situ mrna hybridisation the transcripts were found predominantly in restricted neuronal populations. in order to investigate systematically the effects of geometrically defhied abrupt tissue expansion on the characteristics of impulse propagation, we constructed expanding cellular structures in vitro using patterned monolayer cultures of neonatal rat heart cells (photolithographically patterned substrates.) the preparations, which consisted of narrow cell strands ( - tim wide) inserting into large reetangular cell areas (side lengths > ~m), were stained with the voltage-sensitive dye di-g-anepps and were imaged onto a x photodiode matrix array (maximal spatial resolution .tin x am in the object plane). while impulse propagation was uniform in linear cell strands (average conduction velocity . m/s), a transient decrease in conduction velocity hi the transitional region was invariably observed in the suddenly expanded structures. this decrease was accompanied by marked distortions of the local action potential shapes due to bidirectional electrotonic interactions across the transition: upstream from the expansion, the repolarization phase was interrupted, a few ms after its inception, by a small secondary depolarization ("spike and dome" configuration), while, downstream, the action potential upstrokes were preceded by prominent prepotentials. in those cases where conduction failed at the tlansition, action potential durations upstream were dramatically abbreviated. these findings showed that phenomena similar to those recorded in intact purkinje-fiber-ventricular preparations can be observed in cell ensembles in vitro having comparable geometlins but consisting of cells with uniform active membrane properties. supported by swiss nsf grant a- (sr) and usphs grant ns ms). it is a noncollagenous glycoprotein with a molecular mass of kda consisting of three identical subunits. cmp is selectively extracted with edta-containing buffer what indicates a divalent cation dependent anchorage of the protein in cartilage matrix. electron microscopy revealed the presence of three compact globular subdomains and sequence analysis indicated the presence of a coiled-coil c~-helical assembly domain formed at the c-terminal end of each subunit. the trimeric structure was retained after complete reduction under native conditions which reveals that the subunit structure of cmp is not only stabilized by disulfide bridges but also by the coiled-coil assembly domain. tissue distribution studies in mouse revealed that cmp is selectively expressed in some but not all cartilages. adult rat cardiomyoeytes (arc) in culture degenerate the myofibrillar apparatus and after attachment to the substrate they grow and reassemble new rnyofibrils. the appearance of new, well organized rnyofibrils can be observerd in several distinct regions. they appear close to the membrane proximal to the culture substrate and in the perinuclear region close to the substrate. previous studies have shown that embryonic cultured cells assemble new myofibrils which insert in adherens junctions at sites of cell-cell contacts and in sub-sarcolemmal adhesions plaques (saps) at sites of cell-substrate contacts. this saps are thought to function as the nucleation sites for myofibril formation. we have investigated the formation of this saps in cultured adult rat cardiomyocytes. using confocal light microscopy we can show that the interaction between rnyofibrils and membrans occurs close to the membrane proxirnal to the substrate and that this saps are flanking both nascent myofibrils and sfls, the latter structures being believed to serve as scaffold for myofibrillogenesis, additionally we have used electron microscopy to investigate the formation of this structures at higher resolution. $ - muscle satellite cells (sc) are quiescent myoblasts, ready to be activated and to regenerate new muscle fibers in case of muscle damage. in vitro, single human muscle sc, cultivated as clones, give rise in proliferating conditions to two subpopttiations of cells, cells expressing both ~-striated muscle actin and desmin (c~-sr+dsm+), and cells expressing desmin alone (dsm+). in culture conditions promoting differentiation, a clone .typically gives rise to a fusing progeny, yielding c~-sr+dsm + myotubes, and to non fusing myoblasts (nfmb). the latter are dsm +, a phenotype similar to quiescent sc found in situ. in order to determine whether nfmb are the in vitro equivalent of in situ quiescent sc, this first generation of nfmb were selectively collected and subcultured. in proliferating conditions, nfmb were able to resume proliferation and to give rise to a progeny with both c~-sr+dsm + and dsm + cells. when cultured in differentiating conditions, nfmb formed ct-sr+dsm + myntubes, and a new generation of dsm + nfmb. when nfmb of the second generation were again selectively collected and subculture& they resumed proliferation and ~re again able to yield both myetubes and a third generation of nfmb. nfmb of the first generation were also cultivated as clones, and to % of them were able to resume extensive proliferation, yielding in their progeny both c~-sr+dsm + and dsm + cells, which differentiated into myotubes as well as nfmb. these results strongly suggest that sc have the stem cell property, of selfrenewal, yielding in their progeny both cells ready to differentiate into muscle and ceils similar to themselves. extra cellular matrix (ec~ regulates the expression of b-casein in cultured mouse mammary epithelial cells. we have developed a functional ~ epithelial cell strain, which expresses high level of milk proteins, forms alveolar-like structures when plated onto a reconstituted basement membrane and secretes casein unidirectior~lly into a it~aen. we have further shown that fflv~dependent regulation of b-casein occurs mainly at the transcriptional level and that ' sequences play an inloortant role in this regulation. we have located a bp transcriptional enhancer (bcei) within the ' flanking region of the b-casein gene. using functional assays, we show that bcei contains responsive elements for ~ and prolactin-dependent regulation. bcei placed upstream of a truncated inactive b-casein promoter reconstitutes a promoter even more potent than the intact prcmoter, which contains bcei in its natural context more than . kb upstream. this small fusion promoter reconstitutes the nonaal regulation pattern. we show that bcei mediates f/ dependent regulation even when linked to a het~rologous viral promoter. purified satellite cells (sc) were obtained from human skeletal muscle biopsies following cell sorting by flow eytometry. a chemiluminescent assay for acetylnholine (ach) revealed the presence of , nmoles/weu of . sc. in elonal cultures of proliferating sc, this intracellular level of ach was .l nmoles/well. when cells were cultured in the presence of an esterase inhibitor ( ixm phospholine), the ach amount was enhanced fold. conversely, cultivating the cells in presence of a potent inhibitor of choline acetyl transferase ( gm bromoach), gave a % decrease of ach content finally, the release of ach was detected in the supernatant of cultured sc, following a min incubation, and the measured level of ach was pmoles/well/min. the presence of an ach-like compound in myogenic cells in both freshly isolated and in proliferating sc suggests that sc may contain ach in situ. in additiou, the fact that ach was present in the extracellular medium suggests that ach can be released spontaneously by the cultured sc. type xii collagen is an extracellular matrix protein associated with collagen fibrils in vivo. as observed for several other extracellular matrix proteins, the synthesis of type xii collagen by chick fibroblasts cultured in . % fcs is stimulated by tgf-i~i. two splice variants of type xii collagen are known, with subunits of either kda or kda. by using antibodies specific for the large ( kda) form of type xii collagen, we could show a more restricted distribution of the large variant compared to the smaller form in the embryo. while only the large variant carries chondroitin sulfate, both variants are identical in their collagenous domain. it is thought that via this domain, type xii collagen binds to collagen type i fibrils. we demonstrate here that type xii collagen can bind to collagen i fibrils also in vitro. by neutralizing acid soluble collagen type i, we could coprecipitate type xii collagen together with newly formed collagen type i fibrils. this interaction occurs in physiological saline but is highly salt dependent. in further studies we investigated wether s-methionine labeled type xii collagen treated with chondroitinase abc, ct-chymotrypsin, or collagenase can still be precipitated together with type i fibrils. in addition, we found that type xii collagen also affects the rate of type i collagen fibril formation. h.u. keller department of pathology, university of bern, ch- bern locomoting blebbing cells colchicine ( -sm) can induce locomotion associated with blebbing in walker carcinosarcoma cells. blebs expand at a rate (about ~m/sec) which is much faster than known rates of actin elongation. this suggest that the membrane is directly pushed forward by forces other than actin elongation, possibly by hydrostatic pressure. this interpretation is suggested by the observation that there is significant f-actin staining all along the cell membrane but not with the cytoplasmatic content the blebs. blebbing is suppressed by . m sorbitol. the finding that cytochalasin d suppresses blebbing shows that actin polymerization is nevertheless indirectly instrumental in bleb formation, possibly by generating a high intracellular pressure. a tentative model explaining locomotion of blebbing cells is presented. osteopetrosis encompasses a family of diseases with different causes and the common phenotype of impaired bone resorption. the osteopetrotic mouse mutant oo/oo is deficient in csf- , the growth factor for the cells of the mononuclear phagocytic system. the phenotype of oo/ou mice is characterized by a low number of peritoneal macrophages and peripheral monocytes, and a virtual absence of osleoclasts, leading to the osteopetrotic phenotype. injections of csf- into op/oo mice reversed the osteopetrotic phenotype, proving the requirement for csf- during the process of osteoclastogenesis. by in situ hybridization, expression of csf- was demonstrated during bone development. osteoclast precursors and mature osteoclasts were identified as putative target cells for the growth factor, since these cells express csf- receptors, which is encoded by the proto-oncogene c-fm~. subsequently, specific binding of csf- to osteoclasts was shown. expression of c-fms parallels the expression of csf- in bone both in time and place, suggesting a local action of this growth factor in osteoclast formation, but also in the modulation of osteoclastic activity. different transcripts, raised by alternative splicing of a commmon nuclear rna precursor, have been found to encode a secreted and a membrane associated forms of csf-i. the secreted form can be modified by attachment of a glycosaminoglycan side chain, which serves as an anchor to integrate the peptide into the extracellular matrix. investigation of the role the different csf- forms play during recruitment of osteoclasts and regulation of their activity will provide further insights into the mechanisms governing these processes. comparative sequence alignments of known voltage-gated k + channels revealed two conserved cysteine residues in the putative transmembrane segments $ and $ . if the cysteines were connected by a disttifide bridge, it would put a structural consllaint on possible channel models by placing $ next to $ . we used site-directed mutagenesis to study systematically the potential roles of these invariant cysteines. fourteen of substitutions in $ (c ), and of substitutions in $ (c ) maintained channel function in xenopus oocytes microinjected with mutant crna. therefore, the conserved cysteines are not essential for k + channel expression in xenopus oocytes. however, electrophysiological characterization of the cysteine replacement mutants revealed distinct roles for the two cysteines. inactivation, deactivation, and ion permeation did not considerably change in $ mutants. in $ , in contrast, cysteine replacement by leucine, asparagine, glycine and valine accelerated inactivation and deactivation kinetics substantially, whereas serine and threonine showed opposite effects. furthermore, the voltage dependence of deactivation was differently affected. in summary, it appears that the side-chain at position in $ of kv . is involved in channel gating by participating in the transitions from the open to the closed and inactivated state of the channel. (supported by grants from the nih and mda to rhj, and from the snf and the swiss foundation for medical-biological grants to rdz.) the segment between $ and $ of voltage-gatedk + channels, called h or p region, has been implicated to form part of the ion conduction pathway. little is known about the conforroation of this region although various models have been proposed including a j barrel-like structure formed by four adjacent antiparanel j sheets. to gain insight into the secondary structure of this region, we used cysteine substitution mutagenesis and sulfhydryl-specific, membrane-impermeant reagents to probe the accessibility of amino acid side chains in and around the p region of kv . (drki). twemy-eighi positions from k to t were each mutated to a cysteine. after expression of mutant k + channels in xenopus oocytes, cysteine side chain accessibilities were probed by superfusion with ch so sch ch nme + (mtset), mtset can form a mixed disulfide with an accessible cysteine, and this may lead to current reduction if the covalently modified side chain is in or close to the ion conduction pathway. thus far, we have identified four mutations that showed k + current reduction after superfusion with mm mtset. the mutants p c, i c, y c, and k c showed %, %, %, and % reduction in current amplitude, respectively. in coutrast, mutants $ c, a c, t c behaved like wild type kv . with less than % current reduction. our results suggest that the side chains of p , , y , and k are directly accessible from the extracellular environment. taken together, these residues may, therefore, face the lumen of the ion channel pore. furthermore, the degrees of inhibition are in agreement with a model in which the ion conduction pathway narrows from residue k to y to i . (supported by grants from nih and mda to rhj, and from the snf and the swiss foundation for medical-biological grants to rdz.) motejlek k., h,,iuselmann r., and liig:her b.; pharmakologisches institut der universitiit ziirich, winterthmtr. , ch- zlirich comparison of ' flanking sequences of three gabaa-receptor subunit genes (atl, ~, ) revealed a novel conserved purine sequence dement with the consensus sequence gagaggggagaoga gagag(gg/aa)g. this element is present once each in the (zl and subunit gene promoters and in seven tandem copies in the gene promoter. a novel dna binding activity (bsf ) was identified that binds to various versions of this purine element. bsf was found ordy in brain but not in any other tissues tested. furthermore, the factor is distinct of beta, another brain-specific dna binding .protein with a purine-rich dna recognition sequence. the expression of bsf during differentiation of eerebeuar granule cells in vitro correlates with the expression of gabaa-receptor subunit genes. this correlation suggests a role for bsf as a transcriptional activator of neuronal genes. therefore, bsf may be important for cell ty~specific regulation of gabaa-receptor gone expression. we also found that bsf binds to wheat germ agglutinin, suggesting that this protein is glycosilated. taking advantage of this property, we are in the course of purifying bsf , the question, whether bsf is indeed a transcription factor is being investigated in vivo and in vitro. to produce a simple method of estimating the free magnesium concentration ([mgzq) in solution, we have manufactured dip cast mg + macroelectrodes using the neutral mg carrier eth .we have used the elecmides to measure the dissociation constant (k~) for mgatp in a background solutions mimicking the intracellniar milieu. for the mcasmement of the big atp dissociation constant, the background solution contained mmol/l n%atp. this solution was titrated with mgci and the changes in [mg ~] above . retool/ monitored with the maeroelectrode. during the titration the ph was maintained constant. fitting such titration curves with the standard hyperbolic binding equation, after correction for zero drift, gave the following mean+sd values for the ka (pmol/ at ~ ph . , . -+- , (n= ); ph , . :k- . (n= ) and ph . , .g~ . (n= ). elevation of the [naq to m~l/ and reducing the [kq to ( mmol/l was without effect at ph . (n= ). reducing the temperature to ~ increased the k a at . to . :k . (n= ). mg =+ macroelcctrodes provide an easy way of measuring [mg z § in solution. [mg ~] can also be measured in a background solution containing i mn~i/l ca, although the electrode response is reduced. interference by protein to date prevents their use in plasma. similarily to the block by zn + and ca +, protons only partially block the channel. the ability for all mutated channels to. conduct na + current, and the partial block induced hy zn +, ca + or h + indicate that y is not located deep in the pore but rather in the extracellular mouth of the channel. progesterone modulates the activity of glycine receptor expressed in colliculli neurons of neonatal rats. maury, k. & bertrand, d. dpt of physiology, cmu, geneva . steroids are potent narcotics and have been shown to act on ligand-gated channels activated by gaba or nicotine. however, little is known about their possible actions on other receptor types. for instance, while it was demonstrated that progesterone inhibits glycine receptors expressed by spinal cord neurons isolated from chick embryos, nothing has yet been reported for brain receptors. in this study, we have determined the properties of glycine channels expressed by brain neurons of neonatal rats using the whole-cell voltage-clamp technique. in inferior colliculi neurons, glycine elicited little-desensitizing inward currents which reversed around - mv. these currents were blocked by strychnine in the nanomolar range. surprisingly, however, we found that progesterone, in the micromolar range, potentiated the glycine evoked currents. these results, contrasting those obtained in chick spinal cord neurons, suggest that progesterone enhances or antagonizes glycine currents depending on the subtype of the receptor. the mammalian auditory organ (organ of corti) relies on two groups of sensory cell for its normal hearing: inner hair cell & outer hair cell. ihcs are mainly innervated by afferent fibers while the ohcs essentially receive efferent fibers. the ohcs possess unique electromofile properties which are thought to enhance the motion the basilar membrane and to refine the mapping of the sound frequency. we found, using whole ceil recordings, that - % of the fleshly dissociated ohcs displays fast inward currents. the magnitude of peak currents which can be as large as na vary from cell to cell. further experiments have revealed that this current is sensitive to ttx and strongly reduced when extracellular sodium is replaced by choline. its kinetic is relatively slow compared to that of sodium current of typelspiral ganglion cell, and has a more negative inactivation. other voltage activated currents and ligand-gated currents are under inverstigation. these results will provide further insights in the hearing transduction mechanism. the formation and the properties of homotypic and heterotypic gap junctions were studied using two types of insect cells, c / and sf . two single cells were pushed against each other and the subsequent de novo formation of gap junction channels was assessed by means of the dual voltage-clamp method (bukauskas and weingart: pfl~g. arch. : , it was symmetrical for homotypic junctions. the vj-sensitivity was less pronounced in sf cells. hence, the relationship for heterotypic junctions was asymmetrical. all pairs examined revealed a s-shaped relationship between gj(ss) and v, which was virtually superimposable (gj(ss) declined upon depolarization). each channel contains a vm-gate and two ~-gates. the vj-gates are operated independently. they close when their intracellular aspect is made positive. shaped curve compatible with a two-state boltzmann process. half maximal inactivation was reached at - mv and + mv, implying an asymmetrical gating behavior. in case of an asymmetrical protocol (~ and vewas stepped simultaneous, but in opposite direction), the q-dependent asymmetry disappeared (half maximal inactivation at - mv and + mv). the asymmetry in gj-gating, attributable to the pulse protocol, was also reflected in the time constants of ij inactivation (r cerebellar granule cell cultures were used to study the regulation of the nmda receptor expression. we have shown previously that chronic membrane depolarisation ( him k +) or treatment with nmda {i ~m) promotes the functional expression of nmda receptors, as assessed by nmda-evoked ca + influx. we have now developed a rnase protection procedure for the quantitative determination of the mrna levels of the nmda receptor subunits known so far (nr-i, a, b, c, d). the growth conditions mentioned above failed to alter the mrna levels of the different subunits. at the protein level, nmda receptors were evaluated quantitatively by the labeling pattern obtained by the new photoaffinity ligand i-cgp a. the intensity of the phctolabeled bands, thought to represent nr-i and nr- a subunits, was increased by treatment with high k + or nmda compared with control cultures. this result suggest an increase in receptor protein by high k + or nmda treatment. thus, posttranscriptional mechanisms seem to play an essential role in the modulation of the nmda receptor activity. the gene encoding the pore-forming subunit of the rat epithelial na + channel (~renac) was cloned recently and shown to belong to a novel gene family coding for cation channels (nature , p - ( ) . transport of na + ions through these channels is the rate-limiting step of na + absorption and thereby controls osmotic balance of body fluids and secretions. in order to study the implication of ~renac in regulating sodium balance, blood volume and blood pressure and therefore its involvement in hypertension, we expressed ~renac under the control of the human cmv promoter in transgenic mice. several lines of mice showing expression in different tissues were obtained. progress in the analysis of these mice will be discussed. activation of metabotropic glutamate receptors increases the excitability of neurones in the lateral septum of the rat. to elucidate the mechanism(s) of this action, we used whole-cell patch-clamp recordings from coronal brain slices of young animals. in voltageclamped cells, the selective agonlst (is, r)-acpd, at - /~m, had the following effects, a) it evoked a sustained inward current, which was resistant to ttx and to cadmi~am and which persisted in neurones loaded with bapta, a calcium chelator. this current displayed inward rectification and was reduced, or suppressed, when extracellular sodium was partially replaced with n-methyl-d-glucamine. b) it elicited a ttx-insensitive, voltage-dependent inward current, which activated at - /- mv and which reversed at aound mv. this current was suppressed in a low-calcium/high-magnesium perfusion solution and was undetectable in the presence of intracelhilar bapta. we suggest that acpd exerts a dual action on lateral septal neurones. it causes a steady depolarization by generating a sodium-dependent current and it triggers transient plateau potentials by inducing a calcium-dependent cationic current. interaction between these currents results in a powerful, self-reinforcing neuronal excitation. we have investigated the effects of protein kinase c (pkc) activators ( [~-pma, oag) and of phosphatase inhibitors (okadalc acid, calyculin a) on voltage-gated ca + and k + channels in ngf-differentiated pc ceils. whole-cell ba + and k + currents were recorded with the patch-clamp technique. by using specific ca + channel blockers (co-conotoxin (cgtx), isradipine) we found types of ba + currents (iba): a) a ~cgtx-sensitive iba; b) an isradipine-sensitive iba; and c) a t -cgtx plus isradipineresistant iba. [~-pma and oag specifically down-modulated the isradipine-sensitive iba. the inhibition of iba was prevented by staurosporine and pkc ( - ) ( pk inhibitors). the delayed rectifier k + current was unaffected by pkc activators. applied externally, okadaic acid and calyculin a inhibited the total iba by affecting several components of the ba + currents. however, the ~-cgtx plus isradipine-resistant iba was unaffected by okadaic acid. in conclusion, our results suggest a differential modulation of voltage-gated ca + and k + channels by the pkc signalling pathway in ngf-differentiated pc cells. agrin, a protein isolated from basal lamina extracts of the electric organ of the marine ray, is thought to mediate the motor neuron-induced aggregation of acetylcholine receptors (achrs) in muscle fibers at their neuromuscular junction. recent cloning in the marine ray, rat and chick has revealed that agrin can be alternatively spliced at two sites in its c-terminal half. site a encodes either or amino adds and site b encodes either , , or ( + ) amino acids. studies of agrin mrna expression indicate that early in synaptogenesis, chick motor neurons contain high levels of a b . in contrast, non-neuronal cells and muscle calls contain a b and aob mrna. in the adult ray, electric lobe motor neurons that innervate the electric organ contain a b , whereas agrin mrna in the electric organ again lacks both sites (mcmahan et al. ( ) , curr. up, cell biol. : - ). to investigate the functional properties of agrin isoforms in more detail, we have now compared their specific activity to aggregate achrs on cultured chick myotubes. heterologous expression of the c-terminal half in cos- and cells revealed that chick agrin isoforms a b and a b were highly active, while a bll was up to fold lower in activity. no activity could be detected for the a b and aob isoforms. in agreement with these findings the a b isoform of the marine ray also showed high achr-clustering activity. therefore, we conclude that motor neurons throughout development synthesize highly active agrin isoforms while the postsynaptic target ceils do not play a primary inductive role in the formation of synapses. we have used whole-cell patch-clamp recordings and hypothalamic slices in order to characterize the effect of n-methyl-d-aspartate (nmda) on suprachiasmatic neurones. in ceils clamped at or near their resting membrane potential, nmda ( - #m) generated an inward current of - pa, which was insensitive to ttx and which reversed at about inv. the nmda current-voltage (i-v) relation contained a region of negative slope conductance. the nmda current was reduced or suppressed by d(-)- -amino- -phosphonopentanoic acid (d-aps) or by mk- . it was potentiated by reducing the extracellular magnesium concentration from to . ram, or by adding glycine ( /~m) to the perfusion solution. in a majority of neurones, lowering the extracellular calcium concentration from to . mm caused a . to -fold enhancement of the nmda current. i-v relations indicate that in the low-calcium solution, the region of negative slope conductance was attenuated. this effect was due to extraceuular calcium, since it persisted in neurones loaded with the calcium chelator bapta. we conclude that nmda channels present in suprachiasmatic neurones may be modulated by extraeellular calcium. institut, and abt. pharmakologie*, biozentrum, basel. during innervation of skeletal muscle, the myonuclei underlying the synapse begin to express acetylcholine receptor (achr} ~-subunit gene and they remain activated after the nerve is removed. our experiments show that this is due to a factor in the synaptic portion of the muscle fibre basal lamina (bl). one candidate for this factor is agrin, which regulates ache clustering in the synaptic membrane: i) unlike in untreated muscle, the level of s at synapses was strongly reduced in cultured rat muscle fibres after proteolysis of synaptic bl. ) conversely, when rat myotubes were cultured on bl isolated from adult muscle, they preferentially expressed achr accumulations and ~-mrna at sites where they contacted the synaptic portions of the isolated bl. ) when various substrates were impregnated locally with agrin a big, an isoform expressed by motor neurones in fetal spinal cord, it locally induced in the myotubes the expression of s-mrna. art increase of functional nicotinic acetylcholine receptors precedes the fusion of cultured human muscle satellite cells. r. m. kranse, c.-r. bader and l. berrtheim. department of physiology and division of clinical neurophysiohigy, university medical center, geneva , switzerland nicotinic acetylcholine receptors (nactlr) are expressed on embryonic myoblast and acb may play a role in mechanism of fusion. our study focuses on the appearance of functional nachr in freshly isolated and cultured satellite cells (sc) from normal human muscle biopsies. sc cart be conditioned to either proliferate or fuse to form myotubes depending upon culture media. presence of ach-activated current was investigated using the whole-cell and single.channel patch-clamp technique. in freshly isolated sc (whose properties should be close to the quiescent in vivo sc/no nachr were observed (n= ). in the proliferating state, % (n= ) of the satellite ceils (which are also called myoblasts) displayed a small ach-activated current ( pa/pf) and, as expected, after fusion of salellite cells into myotabes, % of the ceils displayed an ach-activated current (n= ; pa/pf). to examine, whether the increased expression of nachr precedes sc fusion, sc were cultured in a medium which promotes fusion, but were prevented from fusing by keeping them at a low density. in this culture condition, % of the sc (n= ) displayed an ach-activated current and, in addition, these cells expressed a times higher ach-current density. ( pa/pf) than the proliferating sc. we also observed that, in high density cultures, a small population of sc do not fuse even atter months in the medium promoting fusion. these non-fusing myoblasts (nfmb) had no nachr. our results suggest that the appearance of nachr may be related to the process of sc fusion as i) quiescent sc in vivo do not express nachll ii) nachr expression increases in conditions promoting fusion and iii) no nachr were observed in nfmb. the latter cells may be equivalent of quiescent sc in vivo. $ - we have investigated the sensitivity of neuronal nicotinic acetylcholine receptors (nachrs) of known subunit composition to various cholinergic antagonists. neuronal nachrs were expressed in xenopus oocytes after injection of pairwise combinations of (x or c~ with either ~ or ~ crnas. the two-electrodes voltage-clamp technique was used to measure currents induced by rapid application of low concentrations of acetylcholine (ach) together with increasing concentrations of antagonists. the response of ct ~ neuronal nachrs to ach was halfmaximally inhibited by following antagonist concentrations (ic ): hexamethonium ( . i.tm), mecamylamine ( . p.m), pentolinium ( . i.tm) and trimetaphan ( . ~tm). with (x nachrs the ic s of the same antagonists were about times higher. finally, (+)-tubocurarine was a conventional competitive inhibitor of ach in ~ and t~ ~ nachrs. in contrast, low concentrations of (+)-tubocurarine increased the response to low ach concentrations in a ( and t~ ~ nachrs. these results further underline the importance of [l subunits in determining the functional properties of neuronal nachrs. supported by the hochschnlstiftung and nf grant . . to abc. previously, the role of the transglial tubular system was shown for the squid giant axon: in na + free (tris) solutions the series resistance increased in correlation with a decrease of the tubular opening density (tod). in the crayfish giant axon, which show similar tubules, we correlate the change in tod, measured in freeze-fracture preparations, witl] the conduction velocity. the control to'd was estimated to be per #ms. after and rain tris-inkubation the tod decreased to , and , , respectively. this reaction was reversible when these axons were reincubated in na~-c ntaining solution. after rain reincubatiou the tod was . per tam'z. to determine the influence of decreased tod on the impulse conduction velocity, the nerve was incubated for min in tris solution followed by reincubation. impulse propagation then recovered in two phases. during an initial fast phase with a short time constant of - rain na + diffused back into the periaxonai space and allowed impulses to occur with a low conduction velocity. in the second phase with a long time constant of min, conduction velocity recovered slowly to normal values. this time course was comparable to the recovery of tod, as estimated from recent freeze-fracture preparations after short reincubation. these results indicate that the normal tod is a necessary condition for the normal excitability of the axon and determines its conduction velocity. most spinal cord neurons respond to the inhibitory action of gaba and glycine, suggesting co-expression of gaba a-and glycine receptors n individual ceils. while glycine-receptors are exclusively found in post-synaptic densities, the cellular localization of gaba areceptors has not yet been characterized. in this study, the distribution of gabaa-receptors in the spinal cord was analyzed with an antiserum recognizing the (xl-subunit. double-and tripleimmunofluorescence staining were employed to identify synaptic receptors apposed to gabaergic terminals (immunoreactive for glutamic acid decarboxylase) and to assess their co-localization with glycine-receptors (visualized with an antibody to the kda protein gephyrin). staining for the gabaa-receptor ctl-subunit decorated the soma and dendrites of numerous neurons in laminae iii-viii and x, revealing their morphology in clear detail. by contrast, laminae i and ix contained little immunoreactivity for these gabaa-receptors. most gabaa-receptor-positive cells also exhibited a prominent glycine-receptor immunoreactivity. both types of receptors had very similar distribution patterns and were frequently co-localized in sites apposed to gabaergic boutons. these results indicate that gaba aand glycine-receptors may co-exist within single post-synaptic densities, suggesting a possible synergism between gaba and glycine neurotransmission in spinal cord neurons. prenatal benzodiazepine (bdz) exposure changes both behavioral and neuiochemical parameters of developing iats. these changes are not a consequence of remaining bdz in brain tissue, but rather indicate alterations in bdz receptol binding and function. since the bdz binding site is located on the gaba~ receptor complex, it is possible that prenatal bdz treatment influences the expression of gaba~ receptor subunits. to evaluate effects of pienatal bdz exposure on mrnas expression specific for several gaba~. receptor subunits (~i, ~ , ~ & y ), we treated plegnant rats with diazepam (l. mg/kg/d; s.c.) from gestational day to . we then analyzed specific mrna levels in offspring at four diffeient developmental stages (gd , pns, pni & adult) by in situ hybridization. pleliminary data flom optical density measurements indicate a deciease in expression of mrna in particular for ~-subunit of gab~ receptors in different brain reglons of plenatally diazepam-treated zats. processing of sensory information within the auditory system has been well characterized using histological and eleetrophysiologlcal techniques. however, relatively little is known about the neurotransmitters involved. the present study was performed to elucidate the possible role of excitatory (eaa] and inhibitory (i.e., gaba) amino acids in neurotransmission within the primary auditory cortex (all the main afferent to the ai, the ipsilatera} medial geniculate body (mgb), was electrically stimulated and evoked responses were recorded intracellularly in the ai region in halothane anesthetized cats. a seven-barrelled iontophoresis pipette glued alongside the recording electrode allowed localized application of eaaergic and gabaergic compounds onto the recorded neuron. in most ai neurons, mgb stimulation evoked a short latency, short duration epsp followed by a long-lasting ipsp. pattern, duration, and amplitude of synaptic potentia{s were highly variable and strongly dependent on stimulation intensity, reduction of gabaa receptor-mediated inhibition by iontophoretic application of either bicuculline or sr markedly enhanced mgb stimulation-evoked epsps. only the late component of this enhanced epsp was reversibly blocked by the competitive nmda receptor antagonistl ap at currents which selectively blocked neuronal excitation induced by iontophoretic application of nmda, our results demonstrate that in the eat ) nmda receptors in ai are activated following mgb stimulation and that ) a dominant gaba~ receptor mediated inhibition usually masks this nmda receptor activation under our experimental conditions, a highly purified and specific cell wall degrading endo-l, -u-l-arabinanase was isolated from an a. niger pectinase preparation by low and medium (fplc) pressure column chromatographic separation methods. the isolated enzyme was most active on linear , -~-l-arabinans (- i.u./mg), whereas branched arabinans from sugar beets were degraded to a lesser extent (- i.u./mg). the enzyme was shown to be electrophoretically pure after silver staining (sds-page) and its identity was confirmed through specific binding to an antiserum directed against endo-l, -~-l-arabinanase. the major physico-chemical characteristics of the enzyme were the following: mr ' , iep ~ . , ph optimum . , temperature optimum ~ ph stability . - . , temperature stability s oc, km = . mg/ml, vmax = . . - ~unol/min. zn and hg showed potent inhibitory effects. . ) is a specific enzyme of the glyoxylate cycle, which plays a key role in the initiation of gluconeogenesis in germination oilseeds, this pathway is localized in glyoxysomes, single membrane organelles which are converted into peroxisomes when lipid reserves are depleted. the glyoxylate cycle enzymes have been the subjects of numerous conflicting reports typically addressing the problems of their synthesis (on free or bound ribosomes), targetting and suborganelar localization (membrane bound or matrix proteins). recent biochemical studies have shown that ms from germinating soybean cotyledons is an interconvertible enzyme (membrane bound, aggregated or soluble fomls) which is significantly affected by its ionic environment (henry et al., , plant science : - ) . microscopy studies have now been carried out using immunofluorescence staining or immunogold-silver staining for light microscopy, and immunogold labelling for electron microscopy. all results consistently characterize ms as a glyoxysomal mawix enzyme. chorismate mutase (ec . . . ) catalyzes the first step in that branch of the shikimate pathway which leads to the aromatic amino acids phenylalanine and tyrosine. we have isolated a cdna for this enzyme from the higher plant arabidopsis thaliana by complementing a yeast strain (aro ) with a cdna library from a. this is the first chorismate mutase cdna isolated from a plant. the a. thaliana chorismate mutase expressed in yeast revealed allosteric control by the three aromatic amino acids as previously described for plastidic chorismate mutase isozymes. an attachment of rubisco to chloroplast membranes under cu++-stress has been described for wheat (mehta et al., j. biol. chem. , - . the displacement to the membranes has been discussed as a possible step in the degradation of this predominant stromal protein, e.g. during leaf senescence. in our recent experiments it became evident that such interactions are not restricted to rubisco. several other stroreal, and even a peroxisomal enzyme (glycolate oxidase), were also detected in the membrane fraction after to hours of oxidative stress induced in wheat seedlings by a treatment with mm cuso . the velocity and the degree of membrane attachment varied for different enzymes. based on these results it was interesting to check the solubility of rubiseo and its previously described kd fragment which accumulates during the incubation of bean leaf discs under oxygen deficiency (hildbrand and feller, experientia , a ) . neither rubisco nor the breakdown product were found to accumulate in the membrane fraction. thus, at present, the steps involved in rubiseo catabolism cannot be generalized. different mechanisms depending on the metabolic situation and on environmental conditions should be considered. overexpression of the four parsley phenylalanine ammonia-lyase (pal) isoenzymes in e. coli. characterization of the enzymes and kinetic analysis of the inhibition by -aminoindan- -phosphonic acid. christoph appert, j/irg schmid, jerzy zorn* and nikolaus amrhein institute of plant sciences, eth-z/jrich, zijrich. *technical university wroclaw, - wroclaw, poland. all four phenylalanine ammonia-lyase (ec . . . ) isoenzymes of parsley (petroselinum crispum nym.) were expressed as glutathione s-transferase fusion proteins in e. coil after affinity purification, the glutathione stransferase moieties were cleaved off with factor xa and the phenylalanine ammonia-lyases were characterized. the proteins form enzymatically active tetramers, even as fusion proteins. the four isoenzymes have comparable km values for l-phenylalanine ( gm to . /.tm), similar temperature ( ~ and ph optima ( . ). the aminooxy-and phosphonic-analogues of l-phenylalanine are competitive inhibitors of the enzymes. -aminoindan- -phosphonic acid (j. zo{a & n. amrhein, liebigs ann. chem. , ) was found to be a potent slow-binding inhibitor of these and other phenylalanine ammonnia-lyases, both in vivo and in vitro. chlorophyll a fluorescence has been used to compare the drought resistance of two varieties of solanum tuherosum (avr/i)c- st- and sibtema). fast fluorescence rise kinetics were measured during the first second of illumination with a time resolution of l ms and z bits in fluorescence intensity. the rise kinetics shows the typical steps called fo -j -i -p. with this values different indexes have been defind with the goal to compare the behaviour of these two varieties of potato. salicylic acid (sa) was proposed as a putative signalling molecule for the induction of systemic acquired resistance (sar) in infected plants. we studied its biosynthesis as follows. cotyledons of cucumber plants were injected with a solution containing pseudomonas lachr~ans and fed with radioactive sa precursors by injection of c-benzoic acid ( c-ba) or cphenylalanine ( c-phe). alternatively, leaf of cucumber plants were inoculated with tobacco necrosis virus (tnv) and leaf disks were then vacuum-infiltrated using c-ba or c-phe. free and bound c-phenolies were quantified by hplc. incorporation of c into sa was found in the two plant-pathogen systems. i~c-ba gave mostly c-sa and an unknown polar compound. c-phe gave also some c-sa, in addition to c-labelled lignin precursors like ferulic and p-coumaric acids. the biosynthetic pathway of sa from phe through cinnamic acid and ba will be discussed. after infection with a necrotizing pathogen, systemic acquired resistance (sar) is often observed in plants. in cucumber, salicylic acid (sa) increases in the lower infected leaf as well as in the upper uninfected leaves. sa was also found in the phloem sap of infected cucumber plants and was proposed as a signalling molecule for the induction of sar. we intend to clarify whether sa is translocated from the site of infection to the upper leaf, or wether it is made in the phloem tissue upon the action of a primary systemic signal. one cotyledon of cucumber plants was first infected with pseudomonas lachrymans and then fed with c-sa, c-benzoic acid (ba) or c-phenylalanine. after different times, cotyledons and first leaves were collected and the radioactive ~henolics were analyzed by hplc. in some cases, c-sa was found in the first leaf. in addition, two unknown radioactive compounds were detected in leaf after treatment with c-sa and c-ba respectively. we will discuss signalling processes for the induction of sar in cucumber. one modern approach of creating virus resistant grapevine plants is the introduction of resistance genes into existing grapevine varieties by genetic engineering. the goal of this work is to use the coat protein (cp) mediated strategy to induce resistance to nepovirus in vitis spp. as a first step, several chimeric nepovirus cp genes, were constructed by addition of various promoter regions upstream of the gflv and armv cp regions. their ability of conferring resistance to nepovirus infection in transgenic nicotiana benthamiana and n. tabacum plants will be presented. the best constructions will be used to transform several grapevine varieties in order to create nepovirus resistant grapevine plants. $ - the bctalains axe a class of natural pigments found only in plants of the order caryophyllales and in some fungi. the first step in betalain biosynthesis is the conversion of tyrosine to dopa. subsequently , dopa is transformed to betalamic acid, the bctalain chromophorr through the action of dopa- , -dioxygenase. we have purified and characterised a copper-containing enzyme of - kda from amanita muscaria pileus that catalyses the hydroxylation of tyrosine to dopa. considering that the enzymatic activity was restricted to the coloured parts of the mushroom, we postulate that it is involved in betalain biosynthesis. the enzyme featured a broad substrate specificity, and also oxidised the diphenols to their corresponding ortho-quinones, a reaction typical for tyrosinases. the implications of our findings on betalain biosynthesis axe discussed. chlorophyll a fluorescence was measured under steady state conditions of pea and tomato leaves adapted to low light intensity ( wm- s -i) at different temperatures (havaux and strasser, z. naturforsch. c, - , ). when leaves were exposed for a short period (i min) to heat ( to ~ in darkness, the level of variable fluorescence decreased. when the heat stress was imposed in presence of low light, the variable fluorescence was much less affected and virtually no effect of heat treatment was observed until ~ this protecting mechanism by low light was absent in algae and mostly absent in submerged water plants but fully present in free floating plants on the water surface. we conclude that a mechanism has been developed for higher land plants during evolution which protects the plants against heat damage on warmer days. caryophyllales, e.g. portulaca grandiflora, and in a few fungal species, e.g. amanita muscaria. we analysed the similarity of a key enzyme of the pathway, dopa- , -dioxygenase, in plants and fungi both at the protein and the dna level. antibodies against purified dopa- , -dioxygenase from the fungus a. muscaria crossreacted with protein from p. grandiflora petals and two cdna clones were obtained from this plant. their similarity with fungal sequences and with other dioxygenases is discussed. kinetics of prollne hydroxylauonj intrucellnlur transport and c-terminal processing of the tobacco vacuolar cbltlnase. ernst frcydl, thomas boiler and jean-marc neuhaus botenisehes instimt, abt. pflanzenphysiologie, hehoistxasse , ch- basel the vacuolar chitinase a of tobacco (nicotlana tabacum) is syndietlzed as a preproprotein with ma n-teaminal signal peptide which causes its synthesis in the er mad a c-termlnal extension, which has been idcmified as the vacuolar targeting peptide (vtp) ( ) and whleh is cleaved off from the maybe chitinase. it has recently been shown that mature chltin:~e a contains several hydi'oxyprolines within the short peptide spacer that links the n-terminal cysteine-rieh chitln-bindlng domain to the catalytic domain ( ). we received specific antibodies against mature chitinasr (a kind gift from dr. f.meins. f/vii) and raised antihodiea against a synthetic vtp. we performed pulse-chase experiments and cell [ractlonation with ) stably transformed tobacco plants expressing chitinase a or mutants lacking either the chitin-binding domain and spacer (chitah) or the vacuolar targeting poptid (chitavtp) and ) transient expression of the same conswacts in nicotiana plumbaginifolia protoplasts. in both systems, proline hydroxylatino in chltinase a was detectable after vain. and complete after - rain. the ehltinase intermediate forms were detected in the mlcrosomal and soluble fractions for up to - rain. the mature chitinase continuously accumulated only in the soluble fraction. in both systems chitah showed the same kinetics of intraceilular transport and processing. whereas the transport of cbitavtp seemed more rapid.these results indicate that in vivo cbitinase a is synthetized as a proprotein that is than modified by proline hydroxylation. this second intermedinte form is then transported to the oolgi apparatus and sorted to the vacuole. c-terminal processing occurs late in this pathway or in the vacuole. infection of bean roots with the soil-borne phytopathogenic fungus fusarium solani f.sp. phaseoli leads to a rapid increase of chitinase activity (~five-fold). part of this increase in enzyme activity is due to transcriptional activation of the basic class i chitinase isoenzymes. semi-native polyacrylamide gels stained for chitinase activity using the substrate glycol-chitin revealed the appearence of three additional chitinase isoenzymes that are absent from uninfected control roots. conventional protein purification techniques showed that fusarium solani infected bean roots contain two basic and two acidic chitinase isoenzyrnes. their physiochemical properties and possible biological function will be discussed. proteasomes are multicatalytic protease complexes that function as a major nonlysosomal proteolytic system. they exhibit multiple endopeptidase activities that promote the intracellular turnover of abnormal polypeptides and short-lived regulatory proteins. moss proteasome has been purified from protonemata by successive chromatography on macroprep q, bio-gel a- . , bio-gel a- and ha. the molecular mass was estimated to be , kd by gel filtration. gel electrophoresis of proteasome under nondenaturing condition gave a single band and two-dimensional gel electrophoresis identified the moss proteasome to be composed of different subunits with molecular weight in the range - kd. electron microscopy in aqueous solution showed that it is a cylindrical particle composed of four stacked rings with a diameter of nm and a height of rim. end-on projection established a -fold symmetry of the outer disks. substrate specificity of proteasome indicates that it contains endopeptidase activity against substrates bearing hydrophobic, basic, acidic and glycine residues immediately preceding the cleavage site. antibodies raised against moss proteasome reacted with proteasomes of other eukaryotes, including human. $ - hunger r.e., hess m.w., laissue j,a,, mueller c. the nod (non obese diabetic) mouse, a widely used animal model for insulin dependent diabetes mellitus, shows in addition to mononuclear cell infiltration of the islets of langerhans, massive cellular infiltrates of the submandibular and lacrimal glands with considerable tissue damage. several lines of evidence suggest that both insulitis and sialadenitis represent autoimmune disorders, to investigate a possible role of tumor necrosis faetor-a (tnf-c and activated cytotoxic cells (nk cells, t cells) in the inflammatory process, tissue sections of nod mice were hybridized with radiolabeled rna probes specific for the detection of mrna for tnf-r and the serine proteases granzyme a and b (gra and gr ) and perforin which are expressed in activated cytotoxic cells. tnf-e expressing cells were mainly located in infiltrates and are absent in non affected glands, whereas gra, grb and perforin expressing cells were distributed over the whole section with highest densities in the zones adjacent to parenchyma. the finding that activated cytotoxic cells are present in early stage of disease development indicates that cell mediated cytotoxicity may play a crucial role in initial tissue destruction. the role of tnf-a in autoimmune diseases is not known yet. the appearance, however, of cells expressing this gene in the infiltrate indicates a possible role of this cytokine in the progression of the inflammatory reaction, e.g. by increasing the lymphocyte traffic to this organ. we further demonstrated that the induction of inos was at the transcriptional level. in order to understand the underlying mechanisms we characterized the promoter region of the rat nos gene. a enomic rat library was screened using a cdna-fragment coding for the if-end of the inos cdna (nucleotides - ). a , kb hinc-ii fragment containing the "-flanking region of the inos gene was characterized by dna-sequencing. the transcriptional start site was determined by primer extension. sequencing analysis revealed a multitude of possible cis-acting elements homologues to consensus sequences for the binding of different transcription factors including ifn~' response element (ire), nuclear factor-kb (nf-~b), tumor necrosis factor response element (tnf-re), f-activated sites (gas) and one x-box. nf-kb, a transcription factor involved in signaling and immediate early gene activation during inflammatory processes was induced by treatment of mesangial cells with nm of il- . this was shown by the appearence in nuclear extracts of the dna binding activity of nf-~b using electrophoretic mobility shift assays (emsas) with a p-labeled ~r dna probe. pyrrolidinedithiocarbamate (pdtq), an inhibitor of nf-kb, suppressed the expression of inos mrna in mesangial cells without affecting the dibutyryl-camp-triggered increases in nos mrna levels. this data suggest that the il-i ~ signalling pathway responsible for nos expression requires nf-k activation and is definetly different from the signalling cascade directed by camp. recent findings indicate that the multifunctional cytokine il- , which plays a key role in irm-nune and inflammatory responses, also exerts specific effects in the central nervous system (cns). for example, il- promotes neuronal survival, induces differentiation and modulates neurotrophin production. using the very sensitive technique of reverse transcription combined with polymerase chain reaction the developmental profile of il- and its receptor mrnas was analyzed in various rat brain regions. our results indicate that both genes are expressed in a region-specific manner and are developmentally regulated. these findings support the hypothesis that il- is involved in differentiation and maintenance of neuronal subpopulation in the cns. interleuldn (il- ) expression is strictly dependent on a signal transmitted by the t cell receptor upon antigen stimulation. this signal can be mimicked in vitro with phorbol esters and calcium ionophores. we have found that stimulation with ca-ionophore alone induces expression of il- mrna, but does not induce secretion of il- . in polysome gradient fractionation of cells stimulated with phorbol esters and ionophore, the fractions containing il- rrtrna are found at the bottom of the gradient, bound to polysomes. however, in ionophorestimulated cells the fractions containing il- mrna are clearly shifted to the top of the gradient, and therefore are not lzanslated. this effect is specific for il- , as other mrna's like ~ -microglobulin are not regulated. this data suggests that il- gene is translationally regulated. in addition, in vitro experiments have shown that the lack of translation of il- mrna in ionophore-stimulated cells is due, most likely, to the presence of a translational regulator that specifically inhibits translation of il- mrna. assuming the presence of a translational regulator that specifically represses il- mrna translation, we can predict that, if the represser is in excess, even upon a second stimulus that is by itself able to induce secretion of il- , there will be no translation of il- mrna. the resuhs of such an experiment confirmed our prediction. polysome gradients showed that after ionophore stimulation, il- mrna gets "stacked" with one ribosome, and no further ribosome loading takes place. if malaria is highly endemic, every febrile child is a presumptive malaria case, and there is no differential diagnosis based on clinical, immunological or parasitological grounds. we evaluated the diagnostic usefulness of interleukin- receptor (il- r), tumor necrosis factor-receptors (tnf-r ) and (tnf-r ) . sera came from tanzanian pediatric fever patients and controls, all enrolled in the kilombero malaria project. we found that all receptors marked pediatric fever episodes, whereas stability of observed levels was highest for tnf-r and lowest for tnf-r . tnf-r levels reflected severity of the fever attack. regardless of the presence of a febrile illness, tnf-r levels were strongly associated with parasite density. immunological markers can be applied in rapid pre-and post-intervention morbidity assessments, validation of health interviews and monitoring of convalescence. we have recently shown that human eosinophils produce mrna for interlenkin (il)- when stimulated with calcium ionophom (eur. j. immunol. ( ), ). we now present evidence that human eosinophils contain preformed il- protein although they do not express mrna for il- as measured by rt-pcr. il- protein expression was determined using specific anti-human il- monoclonal antibodies by western blotting, facs analysis and immunohistochemistry. moreover, preformed il- was released into supernatant by % pure eosinophils after priming with gm-csf or il- and subsequent -min stimulation with paf, rantes, ionomycin or pma, however not with il- or il- , as measured by elisa. this observation implies that activation of protein kinase c and/or inwacellular calcium mobilization am necessary events for il- release in human eosinophils. the determined amounts of preformed il- protein was higher in patients with asthma compared to normal individuals suggesting a role for eosinophil derived il- in asthma. since the eosinophil is the predominant cell in the asthmatic airways, we determined l- concentrations in bronchoalveolar lavage (bal) fluids from normal individuals and asthmatic patients. indeed, il- concentrations in bals from both groups were similar to those seen in the supematants released by activated eosinophils. the role for i-,- in asthma remains to be determined. to study the effect of canthaxanthin in vitro, the hydrophobic compound was introduced in vivo into chick high density lipoproteins (hdl) by canthaxanthin feeding. the effects of hdl and hdl associated canthaxanthin on two types of cultures have been tested: flat sedimented cell cultures of embryonic chick neuronal retina, retinal figment epithelium (rpe), brain and meninges, and in reaggregate cell cultures of the neuronal retina. at high canthaxanthin concentrations the formation of colored, birefringent entities were induced in the neuronal retina in vitro. in line with human data, parameters of cellular function and cell differentiation remained unaffected by canthaxanthin at these concentrations. by contrast, fidl was found to induce various cell type dependent effects. proliferation of meninges cells was decreased at low hdl concentrations as concluded from.light microscopic examination and indicated by protein content, and lysosomal and mitochondrial activity of the cultures (ic : - mg hdl apoprotein/l medium). these parameters, however, were increased in brain cell cultures, while they remained unaffected in cell cultures of neuronal retina and rpe. concentrations above . g. hdl apoprotein/l caused a reduction of glial cell differentiation. $ - tnf-c~ had close-dependent antiproliferative activity on hormone-dependent human breast cancer cells that represent an early stage of the disease, whereas hormone-independent cells representing a late stage were not affected. however, in the presence of u/ml interferon ?(inf), high concentrations of tnf-c((lnm) were able to inhibit the growth of hormone-independent cells. using specific monoclonal antibodies in ftow cytometry, no significant changes of either the p or the p tnf receptors were observed upon inf treatment of hormone-independent cells. this indicates that the increased responsiveness of these cells for tnf-c~ is not due to a change in available tnf receptors. the affinity of the receptors for tnf-r are one order of magnitude different for the two cell types. inf treatment shifted the ecs of hormone-independent cells towards the ecs of hormone-dependent cells. thus, the basis for the increased tnf-sensitivity of hormone-independent cells in the presence of inf might be the interconversion of tnf receptors from low-to high-affinity. the adhesion of tumor cells to endothelium is the first step in the processus of metastasis, and is frequently dependent of cytokinemediated activation of endothelial cells. cytokines are also involved in nitric oxide (no) production by various cells. these experiments were designed to evaluate no production during tumor cell adhesion to endothelium. rat brain-derived endothelial and rat colon carcinoma cell lines were used during these experiments. no was evaluated with the griess reagent. activation of endothelial cells with cytokines (tnf-d, and ifn-~ ), only slightly increased ( - %) the adhesion of tumors cells to endothelium. activation of endothelial cells with tnf-ol and ifn-~, induced a - fold increase of no production. however, addition of tumor cells to the endothelial monolayer, either directly or in a semi-permeable membrane, decreased the cytokine-induced no production. these results indicate that no production is modulated during the proeessus of adhesion of tumor cells to endothelium. is induced in rat mesangial cells by inflammatory cytokines such as interleukin ] (il- ~) and requires bh as a cotactor. , -diamino- -hydroxypyrimidine (dahp), a selective inhibitor of gtp cyclohydrolase i, the rate-limiting enzyme for bh synthesis, potently suppressed il-i~induced nos activity, measured as nitrite production. inhibition of no synthesis by dahp was reversed by sepiapterin, which provides bh via a salvage pathway. sepiapterin dose-dependently augmented il- i],-stimulated no synthesis, indicating that availability of bh limits the production of no in cytokine-stimulated mesangial cells. n-acetylserotonin, an inhibitor of the bh synthetic enzyme sepiapterin reductase, completely abolished il-lj~-induced no formation whereas methotrexate, which inhibits the pterin salvage pathway, displayed only a moderate inhibitory effect, thus suggesting that mesangial cells predominantly synthesize bh tom gtp. in conclusion, these data demonstrate that bn synthesis is an absolute requirement for cytokine induction of nos in mesangial cells. inhibition of bh synthesis may provide new therapeutic approaches to the treatment of pathological conditions mediated by no. - [ ) can induce a macrophage-type of nitric oxide synthase (inos). northern-blot analyses of inos mrna-levels and nuclear run-on experiments of control and il- ~ stimulated mesangial ceils revealed that the induction of inos was at the transcripuonal level. here we demonstrate that platelet-derived growth factor bb (pdgf-bb) can suppress the il- dependent inducuon of the inos gene. nortllern-blot analyses of cellular rna isolated from mesangial ceils that were coincubated with il- [ ( nm) and pdgf-bb ( ng/ml, ng/ml and ng/ml) revealed a dosedependent suppression of inos mrna-levels. using run-on experiments with nuclei from mesangial ceils after stimulation with il-i~ ( nm) and pdgf-bb ( ng/ml) we demonstrate that the inhibition occurs at the transcriptional level. basic fibroblast growth factor (bfgf), on the other hand, potentiates the il- dependent stimulation of inos. coincubation of mesangial cells with il- ( nm) and bfgf ( ng/m[, ng/m[, ng/m[, ng/m dose-dependently superinduces inos mrna levels as shown by northern-blot analyses of total cellular rna form control and stimulated cells. run-on experiments with nuclei from mesangial cells stimulated with il- ( nm) and bfge ( ng/ml) revealed an enhanced transcriptional activity of the inos gene. thus, pdgf-bb and bfgf differentaity modulate the il-i~ dependent expression of the inos gene and are therefore an excellent model system to study the crosstalk between signal transductjon pathways. we report that ifnyr-/-mice have an increased resistance to lipopolysaccharide-induced toxicity (lps). lps-induced lymphopenia, thrombocytopenia and weight loss seen in wild type mice were attenuated in ifntr-/-mice. ifntr-/mice survived in the d-galactosamine-lps model when conditions were % lethal for wild type mice, correlating with serum tnf levels of up to fold higher in wild type mice. bone marrow and splenic macrophages from ifnyr-/-mice had a to fold decreased lpsbinding capacity, with serum from these mice lowering macrophage lpsbinding by a further %. thus, depressed tnf synthesis, diminished expression of lps receptors and low plasma lps-binding capacity in the mutant mice likely combine to manifest in the resistant phenotype of ifny r-/mice to endotoxin. in a complementary approach we have screened the ' portion (- . kb/+ . kb) of the gene for tissue specific and/or inducible dnasei hypersensitive sites. in chromatin from fibroblasts or kidney epithelial cells this segment of the il ra gene does not contain any dnasei hypersensitive sites. in contrast, in resting t-and b-lymphocytes, as well as in activated t cells, two sites (dhi, - . kb; dh , - . kb) were found. a third site (dh , - . kb) is detectable in t cells that have been activated with concanavalin a and il . this site maps to the same position as cisacting regulatory sequences that are required for the response of the il ra gene to signals from the il receptor. interleukin- (il- ) production in cultured human dermal fibroblasts can be stimulated by interleukin-i (il-i} added to the culture medium. absence of fetal calf serum and of growth factors (insulin, egf, t ) reduced the stimulation by more than %. egf and t and even more choleratoxin increased the il- production in absence of fetal calf serum. the effect was dose dependent. % human ab serum substituted for i % fetal calf serum. pretreatment of the cultures with serum or growth factors prior to stimulation with il had a stimulatory effect (serum, t , egf) or an inhibitory effect (hydrocortisone, choleratoxin). our results suggest that the il-i signal transduction to il- is modulated by serum components and growth factors as well as through c-amp produced by choleratoxin. interleukin- (il- ) is produced by a variety of cells and plays a central role in host defense mechanisms. its function include induction of il- and il- receptor expression, proliferation and differentiation in t-cells. in cocultures of human dermal fibroblasts and allogenic human t-cells a cell number dependent to fold increase of the il- production could be demonstrated after h and h. conditioned medium from tcells and from fibroblasts failed to induce il- secretion in fibroblast and t-cell cultures, respectively. no up-regulation of il- production was found in cocultures of fibroblasts with tcells killed by ethanol and in t-cell culture incubated with recombinant hll-lc~. after separation of the cells il- production in fibroblast and t-cells returned to precoculture levels. our results indicate that cell-cell contact more than soluble factors must be involved in the up-regulation of il- synthesis in cocultures of t-cells and fibroblast. using cocultures of autologes human t-cells and fibroblasts, we are currently investigating whether the cell contact essential for the il- production depend on immunolcical interactions of t-cells and fibroblasts. rapid growth of tumor often results in oxygen and nutritional deprivation leading to extensive necrosis, which is one of the characteristics of glinblastomas (gbl). neoplastic cells surrounding necrotic areas often present a peculiar arrangement of cells called "pseudo-palisading" -as a hallmark of this entity, together with abundant neovascularization. a special biological milieu is probably formed in these palisading areas by certain unknown cytokines and/or growth factors induced by the necrotic process. plate et al. ( ) reported the expression of vascular endothelial growth factor (vegf) in the palisading cells. we previously demonstrated that gbl cells produce il- , which is known to be an angiogenic factor. the aim of this study is first to assess if il- is expressed in the palisading ceils, and second to determine if hypoxia can trigger il- gene expression. in rive observation using in siru hybridization and immunohistuchemistry showed that il- is highly expressed specifically in the palisading cells. we also demonstrated the mrna expression of il- receptor in the endothelial cells adjacent to necrotic area. to simulate the in vivo condition, two in-vitro models are currently developed to determine if il- is induced by hypoxic insult on cells. first, gbl cell lines are cultured in the absence of oxygen and il- expression is assessed by northern blot analysis. preliminary results have demonstrated the induction of il- by hypoxia. secondly gbl cells are grown in spheroids until development of central necrosis. the il- expression will be assessed by in situ hybridization combined with immunohistochemistry to determine if the ceils surrounding necrosis express il- . it has been proposed by taniguchi and his colleagues that activation of the interferon (ifn)-i gene by virus or double-stranded rna involves induction and modification of irf- . overexpression of irf- can induce expression of the ifn-b gene in certain cells. a role of irf- in the activation of ifn-a genes has also been proposed. furthermore, irf- has been claimed to play the role of a tumor suppressor gene. we have generated mice in which both irf- alleles were disrupted and found them to develop normally. no spontaneous tumors have been observed so far. injection of poly(i)-poly(c) resulted in the same levels oflfn in blood of wildtype and null mice, and equal ifn-ct mrna levels in spleen. induction of wild type and irf ~ embryo fibroblasts (mefs) with virus resulted in the same levels of ifn-a and ifn-i] mrna respectively. in the case of polyo)-poly(c) induction, the levels of both fn mrnas were higher in wild type than in mutant cells; this difference was abolished if the cells were first primed with type i ifn. we conclude that irf- does not play an essential role in the induction oflfn genes. mitsuhiro tada, annie-claire diserens, isabelle desbaillets, marie-france hamon, nicolas de tribolet, erwin van meir; chuv, lausanne there is increasing evidence that a variety of cytokines produced by glioblastoma (gbl) cells modulate the tumor growth by affecting the host's immune response and by stimulating neovascularization. cytokines such as il-i, il- , il- , mcp-i, il- and tgf- , affect each other's production and receptor system and seem to form a cytokine network. among them, il- may play a pivotal role, since il-i can induce or increase the production of other cytokines (il- ,il- ,mcp-i,tgf- ) and modulate their receptor expression. to test this hypothesis, we investigated co-expression of cytokines and their receptors in gbl cell lines and gbl tissues using rt-pcr, northern blot analysis, immunnhistnchemistry and elisa quantification. a majority of the gbl cell lines and gbl tissues expressed il-i~, il- , type i and type ii receptors, indicating the presence of an il- autocrine loop. il- stimulated growth of some of the cell lines. a positive correlation of il- and il- expressions with the presence of an il-i autocrine loop in the cell lines was found. immunohistochemical studies showed the in rive co-expression of the il-i family and the secondary cytokines (il- , il- ) in gbls. antisense oligonucleotide to il-lc~ and/or il- partly suppressed this secondary cytokine expression. these results demonstrate that the il- autocrine loop plays a central role in the formation of the cytokine network in gbls. double-stranded rna-dependent protein kinase (pkr) is induced by type i interferon (ifn) and is implicated in the establishment of the antiviral state. upon activation, pkr phosphorylates the eukaryotic initiation factor eif- , thereby throttling protein synthesis. it was suggested that pkr may be essential for the induction of ifn- gene expression by both virus and double-stranded rna and for the activation of at least some ifninducible genes. recently it was proposed that pkr is a tumor suppressor gene because t ceils overexpressing inactive human pkr gave rise to tumors in nude mice (koromilas et al., ; meurs et al., ) . we have produced homozygous pkr knockout (pkr ~ mice and found that they develop normally and have no striking phenotype. induction of ifn-c~ and ifn- gene transcription by virus was unimpaired in fibroblasts derived from pkr ~ mice, as was the induction of several ifn-stimulated genes. so far, no spontaneous tumor formation was observed. pkr o/o embryonal stem ceils showed no increased malignancy in nude mice as compared to their wild type counterparts. we conclude that pkr does not play an essential role in viral induction of the ifn genes and that its tumor suppressing effect, if any, is redundant. tgf- plays an important role as a negative regulator of hepatocyte proliferation in liver regeneration. exposure of rodents to nongenotoxic carcinogens like cyproterone acetate (cpa), thioacetamide (ta) and phenobarbital (pb) causes abnormal hepatocyte proliferation and liver tumors after long term treatment. this suggests that these chemicals have either a direct mitotic activity or impair the negative regulatory system for growth. therefore the inhibitory activity of tgf- on dna-synthesis induced whith cpa was investigated in cultured rat hepatocytes. after a h exposure to cpa, a dose dependent increase in dna synthesis ( h-tdr incorporation) was observed. the maximum effect was attained with pm cpa (up to . fold) which was stronger than with ng/ml egf (up to . fold). tgf- and tgf- inhibited this response dose dependently (idso = . ng/ml) and reduced it to control levels at ng/ml. these findings show that the tgf- mediated pathway for negative growth control in hepatocytes is still responding after cpa treatment. mouse mxl protein is an interferon (ifn) induced gtpase with intrinsic antiviral activity against influenza a viruses. it accumulates in the cell nucleus and inhibits the transcription of influenza virus genes, recently, we have shown that mxl exerts its inhibitory activity by interfering with the function of the viral polymerase subunit p . pb is responsible for recruiting " ends of cellular mrnas as primers and for the elongation of nascent viral transcripts. to elucidate which pb dependent step of viral mrna synthesis is the target of mxl action, we examined the activity of recombinant mxl protein in an in vitro transcription system of influenza virus. first, we expressed wildtype and mutant mxl proteins, carrying a hlstidinetag at their n-terminus, in e. coil and purified them under nondenaturing conditions by ni-chelate affinity chromatography. mxl efficiently inhibited viral transcription in vitro, while mutant mxl proteins, lacking antiviral activity in vivo ,were inactive. moreover, the use of capped synthetic rna primers revealed, that mxl almost completely abrogated the elongation of viral transcripts, whereas the the cap-binding and endonuclease activity of pb was not affected. a $ - neutralization rates of tnf-a from eight animal species by a monoclonal antibody against human tnf: implication for receptor action? we have recently developed a sensitive bioassay for measuring porcine tnf-a based on homologous pk( ) cells. this bioassay is -to -fold more sensitive than the widely used l bioassay, also, human tnf-a and human tnf- were detected with a slightly higher sensitivity in the new test compared to the l bioassay, we now show that also canine, ovine, bovine, equine and caprine tnf-a can be detected with the pk( ) bioassay. we tested a murine monoclonal anti-human tnf-a antibody for its capacity to neutralize tnf-a from eight different animal species. the action of this antibody revealed that neutralization of tnf bioactivity is a complex phenomenon, indicating species-specific differences in the interaction with tnf receptors. to study this differential neutralization, we are cloning the porcine tnf receptors for expression in a heterologous cell system. administration.of high dose r-tnf(~ in combination with ifn in isolation and perfusion of the limbs in melanoma patients has shown to be very promising with a response rate greater that %. this observation prompted us to investigate the possible expression of the tnf receptors in melanoma cells using the monoclonal antibodies utr- specific for the tnf type a ( kda) receptor and htr- specific for the tnf type b ( kda) receptor. flow cytometric analysis of cultured melanoma cells showed the presence at low level of the tnf type a and to a slightly higher level of the type b receptor. similar results were obtained in vivo by immunohistochemistry on fresh tumor raateriai, treatmem of melanoma cells in culture with the-amp induced up to a fold increase in the number of type a tnf receptors with only a minimal change in the number of type b receptors. this increased expression of tnf receptors is conflrmed by direct binding experiments using i-labeled tnftx. the number of cpm's bound on dbc amp treated cells was about . - fold higher than on untreated control calls, likewise incubation of melanoma cell lines with ifny increased the specific binding of subsequently added i-labeled tnfct. from flow cytometric analysis using the two anti-tnf receptor antibodies it became evident that flint was able to increase the expression of both type a and b receptors depending on the cell line used. characterization of a murine tumor necrosis factor (x-lacz reporter construct tumor necrosis factor ot (tnfcq is a prominent mediator of a variety of different pathologies such as endotoxic shock syndrom and rheumatoid arthritis. it also plays a crucial role in host defence against bacterial and viral infection. up to now, only few data about signal pathways leading to tnfo~ induction are available. an easy to handle tnfc~ -lacz reporter assay will represent a useful tool to study signal transduction on the one hand and provide data about compounds with potential tnfo~ inducible activity on the other hand. in order to develop a routine macrophage cell line capable to easily report tnfc~ induction, different tnfe-lacz reporter constructs were assembled. data about functionality of the different constructs in routine macrophages will be presented in the context of tnfot expression. cloning of a novel protein binding to lymphokine, fos and myc mrna with unexpected enzyme activity j. nakagawa, h-p. waldner, s. meyer-monard, and ch. moroni inst. medizinische mikrobiolegie, univ. basel an au-rich sequence in the ' untranslated region of lymphokines, c-los, and c-myc proto-oncogene mrna is responsible for their rapid decay. we have purified a kd protein by an auuua affinity column from human brain. partial amino acid sequence was obtained and the corresponding edna has been cloned. unexpectedly the edna sequence exhibited significant homology to enoyl coa hydratase and bacterially expressed recombinant protein indeed showed the activity. while rat hydratase did not show rna binding activity, the recombinant protein bound to cfos, c-myc, auuua cluster of il- mrna, and adeno virus iv, but not to mutated ad-iv, nor irrelvant transcript. the binding was competed out by poly u. interestingly the protein seems to be processed from a larger precursor. we suspect that the protein plays a role in mrna turnover and perhaps in oncogenesis. institute for medical microbiology, university of basel in t cells, the immunsuppressive agent cea is known to inhibit ca + dependent induction of lymphokine mrna transcription. in contrast, the immunsuppressive drug rapamycin inhibits the lymphokine induced proliferation. we have analysed the effect of these drugs on a v-h-ras induced autocrine mast cell tumor line which expresses il- constitutively. csa and rapa inhibited autocrine growth by different mechanisms,, because added il- could antagonize inhibition by csa, but not by rapa. whereas csa acted by downregulation of il- mrna expression, rapa blocked il- induced proliferation. cea also inhibited il- superinduction by ca-ionophore, whereas rapa did not. nuclear run on assay indicated that the mechanism is posttranscriptional. therefore, we have introduced il- transgenes with and without the au-rich region in the ' utr of the mrna into a tumor line. in contrast to the wild type, the expression of the mutant construct was insensitive to csa. since the mutant lacks sequences known to regulate rapid mrna decay, we suggest that csa affects the regulation of il- mrna stability. some non-hematopoietic cell lines produce both scf and its receptor, the c-kit protein (c-kit). testing the hypothesis that scf may be involved in secondary growth of nb bone marrow metastasis, we found and previously communicated (beck d. et al, proc aacr, , , ) a low or absent expression of c-kit in nb cells. the mrna and surface expressions of scf gene in nb cell lines grown in chemically-defined medium were evaluated by northern blot analysis and flow cytometry. the scf antigenic concentrations in culture supernatants were determined by elisa and growth-inhibition experiments performed by pre-incubating nb cells with anti-scf antibodies prior to h-thymldine uptake assay. the scf mrna was expressed in of tested lines but no membrane-bound antigen could be detected on those cells. detectable levels of scf in the supernatants were found in / lines (range : - pg/ml). blocking experiments resulted in a decrease of dna synthesis in i out of lines only. from these and previous results, we conclude that scf is synthesized and released by many nb cells in vitro but the functional ~ole of it remains undetermined since scf does not appear to be usually involved in autocrine or paracrine growth stimulation of nb cells (supported by swiss fnrs grant - ). expression of the v-h-ras oncogene in the il- dependent pb- c mast cells leads to the generation of two classes of il- autocrine tumors in vivo. autocrine il- expression in class- tumors results from increased il- mrna stability due to the loss of negative trans-acting posttranscriptional control. this defect can be corrected by somatic cell fusion to the nontumorigenic parental pb- c resulting in downregulation of oncogenic il- expression and concomitant tumor suppression. expression of the v-h-ras oncogene remains unaffected in class- hybrids. in contrast, class- tumors are characterized by transcriptional activation of the il- gene due to the insertion of an endogenous retroviral element (intracisternal a-particle) which cannot be overcome by ceil fusion (see hirsch et al, , j.exp.medicine , - ) . although v-h-ras is required for generation of either class of tumors, expression of anti-sense ras constructs inhibited only the proliferation of class-i tumor cells. experiments are underway to characterize the target and the nature of the class- alteration. nitric oxide (no) promotes vasorelaxatlon of renal vessels in vitro. the purpose of the present study was to assess the role of no in regulating renal hemodynamics in vivo. renal blood flow (rbf) and glomerular filtration rate (gfr) were studied in anesthetized mechanicallyventilated newborn rabbits both before and after the administration of a no synthesis inhibitor, ng-nitro-larginine methyl ester (l-name), at a dose of pg/kg followed by jzg/kg x rain. such a dose of l-name did not modify mean arterial pressure or pulse rate. by contrast, the renal vascular resistance increased by + % (p < . ) while rbf decreased by -+ % (p < . ). gfr and urine flow rate remained constant. the overall results suggest that in normal conditions no may play a role in decreasing the renal vascular resistance of the immature kidney, without altering systemic blood pressure. interleukin- (il- ) is a t-cell derived lymphokine which, in addition to its effects on the immune system, is also able to suppress the growth of certain tumor cells. il- reduced the growth rate of four human colon tumor cell lines up to % in a dose-dependent manner. three other cell lines showed no significant alteration of h-thymidine incorporation or colony formation in the presence of u/ml il- . responder and nonresponder tumors were immunopositive for il- receptor (il- r) expression in flowcytometric analysis, using monoclonal antibodies raised against recombinant il- r and bound biotinyated il- . responsive tumors expressed more receptors. - binds with high affinity to a kda transmembrane receptor (il- r), through which the extracellular signal has been shown to be transduced. coimmunoprecipitation and chemical crosslinking studies have enabled us to demonstrate il- r target proteins. tyrosine phosphorylation of various proteins between and kda appears to be initiated during il- mediated signaling events in colon tumor cells. the cloning of human il- r target proteins will contribute to the understanding of the il- signal transduction pathway at the initial stage. the renal effects of endothelin- were investigated in anesthetized and meehanleally-ventilated newborn rabbits. each animal acted as its own control. in newborn rabbits, a bolus injection of nmol.kg " of endothelin- caused an initial fall in mean blood pressure (mbp) followed by a gradual but significant increase in mbp that lasted for rain. the dramatic increase in renal vascular resistance (+ -+ %) induced by endothelin led to a fall in glomerular filtration rate (- -+ %) and renal blood flow (- _+ %). in spite of the reduction of gfr and rbf, urine flow and sodium excretion rates increased significantly (+ _+ % and + _+ %, respectively). in additional newborn rabbits, a bolus injection of lnmol.kgaof endothelin- -a dose that usually induces marked renal and systemic vasoconstriction in adult models -did not affect systemic or renal hemodynamics. in conclusion, endothelin induces renal and systemic vasoconstriction, and affects water and sodium homeostasis during the neonatal period. however, these effects occur under higher doses than those used in adult animals, possibly reflecting receptor immaturity and/or interference of high levels of counteracting hormones. ontogeny of the endocrine pancreas in xenopus laevis c, maake , w. hanke and m. reine~ke , institute of anatomy, university of z rich, switzerland and department of zoology ii, university of kaflsruhe, f.r.g. the pancreatic islets of anurans show insulin (ins), glucagon (gluc), somatostatin (som) and pancreatic potypeptide (pp) containing cells. since only limited information exists on the ontogeny and on the presence of insulin-like growth factor (igf- ), we studied the development of the gastro-entero-pancreatic (gep) system in xenopus laevis using immunohistochemical techniques. singular ins-immunoreactive (-ir) cells were observed in the pancreas as early as on stage . at stage , the first pp-ir cells were found in the gep system followed by som-ir and gluc-ir cells. the first pancreatic islets consisting of ins-ir and gluc-ir cells occurred around stage . between stage and , i.e. after the onset of metamorphosis, the endocrine cells decreased in number and the islets partly disintegrated. starting with stage , the islets reformed and the amount of endocrine cells increased reaching the adult status at stage . around stage , igf-l-immunoreactions appeared. igf- immunoreactivity was found in pp-ir and gluc-ir cells but not in ins-ir and som-ir cells. it is assumed that islet-derived igf- may p~ay an important role during metamorphosis in xenopus. t. cathomen and r. cattaneo, institut for molekularbiologie i, universit~t zorich, hfnggerberg, ch- zorich the signals used for synthesis of the measles virus fusion (f) protein are poorly understood: a long (> bases) untranslated region is followed by three in frame augs at codons , and . f is a type i transmembrane protein with a postulated signal sequence of amino acids. we confirmed that the aminoterminal residues contain a signal peptide by transfering them to another protein. with the other envelope protein hemagglutinin, f protein induces virus-cell and cell-cell fusion. mutagenesis of the three augs indicates that proteins initiated at codon , or are all functional, but show slightly different fusion efficiencies. forms translated from aug or appear larger in electrophoresis than forms initiated at aug , suggesting that two different signal peptide cleavage sites are used. we are currently investigating whether both f protein forms are incorporated in viral particles. the production of protein isoforms with staggered amino-termini is common in cytoplasmic and type ii transmembrane proteins, but signal sequences usually preclude a similar organization of type i transmembrane proteins. beguin p., beggah a.t., rossier b.c., jaisser f. and geering k. institut de pharmacologie et de toxicologie de l'universit , ch- lausanne recent experimental evidence suggests that na,k-atpase might be a candidate for regulatory phosphorylation by protein kinases a and c (pka and pkc). to verify this hypothesis, we have mutated several serine and threonine residues in consensus phosphorylation sequences of the ~subunit of bufo marinus na, k-atpase. the mutants were expressed in x~nopus ooeytes and phosphorylation was studied in oocyte homogenates upon stimulation of oocyte, pka and pkc. our results indicate that a unique phosphorylation site for pka is located at serine in the c-terminus of the ~-subunit but its phosphorylation can only be revealed in the presence of detergent. on the other hand, mutations of several serine and/or threonine residues located in consensus sequences for pkc phosphorylation did not or only partially abollsh phosphorylation by pkc. in particular, mutations close to the catalytic phosphorylation site of the ~-subunit influenced phosphorylation by pkc, suggesting that either this region contains a real phosphorylation site or is part of a conformational domain necessary for phosphorylation by pkc. the heat-stable antigen (hsa or mouse cd ) gene is differentially regulated but has a housekeeping promoter. roland h. wenger*, georges kshler and peter j. nielsen. max planck institut f(jr immunbiologie, st beweg , d- freiburg i. bsg. "present address: physiologisches institut der universit&t zi.)rich, winterthurerstrasse , ch- z rich, expression of the gpi-anchored murine glycoprotein heat-stable antigen (hsa) shows tissue-specific as well as developmental regulation. during the maturation of several hematopoietic lineages, hsa expression is generally high in immature precursor ceils and low or absent in terminally differentiated cells. we present evidence suggesting that this regulation of the hsa gene (cd a) occurs at the transcriptional level. in addition, sequence and methylation analysis of the cd a promoter revealed characteristics of both "housekeeping" and tissue-specific promoters, including a methylation-free, hpall tiny fragment (htf) island, multiple putative sp and ap- consensus binding sites and a tata box. functional analysis of a . kb dna fragment containing these elements fused to the cat reporter gone in transient transfection experiments showed activity in both hsa expressing and non-expressing cell lines with a strength similar to that of the hsv tk promoter. large fragments from the flanking region of the cd a promoter did not influence the ubiquitous nature of this promoter, finally, the cd a, cd b and cd c genes were mapped to mouse chromosomes , and respectively. we have cloned, sequenced and characterized the gone for the m__itochondriat nadh-cytochrome b reductase (mcri) of saccharomyces cerevisiae. surprisingly, this gone encodes two mitochondrial isoforms of the flavoenzyme: a kda integral protein exposed on the outer surface of outer mitochondrial membrane (mcrlp ), and a kda soluble protein of the intermembrane space (mcrlp ). the smaller intermembrane space isoform is generated from the larger form by the action of the inner membrane protease i (impi) on the outer surface of the inner membrane. this is the first demonstration that a single gone encodes proteins which are located in different compartments of the same organelle. with special interest in any with a mitochondrial localization. degenerate primers were designed on the basis of high homology between members of the abc family, and pcr was performed on yeast genomic dna. ten dna fragments bearing significant homology to the abc family were amplified, nine of which were previously unidentified. disruptions of five of the corresponding genes were performed. disruption of one gene led to markedly impaired growth on rich medium and cessation of growth on minimal medium. the gene was cloned and found to encode a protein of amino acids, a "half-transporter" which likely forms a dimer. ibi~tlnofluorescence on yeast expressing the gene cmyc-tagged at the c-terminus reveals co-localization of the protein with porin and with mitochondrial dna, visualized by dapi staining. nycodenz-purified mitochondria are enriched for the protein by immunoblotting. additionally,the c-myc epitope is protease-protected in mitoplasts, indicating an inner membrane sub-localization and a probable orientation with the c-terminus in the matrix. the gene, termed atmi for abc transporter of mitochondria, thus encodes the first member of the large abc transporter superfamily to be localized to this organelle. present work is aimed at revealing the substrate of this transporter. cycle in cardiac myocyte. the molecular study of this protein has been difficult even after its over-expression was made possible by the cloning of the corresponding cdna. the chief problem is the lack of convenient domains which could be used in the purification of the molecule. we have previously reported the efficient expression of the cardiac na+/ca + exchanger in mammalian cell lines using the t rna polymerase-hybrid vaccinia virus system. we have now constructed the recombinant virus for the exchanger with a xhis-tag at the c-terminal. this tag has enabled the purification of the protein through a ni-nta agarose column. the expressed tagged protein induced na-dependent ca uptake which was not different from that in intact cells, i.e., cells in which the exchanger did not have the tag, the most important aspects of the purification produced and those of the reconstitution of the expressed protein will be described. $ - the neural cell adhesion molecule l is involved in the formation and specification of cell contacts in the central and peripheral nervous system during development, and thus may also play a role in synaptic plasticity of the adult central nervous system, using extracellular recordings of evoked field potentials in the rat hippocampal slice, we found that locally applied polyclonal anti-l and fusion proteins containing the ig-domains i-vi of l specifically block the stabilization of ltp in the ca region. baseline synaptic transmission was not affected by the presence of the anti-l antibodies. the anti-ll-induced effect was dependent on the antibody concentration and anti-l antibodies had no effect on previously established ltp. ltp was also reduced by an antiserum against the recombinantly expressed ig-domains i-vi of l , whereas controt antibodies against liver cell membranes, which bind to neuronal membranes in the hippocampus, exhibited no effect on ltp. the contribution of l to stabilization of ltp within the ca synapses suggests that members of the ig-supertamily of cell adhesion molecules are involved in the structural modifications which are associated with synaptic plasticity in the mammalian central nervous system. an increasing number of surface proteins are described as being attached to the membrane by a glycosyl-phosphatidylinositol (gp ) anchor instead of the conventional transembranous hydropbobic domain. they are initially synthesized with a coohterminal polypeptide extension which is cleaved in the e.r. and replaced by the preformed glycolipid. this processing event is directed by a signal, located in the c-terminal region of the protein, which requires a group of three small amino acids positioned - residues upstream of an hydrophobic c terminal sequence. we previously transformed the transmembfanous glycoprotein d of herpes simplex type i into a gpi-anchored molecule by deleting its cytoplasmic domain and thus creating a molecule, gdww , which meets the requirements for anchoring via a gpi structure. we now demonstrate, using gpi-deficient cell lines and biochemical studies, that a hybrid protein consisting of the thy-i ectoplasmic domain and of the c-terminal region of gdww can be alternative[y expressed in a gpi-anchored or in a transmembranous form. service de p diatrie, chuv, ch- oll lausanne proteolipid protein (plp) is a major myelin protein in the central nervous system (cns). a number of x-linked dysmyelinating disorders have been identified as mutations in the pip gene. we have identified a novel plp mutation in paralytic tremor (it) rabbit, characterized by body tremor, spastic limb paresis and hypomyelination in the cns. reduced levels of plp protein and its mrnas were observed in the cns as well as in the pns. plp sequence analysis revealed a single nucleotide change in exon which results in the substitution of a histidine by a glutamine at position . histidine is localized on the boundary between the first transmembrane region and the intracellular part of the protein. therefore, its position can be crucial for the efficient plp interaction with other proteins and lipids, and correct incorporation of the plp molecule into the membrane. histidine is also part of a short fragment of ten amino acids which is conserved in all species studied. the same amino acid is altered in another hypomyelinated mutant, shaking pup, stressing its functional importance. furthermore, the pt mutation affects the plp "premyelin" functions, including oligodendrocyte maturation. equilibrium constants for octamer dissociation as well as dissociation rates in vitro increased in correlation to the number of charged residues eliminated, e.g. mutant - , in which all four charged residues in the n-terminal heptapeptide are substituted with uncharged amino acids, showed a -fold higher equilibrium constant for octamer dissociation and a -fold increased dissociation rate compared to wild type. this strongly suggests that the charged amino acids in the n-termlnal heptapeptide of mib-ck, and therefore an ionic interaction, play an important role in forming the oetameric molecule. gangliosides and neutral glycosphingolipids (gsls) cell surface molecules are involved in a variety of biological processes and are assumed to play an important role in the mechanisms of hiv entry into lymphoid and epithelial cells. the total lipid-bound sialic acid (lbsa) content and the composition of gangliosides and gsls were investigated on pbmn cells from hiv+ve and normal donors. lbsa level was quantified by a fluorimetric assay, while gangliosides and gsls were assayed with high performance thin layer chromatography (hf'tlc) after multistep lipid extraction of x g membrane fractions. in normal pbmn cells the lbsa content accounted for . /~g/ cells or nmol/mg protein whilst in pbmn cells from hiv+ donors the lbsa level decreased to . ,ug/ cells or nmol/mg protein. the qaantitation of neutral gsls and gangliosides was carried out by scanning spots revealed on hptlc plates and the integrated areas computed with a dedicated software. our findings showed that the most relevant ganglioside present in normal pbmnc was gm ( - % of total gangliosides) followed by small amounts of od ( - %) and other acidic glycosphingolipids. neutral gsls included gl , gl , gl and gl -hexosylceramide. hiv+ pbmn cells were characterized by a decrease of neutral glycosphingolipids as well as gm content which represented only % of the ganglioside fraction, indicating an alteration in the glycolipid composition in the plasma membrane of infected cells. the neuropeptide y (npy) is one of the most abundant peptides of the mammalian brain. upon stimulation of cell surface receptors, npy generates diverse physiologic responses. npy acts on the cardiovascular system. it is a vasoconstrictor by itself and in addition, it potentiates the action of vasoactive substances such as angiotensin ii or norepinephrine. npy also inhibits glucose induced insulin secretion and is a potent inducer of appetite. to facilitate the development of npy antagonists we are investigating the interaction of npy with its cell surface receptor.we first constructed a series of mutants in which negatively charged residues present in putative extracellular domains of the y receptor were systematically replaced by alanines. the mutant cdnas were transiently expressed in hela ceils using a vaccinia virus derived expression system and the abi}ity of the encoded proteins to bind npy was evaluated. the capacity of the mutant proteins to be recruited to the cell surface was assessed by confocal microscopy. we identified initially spacially clustered extracellular acidic residues of the human y npy receptor essential for npy binding. subsequently, we mutated additional residues. based on these data a detailed computer model of the interactions between npy and its receptor was built. functional activity to energy metabolism. l. pellerin and p.j. magistretti. institut de physiologie, universit de lausanne, switzerland. astrocytes play a central role in brain energy metabolism. for example glucose, the exclusive brain energy substrate, is avidly taken up by astrocytes (pnas : , ) . application of glutamate (glu), the main excitatory neurotransmitter, stimulates in a concentration-dependent manner h- -deoxyglucose ( - ( ) uptake by astrocytes in primary culture with an ec of ~ ~m. the effect is not receptor-mediated since it can not be reproduced by glutamatergic agonists such as (nmda, kainate, quisqualate, t-acpd) nor prevented by antagonists (apv, cnqx, ap ). instead, it involves glutamate uptake since the effect is blocked by dl-threo-j hydroxyaspartate, a potent inhibitor of the glu transporter. replacement of na + in the medium by choline also prevents the effect, suggesting a na+ driven process. finally ouabain, a selective inhibitor of the na+/k + atpase, completely prevented the effect of glu. these observations suggest that when glutamatergic synapses are active, glutamate, taken up by astrocytes, stimulates -dg uptake by astrocytes whose end-feet are known to surround capillaries, i.e. the source of glucose. they also provide a simple explanation for the mechanism linking functional activity to energy metabolism. cloning and molecular characterization of the mouse astrocyte glycogen synthase. g. pellegri, c. rossier, s. van berchem, p.j. magistre~i and j.-l. martin. institut de physiologie, universit de lausanne, switzerland. in the brain glycogen is predominantly localized in astrocytes, although its presence has been detected in certain large neurons, in ependymal and choroid plexus cells. glycogen is synthesized by the enzyme glycogen synthase (glys) from udp-glucose. out of the different glycogen synthase isozymes, only the muscle and the liver forms of glys have been cloned. we have isolated and characterized from a mouse astrocyte ~.zapii cdna library, a cdna clone encoding the mouse cerebral cortical astrocyte glys. the . kb clone isolated contains an open reading frame of bp encoding a protein of amino acids. the coding region of the mouse astrocyte glys cdna shares % and % identity with the nudeotide sequences of the human muscle and the rabbit muscle giys cdna respectively. the cellular distribution of the glys mrna studied by northern blot shows the presence of a single transcript of kb in cultures of astrocytes and, to a lesser degree, of neurons. the distribution of glycogen phosphorylase, which was also examined by northern blot shows the presence of a . kb transcript both in cultured astrocytes and neurons with however higher levels expressed in astrocytes. the functional molecular size of the vacuolar h+-pyrophosphatase (h +-ppase ec . . . ) from maize (zea mays l.) roots was estimated by radiation inactivation, both for substrate hydrolysis and for proton transport. light microsomal vesicles enriched in tonoplast membranes were prepared from young ( days) maize roots using sucrose step gradients. these vesicles were still competent for ppi-dependent proton transport after freeze-drying and rehydration of the membranes. frozen native or freeze-dried samples were irradiated with ~ for various periods of time. after thawing the samples, the activities of glucose- phosphate dehydrogenase (added as an internal standard) and h+-ppase (hydrolysis of pyrophosphate (ppi) and ppi-dependent proton transport) were tested. by applying target theory, the functional size for hydrolysis was found to be around mr when the native or freeze-dried samples were irradiated. no ppi-dependent proton transport activity was measurable after irradiation of the native samples. on the contrary, the freeze-dried membranes were still pumping protons after irradiation and a target size of around mr was measured for the transport activity. these experiments suggest that the native h+-ppase from maize roots may exist as a dimer (at least) of the mr catalytic subunit. $ - the na,k-pump moves na + out and k + ions into the cells. the domains of the protein involved in ion translocation have not been determined. we have studied the role of the n-terminal region in the na + translocation by measuring the kinetics of charge movements under na/na exchange conditions in wild type (wt) and two n-terminally truncated forms of the bufo ~ subunit with deletions of the (t ) and (t ) first amino acids expressed in xenopus oocytes. we have developped a new technique to perform fast (< i ms) voltage clamp on whole oocyte. the membrane on one side of the oocyte is permeabilized by digitonin allowing to measure current flowing across the other half. we observed presteady state ouabain-sensitive currents similar to that reported by other techniques (j.gen. physiol. : , ) . the estimated forward charge translocation rate was lower in both n-truncated mutants (wt • , t z , t z s-l). the midpoint potential of the charge translocation (best fit to boltzman equation) was - , - , and - mv in the wt, t and t groups, respectively. the highly charged nterminus of the q subunit has a significant role in the forward translocation of na + ions. deletion mutants of human small intestinal lactase-phlorizin hydrolase (lph) were constructed to determine the role of protein subdomains in the intracellular transport of lph. the mutant lph mact ( aa deletions), which contains the membrane anchor (ma) and the cytoplasmic tail (ct), was transported to the cell surface in a fashion similar to wild type lph. by contrast, lph lacking the transmembrane domain persisted as a mannose-rich polypeptidc. this suggests that the membrane anchor and/or cytoplasmic tail are implicated in the er-egress of lph. another mutant, lph mact ( aa deletions), was not efficiently transported to the golgi apparatus. epitope mapping with monoclonai antibodies as well as protease-sens{tivity assays provided an evidence that the tertiary structure of lph mact is very similar to that lph mact. in view of the variations in the proportions of the complex glycosylated molecules of the two mutants we conclude that the domain between lph mact and lphi mact may play an essential role along the secretory pathway of lph. containing tyrosine. hussein y. naim, and michael g. roth; dept, of biocherni~try, ut-southwestem medical center, at dallas, dallas,texas-usa we have investigated an artificial internalization signal created by site-directed mutagenesis of the short cytoplasmic sequence of the influenza virus hemagglutinin (ha). mutation of cysteine to tyrosine converted ha from a protein essentially excluded from coated pits to one that internalized at rate similar to some cellular endocytic receptors. to determine whether or not the ha-y signal is a degenerate form of the internalization signal found in proteins such as the transferrin receptor and the mannose- -phosphate/igfii receptor, we have mutated amino acid positions in the ha-y shown to be important for internalization of the two receptors. we have changed amino acids on either side of y to those that either fit, or break the pattern of aromatic and hydrophobic residues proposed as consensus sequence. our results indicate that the ha-y mutant contains a sub-optimum sequence for a tyrosine-based internalization signal similar to those found in the receptors for transferrin, ldl, and mannose- -phosphate/igfil. however, amino acids with side chains having different chemical properties functioned well in positions that are important for the internalization signal, indicating that the consensus sequence for this signal is more variable than previously proposed. n-linked glycesylation is an essential protein modification occuring in all eukaryotic cells. core-oligosaccharides are transferred by the enzyme n-oligosaccharyl-transferase frc~ the donor glc man gicnac -dolichol to selected asparagine . resldues of t~e nascent polypeptlde chazn. the donor is build up in a stepwise fashion at the er-membraneby the products of the alg-genes (asparagine-linked-~lycosylafion) mutants defective in this pathway are available, but often lack a phenotype suitable for isolation of the corresponding genes. we found that double mutants of alg or alg with a wbpl mutation (defective oligosaecharyl-transferase) are severely growth retarded at oc. the double mutant phenotype allowed the isolation of both alg and alg genes which have been refractory to cloning so far. both genes encode potential trarsmenbrane proteins and are able to complement the biochemical defects of the corresponding alg or alg mutants. the na,k-atpase produces the driving force for the regulated transport of na across epithelial ceils. it is composed of an c~-subunit which carries the catalytic and ouabain binding sites and a b-subunit. to test the role of the usubunit in transport regulation, we transfected a cells with the b. marinus al-subunit (txl-tbm). this subunit confers a -fold higher resistance to ouabain (k i = /~m) compared to the endogenous pump. taking advantage of this difference, stable transfectants were selected with ouabain. expression of the cd-tbm subunit was visualized by western blotting. approximately half of the positive cell lines showed a high electrical resistance similar to untransfected cells, when cultured on filters. na-transport activity, measured as isc, was lower even in the absence of ouabain, but the relative increase in response to aldosterone was maintained. functional pumps containing the exogenous or endogenous al-subunit were quantified by equilibrium ouabain binding. interestingly, cells cultured on plastic dishes had a large fraction of low affinity binding sites (txi-tbm), while most sites were of the high affinity type when the cells were grown on filters. we conclude that the ouabain-resistant na,k-atpase cd-subunit of b. marinus can be expressed in a cells and used as a selective marker. however, the results suggest that the expression of functional ouabain-resistant pumps containing the al-tbm subunit could be influenced by the degree of cellular differentiation. in infected cells fully functional na/pi-cotransport was found in a time dependent manner; up to a -fold stimulation of na/p;-cotransport compared to uninfected cells was observed aft r - days. transport of phosphate by infected cells was highly dependent on the presence of sodium, exhibited a kmtd~ of around . mm and was dependent on extraceuular i~h'. in agreement with expressed transport of phosphate, infected cells produced an immunoreactive polypeptide of kda that represents a non-glycosylated form of the to kda mature na/p;-cotransporter. we conclud$ that the protein napi- /rat when expressed in sf cells exhibits na/p-eotransport with similar kinetic characteristics as observdd in prox ma tubular apical na/pcotransport. therefore the napi- protein as expressed h sf cells can be used for further structural and functional studies. talin interacts in vitro with actin, vinculin, integrins and bilayers of acidic phospholipids, and nucleates actin filament growth at the bilayer surface. it is therefore believed to be a key protein mediating aetin-membrane linkage. we have analyzed functional properties of proteolytic fragments of purified platelet talin. using limited proteolysis two fragments of and kd were generated. preliminary results, obtained using viscometry and f-aetin-nucleating assays, suggest that the kd fragment contains the actin binding site. we also assessed the lipid-binding capacity of the fragments. when a mixture of the two fragments was centrifuged in the presence of large multilamellar liposomes containing phosphatidylserine, only the kd fragment, but not the kd domain, co-sedimented with the liposomes, suggesting the presence of a specific lipid-binding site in the kd domain. interestingly, this fragment contains the n-terminus of talin which shows homologies to the membrane-binding regions of protein . . we are now analyzing membrane-binding properties of talin domains in more detail using hydrophobic photolabelling. recent studies suggest a role of the neural cell adhesion molecules l and ncam in mechanisms of memory formation (doyle e, et al, j. neurochem. : - , scholey a.b. et al, neuroscience : - , ; lijthi a. et al. unpublished data and see usgeb ) . in the present study we analyzed the effect of chronic intraventricular infusion of polyclonal antibodies against l (anti-u) or ncam (anti-ncam) on the performance of male wistar rats during the acquisition and retention of a spatial learning task. briefly, rats had to remember the position of a submerged escape platform in a water tank (morris water-maze). when the escape platform was removed after acquisition training (= retention test), the performance of animals chronically injected with anti-l showed an impaired search pattern when compared with that of salineqnjected animals (p> . , mann-whitney). whereas control animals spent up to % of their time searching for the platform in the correct (= training) quadrant, the time anti-l animals swam in the correct quadrant was closer to chance level ( %). although monitoring penetration of antkncam into the hippocampus was almost as efficient as that of anti-l , the performance of the anti-ncam-group was highly variable and did not differ from the performance of controls. these results agree with recent findings on the involvement of neural cell adhesion molecules in other forms of learning. we have isolated cdnas from mouse and from xenopus encoding the ~-subunit of the gastric h,k-atpase, which is expressed in the parietal cells of the stomach mucosa. the gastric h,k-atpase transports h + into the stomach lumen with the counter transport of k +, all at the expense of atp hydrolysis. when xenopus oocytes are injected with crna encoding ~h,k from either species, as well as a gastric ~h,k subunit, the two subunit proteins are synthesized, assemble, and form functional pumps located in the plasma membrane as demonstrated by rb+ uptake. the sensitivity to the gastric h,k-atpase inhibitor sch was determined, with the ki for beth species found to be in the low ~m range. spodoptera frugiperda ceils are often used to overexpress eucaryotle proteins using baculovirus vectors. the atomic force microscope (afm) is a device which allows the observer to obtain high resolution images of biological samples immersed in a fluid medium; this instrument has therefore the potential to visualize overexpressed proteins on the surface of living cells. however, the interpretation of the images is sometimes difficult and requires a good knowledge of the specimen topography, i.e. the "normal" plasma membrane of the cell used for the overexpressisn. on our poster we present afm images of the plasma membrane of both fixed and living spodoptera frugiperda ceils. in additon, we discuss the possibility of achieving high resolution in the imaging of dynamic changes which affect /iving systems. the deduced protein sequence consists of amino acids with about % amino acid sequence identicy when compared to mammalian ~h,k-subunits. data from other species and from the structurally similar na,k-atpase has shown that the ~-subunit is necessary for the formation of a functional pump, which consists of an ~/~ heterodimer. following co-injection of xenopus oocytes with crna for both the ~-and ~subunits, we have observed by rb + uptake h,k-atpase activity in the plasma membrane. we are presently studying the role of the ~-subunit in the maturation and function of the h,k-atpase. the atomic force microscope (afm) is a new type of microscope which allows visualisation of inorganic and organic samples with a very high resolution. a sharp tip fixed at the end of a cantilever is used as a topographic sensor. by scanning the sample with this device, a computer records the minute deformations of the cantilever and computes the topography of the sample. the instrument allows also the measurement of the interaction between the tip and the sample as a function of the distance. the knowledge of these interactions allows us to compute several mechanical properties like indentation, elasticity and stiffness. on this poster we report the results of these measurements on cells (sfg, c ), bacteria (e. coli, b. subtilis) and proteins (actin). in addition we discuss the use of the afm as a sensor for probing the mechanical properties of biological systems at a nanometric scale. neonatal thymectomy of balb/c mice induces aig. we have cloned the ~-and ~subunits of the balb/c h,k-atpase and expressed the proton pump in crna injected oocytes. both and ~ proteins are made, form functional pumps, and are immunoprecipated using auto-immune sera. the ~-subunit can also be in~unoprecipated independent of the ~-subunit when oocytes are injected with ~ crna alone. balb/c mice will be immunized with oocyte membranes containing either the ~/~ or ~ antigens in order to study the role of each h,k-atpase subunit in triggering aig. the prion protein is expressed in both astrocytes and oligodendrocytes of the neonatal brain and is down-regulated in adulthood. m. moser, colello, r, , pott, u, and b. oesch institut for hirnforschung der universit~t zorich, august forel str. , zorich the infectious agent causing transmissible spongiform encephalopathies consists largely or entirely of prp sc, a modified isoform o| normal prp (prpc). prpc is most abundant in the brain. the development of the disease strictly depends on the presence of prpc. and incubation times are shortened by overexpression of prpc. the normal function of prpe is not known and its cellular iocalisation in the cns is poorly characterised, except for its evident presence in neurons. here we describe the expression of prp c mrna in both oligodendrocytes and astrocytes during neonatal development of hamster and rat. in adult animals, expression is down-regulated in gila but not in neurons. our findings provide a link to the previously described accumulation of prpsc in astrocytes and the apparently faster course of prion disease in neonatal hamster. our results argue for a major involvement of non-neuronal cell types in prion diseases. such structures we raised mab against membrane protein fractions. a ~lab against a vitronectin receptor containing fraction was found, which if applied in vitro to bi fi mouse melanoma cells, influenced growth, adhesion and morphology. ~tnen bi fi cells prior to tail vein injection were treated, different outcomes were observed. short treatment (lh) resulted in % inhibition of lung colonization whereas long-ter~ treatment ( weeks) resulted in - times more lung colonies. a candidate molecule likely involved in these observed effects could be identified as a kda cell surface protein. sequence information from peptides revealed in several cases % homology to mouse centrosomin a, i.e.a kda cytosolic protein involved in the organization of the spindle apparatus. these data suggest that we may have found a protein which may participate in a link between the extra-cellular space and the microtubular system. we are currently trying to clone this kda protein from a bi fi c-dna library. wahlberg, j.m., and spiess, m. dept.biochemistry,biozentrum, university of basel. signals for correct basolateral sorting of subunit hi of the human asialoglycoprotein receptor are located in the cytoplasmic tail, involving a tyrosine at position . removal of this residue results in nonpolarized transport of the protein to the surface in mdckii-celis. a deletion mutant of hi, lacking of the normal residues of the cytoplasmic tail, including the tyrosine, has been found not to be transported to the cell surface, but to be accumulated intracellularly. the mutant protein is complex glycosylated and sialylated, indicative of passage into or through the trans-golgi. immunofluorescence analyses show defined juxtanuclear, tubular structures, which do not colocalize with markers for er, early endosomes or lysosomes, but which show similar staining pattern as markers for late endosomes and tgn. to define the determinants responsible for the intracellular accumulation a series of deletions and fusions have been constructed. the results suggest that the length of the cytoplasmic domain is one important determinant for missorting of hi. salerr~ n., medilansld, j., pellegrinelli, n. and eder-colli, l. departement of pharmacology, cmu, geneva in chollnergic neurons the enzyme choline acetyltransferase (chat) exists as cytosolic( - %) and membrane-bound activity ( - %}. are these two activities encoded by a single mrna? is membranebound enzyme preferentially associated with a given cellular membrane? in order to investigate these questions we isolated a fulllength cdna ( . kb) encoding drosophila chat and used it to transfect xenopus oocytes, rat fibroblasts and human neuroblastoma cell lines. triton x- fractionation of transfected cells showed that hydrophilic and amphiphilic chat activity were produced. the sp act. of hydrophflic enzyme reached , . and . ~mol ach/h/rng protein respectively in oocytes, fibroblasts and neuroblastoma whereas the sp. act. of amphiphilic enzyme was . , . and . hinol ach/h/mg protein, respectively. amphiphilic chat was less sensitive to inhibition by the product acetylcholine and to heat denaturation than was hydrophilic enzyme. similar results were observed for the native drosophila chat. amphiphillc enzyme sedimentation was affected by the type of detergent present in linear density gradients of sucrose. transfected oocytes were subjected to subfractionation. besides a large amount of soluble chat activity, a significant proportion of enzyme was found associated with a fraction enriched in endoplasmic reticulum (er). preliminary results using transfected mammalian cells indicate that apart cytosolic chat, plasma mernbranes+golgi enriched fractions as well as er-enriched fraction contain chat activity. we have characterized a drosophila melanogaster gene encoding a very hydrophobic protein. the amino acid sequence shows considerable homology ( - % identity) to neurotransmitter transporters. the gene is, however, not expressed in neural tissue, but in the male germline. transcription and translation occurs in early spermatocytes. antibody staining data suggest the following pathway of the protein during spermatogenesis: the protein is synthesized in early spermatocytes and is transported into the nucleus during prophase of rt i. at meiosis it associates with the mitochondria and it stays associated while they fuse to the nebenkern. during individualization it is stripped off in the cystic bulge. inspection of the putative protein sequence shows that it possesses alle the necessary targeting signals to allow its transfer from the cytoplasm through the nucleus into the mitochondria: three nuclear targeting signals, a mitochondrial import signal and, moreover, two pest-sequences for rapid protein degradation are present. we shall present a, currently tested, working hypothesis. in hg-treated toad skins, water permeability is high or low depending on whether the major anion of the inner bathing medium is so~ or ci. we report here the results of experiments designed to determine if, in skins bathed with so -ringer, net water flow (j~,) could be diminished by agents added to the outer medium. toad skins were mounted inbetween glass chambers and exposed to a standard osmotic gradient of mosm. jw was continuously monitored by automatic, volumetric techniques. exposure to lmm hgclz or chzcihg (external side) led to the typical anion-induced j,, changes. re-exposure to hg caused a mild ( %), although significant (n= , p< . ), fall in jw. since cuso is a sh-reagent like hg, we looked at the effects of cuso (lmm). there was a % reduction of j, (n= , p< . ), an effect totally reversible by simple washing of the skins. likewise, addition of dithiothreitol (dtt, mm) in the presence of cu, caused a % reversal of the cu effect ; there was no change in jw if dtt was given before cu, or if both agents were added simultaneously. finally, in hg-treated, glutaraldehyde-fixed skins, lmm and ram cuso caused % and % jw decrements, respectively, that were totally reversible. in conclusion, cu causes a dtt-sensitive inhibition of jr, in hgtreated skins. further work is needed to establish if the cu effect is exerted on sh-groups of apical aquapories of toad skin. $ - the amino acid sequences of the pc- e-subunits and other cryptomonad biliprotein ~-subunits pose the most intriguing question about the phylogenetic origin of these unique chromopeptides. cryptomonad pc- ~-subunits seem not to be closely related to the known phycobiliproteins, linker polypeptides (lpps), bpe or any other light-harvesting chl polypeptides from the thylakoid. for most of them the number of identical amino acid residues was %- %. sequence alignment of c-pe associated lpps with t-subunits from cyanobacteria and red algae and the cryptophytan ~-subunits however show that the identity between c-pe associated lpps and cyanobacterial x-subunits is still significant, whereas it reaches the limit of significance between cyanobacteril and eucaryotic x-subunits. nevertheless it has to be assumed that x-subunit derive from cyanobacterial c-pe associated lpps as well as cryptomonad ~-subunits may derive from t-subunits. whereas the the phycobiliprotein ~-and subunits remained structurally conservative during evolution (e.g. - % identity between cyanobacterial and red algal pe-subunits) the lpps and t-subunits show drastical changes in their chromophore binding domains. $ - functional and evolutionary relationships of the components of the primary events in photosynthesis have been traced using amino acid sequence comparison. basically, the question was posed regarding the interrelationship of the apoproteins of different photosynthetic organisms which gather light and/or separate charges across membranes. do their protein-chemical structure and organisation decipher us certain clues and parts of the path by which they have been selected and derived from a possible primordial reaction centre polypeptide? within that context the light-harvesting-and energy transfer systems of purple bacteria offered as ideal model components. a data set of more than apoprotein sequences has pointed to some keystructural elements which have been partially verified as such by limited proteolysis and genetic engineering. a careful inspection of the determined bacterial antenna-structures revealed some remarkable similarities to eukaryotic antenna and reaction centre apoproteins indicating possible basic structural motifs for complexing pigment molecules, like chlorophylls or bacteriochlorophylls in very distant representatives of phototrophs. a number of glycoproteins of the nervous and/or immune system that are involved in cellular recognition and/or adhesion share the common lz/hnk- carbohydrate structure. in a large proportion of patients with peripheral demyelinating neuropathy associated with paraproteinemic gammopathy (ppn), monoclonal igm antibodies (m-igm) react with the lz/hnk- determinant of mag and p . we plan to test the hypothesis that anti-mag m-igm autoantibodies may alter the expression of hnk- bearing myelin proteins such as mag, p , mog, ncam or pmp . by using monoclonal antibodies to myelin proteins on tissue sections, preliminary results indicate that mbp protein content seems to be strongly downregulated, whereas galc and $ protein do not appear to be affected by the loss of myelin in ppn nerves. gfap protein expression appear to be upregulated in demyelinating biopsy specimens from ppn patients with anti-mag m-igm gammopathy. in conclusion, the expression of some l /hnk- negative proteins might be disregulated by the presence of circulating anti-mag m-igm in the serum of ppn patients. we have isolated a human liver na+-taurocholate cotransporting polypeptide (ntcp) by screening a human liver edna library with a rat edna probe derived from ntcp (pnas , , ) . the cdna codes for a protein of amino acids which shows % amino acid homology with the rat ntcp. expression of ntcp in x. laevis oocytes resulted in na+-dependent bile acid uptake which exhibited saturability with an apparent km for taurocholate of ~m. ntcp mediated taurocholate uptake into oocytes was inhibited by all major bile acid derivatives, bumetanide and bromosulphophthalein. southern blot analysis of genomic dna from a panel of human/hamster somatic cell hybrids mapped the human ntcp-gene to chromosome . in conclusion, these studies supplement the previously suggested selective mammalian distribution of the ntcp gene family and provide the possibility for direct molecular characterization of human bile acid transporter genes. diverse cell surface molecules of the nervous system play an important role in specifying ceil interactions during development. with the aid of a polyclonal antibody against l , a .skb edna clone closely related to l (ch-l ) was isolated from a ~. gtl library derived from poly(a)* l~qa of day mouse brain. using this edna as probe the remaining cdi'ia ' sequences were cloned out of a plasmid library constructed from postnatal day - mouse brain poly (a)+ p, na. two independent full length clones of . and . kb encompassing the entire coding region of chl] were isolated. these represent putative alternatively spliced mp, nas. the deduced primary structure of chl reveals that, like the cell adhesion molecules (cams) mouse l , chicken ng-cam, and nr-cam (bravo), chl is composed of six ig-like domains, a transmembrane domain, and a short cytoplasmatic region. in contrast to the other l family members, only four and a half instead of five fibronectin type hi-like repeals are found in chl . the fibronectin type [h-like repeats were expressed in e. col[ and the protein fragment was used for immunization. as observed for li, ng-cam, and nr-cam, chl is susceptible to proteolytic cleavage. bands of kd, kd, kd, and kd could be detected bywestern blot analysis. by in situ hybridization, a predominant expression by neurons was found in the adult mouse brain. western blot analysis showed expression of chl protein in brain and heart, but not lung, kidney and intestine. in liver, a kd immunoreactive band was found. the relationship of chl to molecules known to be involved in cell adhesion and neurite outgrowth, combined with its pattern of expression, suggests a role for this molecule in cell interactions dndng neural development. work from this laboratory revealed that in hg-treated, glutaraldehydefixed toad bladders, the water permeability coefficient (pf) is high in sod-ringer and low in ci-ringer ; in all these studies the osmolality of the serosal medium was mosm. as an attempt to determine the mechanism(s) underlying such pf changes, experiments were carried out with bladders whose serosal surface was exposed to [so-, hypo-or hyperosmotic media ; the outer surface was always exposed to a hyposmotic solution ( costa). net water flows were measured continuously with automatic, volumetric techniques. after exposure to mm chzcihg ( ', outer medium) followed by fixation with . % glutaraldehyde ( ', serosal medium), the bladders were thoroughly washed. in sod-ringer, pf (/~m/sec) was as follows (n= ) we wanted to determine the molecular weight (mw) of ntcp and its topology in the plasma membrane (pm) of rat liver. a rabbit antiserum generated against the c-terminus of ntcp was used to determine its mw on western blots and its subcellular distribution in liver and in hepatocytes by immunofluorescene (if). site directed mutagenesis and deletion experiments were used to determine the n-linked glycosylation sites. ntcp encodes a kd protein in rat liver pm vesicles. the basolateral localization of ntcp in liver was confirmed by if. ntop could only be immunostained in permeabilized hepatocytes suggesting an intracellular localization of the cterminus, cnda constructs showed the ntcp is glycosylated at positions and ii. the data show a selective basolateral expression of ntcp in rat liver and they suggest that the two ends of ntcp are located on opposite sides of the pm. zymogen granule membranes (zgm) are known to contain proteins with nucleoside phosphatase activity, but their exact nature and function are unknown. the activities require ca ++ and mg ++ and are sensitive to atpase inhibitors but not to inhibitors of na/k-atpase activity. the aim of this study was to isolate individual proteins of pig pancreatic zgm, to attribute enzymatic activity to individual proteins and to characterize them with various substrates (gtp, atp, amp etc.), inhibitors (amp-cp, amp-cpp, gtpgs etc.) and lectins (maa, dsa, gna etc.). we have subjected highly purified zgm solubilized in chaps to ief on immobiline dry strips | (pharmacia) with s ph range -- . (ist dim.). these strips were then electrophoresed in a polyacrylamide gradient gel containing . % chaps and tris/hci at ph . ( nd dim.). nucleoside phosphatase activity was detected histochemically in the d gel by incubation with substrate in the presence of lead nitrate. focussed strips were also subjected to sds-page for comparison. several proteins showed strong activity to gtp, atp and itp. the role of these phosphatases are discussed in the context of the role of gtp-binding proteins and the de-/phosphorylating events on the zgm in intracellular targeting and exocytosis. the disease-specific isoform of the priori protein (prp sc ) is an essential part of the infectious particle causing scrapie or bse. prp sc differs from prp of normal animals (prp c ) by its relative protease resistance. the physical nature of this difference is still unknown. here we analyzed the protease-resistance of prp sc quantitatively. prp sc was rendered completely protease-sensitive at alkaline ph or in guanidinium thiocyanate (> . m). denaturation in m guanidinium thiocxanate (gdnscn) completely abolished protease resistance of prp bc within min while denaturation in m urea showed a much slower time course. denaturation cuwes were used to calculate the gibbs free energy (as d ) in dependence of gdnscn or urea at different concentrations. the linear relationship between agd and the denaturant concentration is suggestive of a two state model involving the conformational change of a single protein domain. this is also reflected in the small number of side chains ( , ) additionally exposed to the solvent upon conversion of prp sc to its proteasesensitive isoform. our results suggest that minor rearrangements of the structure of prp sc are sufficient to abolish its protease resistance. of physiology, university of zurich, winterthursrstr. , ch- zt~rich in contrast to mammalian kidneys where inorganic phosphate (pi) is reabsorbed unidirectionally, flounder renal epithelial cells both re.absorb and secrete pi. in order to investigate on a molecular level the cellular pi handling we have cloned a re,absorptive nalp i transport system from flounder renal tubules. based on homology with the cloned na/p i cotransporter from rat we have isolated a edna clone (flounder napi-ii, bp) encoding for a protein of amino acids. in the eight transmembrane spanning domains the protein is % identical with the corresponding system from rat kidney, in the hydrophilic regions only %. expression of in vitro transcribed rna in xenopus oocytes specifically stimulated na-dependent pi transport. binding properties of na and pi as well as the ph-dependency were similar to the ones found in mammalian systems (rat, human, ok cells). by northern analysis three transcripts, . , . , and . kb in length, could be detected. rna from flounder renal tubules contained all of the transcripts, intestinal rna only the two lower bands, and non-epithelial tissue from kidney expressed only the . kb transcript. mammalian systems share only the . kb transcript but not the related species. the close functional relationship of flounder napi-ii with the known najp i transport systems and the pronounced differences in primary structure provide the basis for structure function analysis of na/p i cotransport. the amyloid /a protein precursor (app) is a transmembrane protein expressed in different isoforms throughout the organism. our work has consisted in a detailed study of the structure and biological function of app. specifically, we have shown that the secreted form (sapp) of app- is involved in the growth regulation offibroblast% and also stimulates neurite extension in b neurnblastoma cell line. functional mapping studies have shown that the domain responsible for both the growth-regulating and the neurotrophic activities of sapp- is the rerms site, arg- to ser- . the presence ofsapp binding sites on the surface of b ceils (through the active rerms site) indicate that the neurotrophic activity of sapp is mediated by a receptor, and the subsequent activation of a signal transduction mechanism. in addition, we have shown that the rerms site is also involved in the neuronal survival properties of sapp- . finally, we have initiated an in vivo study of app biological function, and shown that a peptide representing the neurotrophic domain ofsapp- can strengthen memory retention in rat through stabilization of synaptic structures in the frontal cortex. this work was supported by grants from the swiss national science foundation ( a- ), and nih (ag ). we previously showed that in smooth muscle cells, the (~i-ar is rapidly phosphorymted following exposure to agonist. to understand this biochemical event, we investigated the phosphorylation of the c( b adrenergic receptor stably expressed in rat fibroblasts ( . pmol/mg prot.). the =( b-ar was solubilized from p-labeled rat cells and immunoprecipitated with antibodies raised against the n-terminus part of the receptor. treatment of ceils with ~.m epinephrine showed a rapid and significant increase in receptor phosphorylation above basal. treatment of cells with a specific pkc inhibitor (ro- ) did not affect agonist.promoted phosphorylation while it completely abolished phorbol esterinduced receptor phosphorylation. this strongly suggests that pkc is not involved in agonist-induced c{ b-ar phosphorylation. the elb-ar contains several putative phosphorylation sites in both its third intracellular loop and c-terminus tail. to investigate the role of these domains, a trunoated receptor (lacking its last amino-acids) was constructed and stably expressed in rat cells ( . pmol/mg prot). this receptor mutant displayed similar ligand-binding properties and abirity to activate phospholipase c as compared to the wild-type receptor. phosphorylation experiments revealed that this mutant is not at all phosphorylated, neither by agonist nor by phorbol esters. agonistinduced decrease in cell surface receptors, measured by binding of the antagonist h-prazosin at ~ was however similar for both the wild-type and mutant receptor. this suggests that loss of phosphorylation sites does not affect receptor internalization. human intestinal lactase-phlorizin hydrolase (lph) is synthesized as a precursor molecule (pro-lph), which undergoes proteolytic processing during maturation. lph expressed in cos- cells was enzymatically active, the electrophoretic properties of lph-species synthesized by transfected cos- cells correspond to those in organ cultured human intestinal explants, in caco- ceils and in transfected mock ceils. they included the pro-lph and a proteolytically processed form, which had similar electrophoretic properties to the mature enzyme. the appearance of the putative mature form was inhibited by brefeldin a, ammonium chloride and chloroquine. in addition, only complex glycosylated pro-lph (pro-lphc) was inserted into the cell surface membrane. these data indicate that in cos- cells pro-lph is inserted into the cell membrane, is internalyzed and enters lysosomes where proteolytic events lead to the appearance of a mature-like enzyme. $ - assembly and transport of paba peptide hydrolase alpha and beta subunits in cos- cells eldedng, j.a., gr nberg, j. and sterchi, e., institut for biochemie und molekularbiologie, universit&t bern. paba peptide hydrolase (pph) (ec . . . ), a metalloendopeptidase from epithelial cells of human small intestinal mucosa, consists of two subunits, ~ and , which form homodimers and oligomers, cdna cloning has revealed homologies with developmentally important proteins from different species and has led to the definition of a new family of metalloendopeptidases, the astacin family (j. biol. chem. , , ) . here, we report on the expression of the cdnas of pphcz and pphj in cos- cells. when expressed on its own, pph(z is synthesized in a core glycosylated form only, suggesting that it is transport-incompetent and not able to exit the er. immune-fluorescence studies have confirmed that pph~ is not expressed on the surface of cos- cells. pphl , on the other hand, matures and is transported to the cell surface. when the two subunits are coexpressed, ppho~ can also be detected on the cell surface, suggesting that a heterooligomeric assembly is necessary for the surface expression of pphtz. truncated forms of both pphc~ and pphi~ could be detected in the medium of transfected cos- cells. in our previous work, we demonstrated by immunocytochemical reaction that sensory neurons expressed nuclear triindothyronin receptors (nt r) in embryonic as well as adult rats. in contrast, in sciatic nerve, schwann cells exhibited only transient nt r immunoreactivity from e to postnatal day . in the present work, we attempt to demonstrate by radioautography that the detected nt r are effectively the binding sites of [ i] labeled triiodothyronin. cryostat sections of dorsal root ganglia (drg) or sciatic nerve were incubated with . nm of l, , , '-[t i]triiodothyronin ci/m mol (nen), while the controls were incubated with a large excess (llxm) of unlabeled trfiodothyronin (t ). in aduit rat drg, radioautographs revealed that silver grains were exclusively restricted to neuronal cell bodies, while the schwann cells ensheathing the axons were free of significant label. in newborn rat sciatic nerve, silver grains accumulated over schwann cells; in contrast in adult rat sciatic nerve, schwann ceils were free of label. in control drg or sciatic nerve sections incubated in large exceas of unlabeled " " , all the radioautographs exhibited only scattered silver grains. in conclusion, our results show that sensory neurons and schwann cells are able to express functional nt r which bind thyroid hormones (snf - - ). characterization of the maturepreeursor glycophospholipid for glycosylphophatidylinositoi anchors of saccharomyces cerevisiae. many attempts to isolate the mature yeast precursor gpi glycophospholipid ready to be attached to newly translated proteins for gpi-anehoring have failed although such precursors were previously identified in mammalian cells and protozoa. here we show that metabolically labeled glycolipids of the expected structure can be observed if the incorporation, their accumulation and their extraction are optimized. two very polar, ( h)-mannose-labeled glyeolipids named cpi and cp were identified and purified. they were structurally characterized using phnspholipase treatments, partial deacylation with methanolie ammonia, hydrofluoric acid dephosphorylation, nitrous acid deamination, acetolysis, exoglycosidase treatments and combinations thereof to produce labeled fragments which could be analyzed by paper chromatography or thin layer chromatography. cp and cp both contain the identical core oligosaccharide mangl, (x->po - )manal, mamxl, (y->)mana-glcn-lansitol, x and y being hydrofluoric acid-sensitive substituents (most likely ethanolamine and phospheethanolamine, respectively). the inositol is acylated although no acyllaositol is present on protein-bound yeast gpi-anchors. cp and cp can also be observed after lableling with ( h)myo-inositol, the lipid moieties of cp and cp can be completely removed by mild alcaline hydrolysis altough the protein-bound gpi-anchors made by the pmi cells under identical labeling conditions are completely mild base resistant. this finding reinforces the notion that the ceramide moiety typically found on the majority of yeast gpi-proteins are added only after addition of the gpi precursor glycolipid to proteins. the distributions of the mannose- -phosphat e/igfll-recept or and a , slalyltransferase in hepg cells: no evidence for co-localization by confocal laser scanning microscopy. the organization of the trans golgi apparatus (ga) with respect to (recycling) man- -p/igfii receptor (mpr) and (resident) c~ , sialyltransferase (st) has been investigated by double immunofluorescence laser scanning confocal microscopy in hepg cells. they were chosen because biochemical evidence suggested sialylation of mpr upon recycling (duncan & kornfeld, j. cell biol. , , - ) implicating colocalization of both proteins. in the steady state st was confined to the ga whereas total (endogenous) mpr was localized to coarse vesicles without evidence for colocalization by confoeal microscopy. endocytosis of surface-labeled mpr was monitored in presence and absence of brefeldin a (bfa): without bfa, early endosomes ( ' of warming up) showed no, late endosomes ( ') little if any colocalization with st. in presence of bfa, the st-compartment disappeared whereas late endosomes persisted with some tubular emanations. under similar conditions, in "absence of bfa, endogenous mpr concentrated in the ga region suggesting colocalization with st. in presence of bfa the mpr compartment formed a redculum whereas the st compamnem disappeared. in conclusion, within the limits of the techniques used, exogenous mpr was not detectable in the st compartment whereas endogenons mpr concentrated in the ga region but obvious co-localization was exceptional. supported by grant - . of the snsf to egb. cultures of dissociated striatal neurons prepared from fetal rats were grown in the presence of neurotrophin- / (nt- / ) as well as the other known neurotrophins, ngf, bdnf and nt- . we found that acute administration of nt- / to seven days old cultures stimulates the hydrolysis of phosphatidyllnositol, an event involved in neurotrophin signal transduction. growth of stdatal cultures in presence of nt- / resulted in increased cell survival as indicated by elevations in total cell number, protein content and number of viable cells. nt- / exposure increased gaba uptake and staining intensity in gaba immunocytochemistry in these cultures indicating atrophic action on gabaergic neurons. to further identify responsive cell populations we analyzed for calretinin, a calcium binding protein known to colocalize with gaba in a number of neuronal cells. nt- / strongly increased the number of calretinin positive cells in cultures prepared from rats of embryonic day , as we]l as calretinin levels determined by western blot analysis. when cultures were prepared from embryonic day rats, nt- / very strongly increased the morphological differentiation of calretinin positive cells. while all effects produced by nt- / were mimicked by bdnf with similar potency, nt- was found to be only marginauy effective, our findings identify nt- / as a potent neurotrophie factor for striatal gabaergic neurons. fusion of cells and organelles is a general patho-biological phenomenon: muscle cells, transport vesicles, langhans cells and virus induced fusion from within (ffwi, during replication) and without (ffwo, by the particles). in vivo and after virus isolation only little fusion can be seen, only after cultivation fusing hsv can be detected; a selective pressure must be released. syn loci are known on the genome; the syn locus is located inside of the cell in the carboxy terminal part of glycoprotein b, all other fusion domains are outside the ceil. fusion inducing mutations are in aa , , , , and and were correlated to fusion from within and without as well as to selective cyclosporin a effects. a model is presented for the syn locus. -by recombination the fusion capacity could be transfected to fusion negative viruses. -hsv penetrates the cellmembrane by fusion by ligand-recepter interactions. ffwo+-strains penetrate at ~ cell/cell fusion was analysed and found to need also receptor-ligand interactions if induced by syn mutations. timing analysis of fusion events and by certain inhibitors allow further conclusions. quenching of lhcii isolated from dark-adapted (lhcii-d) or preilluminated (lhcii-l) rye leaves was similar in both preparations, but reversibility of this process within two hours of darkness was slower in lhcii-d. no differences in fluorescence quenching and full reversibility was observed for both lhcii preparations in reverse micellar solution. xanthophyll/chl concentrations in lhcii were consi-derably decreased under this conditions. excitation difference spectra (before and after illumination) of lhcii in asolectin liposomes showed typical changes of energy transfer between carotenoids and chl. a model is proposed according the xanthophyll cycle controls reversibility of light-driven excitation quenching as well as the xa~a~q~_x~tentof ~,,~nchi~g i/~ lhc~z. myelinogenesis is a complex, developmentally regulated process involving coordinate expression of myellnafion-related proteins. the myelin forming cells are the oligodendrocytes in the central nervous system (cns) and the schwann cells in the peripheral nervous system (pns). we differentially screened a rat postnatal day (p ) spinal cord edna library with probes derived from normal (plus probe) and from myelin-free (minus probe) p spinal cord mrnas. we succeded in the isolation of several novel edna clones whose corresponding mrnas were expressed either selectively by oligodendroeytes or by oligodendroeytes and sehwann cells. here we describe one of the edna clones (tentatively denominated ns ) whose mrna is specifically expressed by oligodandroeytes and schwann cells. the . kd transcript is expressed at highest level during myellnation and at much lower levels in the adult as it is known for other myelin-specific mrnas. sequence analysis of the full length edna sequence showed a % identity to the rat liver udpglucuronosyltransferase family, involved in the detoxificafion system. the amino acid open reading frame encodes a kd protein with a putative signal peptide and two transmembrane domains, whether this ns protein is involved in a detoxification reaction or in glycosilation of proteins and lipids with glueuronie acid is under investigation. neuroblastoma (n ) are sympathetic tumors of childhood characterized by mycn amplification in advanced stages. cd standard (cd s) and splice variants (cd v) represent a group of surface glycoproteins whose expression has been recently linked to metastasis. we analysed the expression of cd s and cd v on n tumors and cell lines by immunohistology and northern blot. results showed high levels of cd s on / nb, / n cell lines and / ganglioneuromas (mature, benign sympathetic tumors). lack of cd s staining was observed on stage iv, mycn amplified tumors only. in cell lines, expression was not detected on cells with a neuronal phenotype while high levels of cd s were expressed by cells with a glial differentiation, independently of mycn amplification. up-regulation of cd s was obtained with agents that induce a glial differentiation, while neuronal differentiation by retinoic acid was not accompanied by a change in cd s expression. no gd v were detected on tumors or cell lines. we conclude that cd s expression is down-regulated in a subset of nb ceils and tumors and that mycn product and additional mechanisms responsible for control of differentiation are involved in this regulation. the glycoprotein gc of herpesviruses is known to be non-essential for virus replication. non-essential herpesvirus proteins are suggested to perform "luxury functions" which may influence the outcome of an infection. we are aiming to analyse the function(s) of ibc specified by bovine herpesvirus (bhv ), bovine herpesvirus hv ) and caprine herpesvirus (caphv ). these viruses are closely related but differ in their pathogenic potential. in a first step we have cloned and sequenced the individual genes. the nucleotide and the deduced amino acid sequence homologies of two hv reference strains was found to be > %. the sequence homologies between bhv and bhv were > % and those between bhv and caphvl were > %. comparisons on the amino acid sequence level revealed that the differences were most prominent in the n-terminal parts, whereby the potential signal sequence region might be concerned. the putative glycosylation sites, however, were not affected, and the transmembrane sequences were similar in size and location. in order to characterize the significance of these differences, we are constructing gc-deletion mutants and recombinant viruses carrying a foreign gc gene. the in vitro and in vivo behaviour of these viruses will be compared to the wildtype virus. $ - o. moullet and j.-l. dreyer, instimt de biochimie, unlversit de fribeurg, ch- fribourg camp has been recently shown to promote a cell survival response by retarding apoptosis (berridge, m.v& el. ( ) exp. hematol. , - ) . on the other hand quinones are widely distributed substances of often potential toxicological significance. we have tested the effects of quinones on adenylate cyclase. the enzyme is rapidly inhibited by pbenzoquinone (ic = - ktm) or dicnlorophenol-indopbennl (/ ' = ilm), but -substituted quinones are inactive. the lc 's are decreased - times after membrane soinbilization but are not affected by gtp, gdp or analogues nor by cholera and pertussis toxins. the inhibition is not mediated by a g-protein or by the activation of a defined receptor. quinone inhibition stoichiometrically competes with forskolin activation of adenylate cyclase, equimolar concentrations of beth quinone and forskolin restoring the enzyme activity to its basal value. the cytotoxicity of benzoquinone, tested in vivo on hep-g , a human bepatocellular carcinoma cell line, could be prevented with forskolin. in plasma membranes quinones tightly bind to only one major and two minor proteins that were purified by page electrophoresis under native conditions. together these observations indicate that quinone action can be attributed to targetting to a limited number of proteins at tile plasma membrane in a highly selective way. by affecting adenylate eyclase, a modulation of the camp pool induces apoptosis as a result of the cytotoxicity via. t.congolense is an african,unicellular hemoflagellate, pathogenic for domestic animals such as cattle.within the host bloodstream,parasites adhere to the wall of the microvasculature,eausing severe organ damage.we have set up and characterized an in vitro assay in order to study the metabolic requirements,reeeptor-ligand interactions and the ultrastructure of this adhesion phenomenon.our findings suggest that adhesion is an active process requiring metabolic energy on part of the parasite, but is independent of the target cells.we also show that t.congolense possess a leetin-like domain localized at specific sites along the surface of the anterior end of the flagellum, which interacts with specific carbohydrate receptors, probably sialic acid and/or n-aeetylglueosamine residues,on the endothelial cell surface. this suggests that adhesion is likely to be mediated by a trypanosomal surface component distinct from the variable surface glycoproteins(vsgs). we also suggest that the cytoskeletal protein aetin is involved in this process. local immune response based upon intestinal parasitespecific t and b cells to giardia lamblia may be impomrant in parasite clearance. experimental infections qf mice (cr:nih:s) with g. lamblia (clone gem-h which e~presses a major kd antigen on its surface recognized b~ mab g / ) have demonstrated that the parasite undergoes surface antigenic variation in rive. experimental i~fection of immu/%odeficient mice (nu/nu mice and sci~d mice) and oontrol nu/+ littermates provided insights into associated immunological mechanisms: dominant surfade epitope-specific slga plays a major role in modulatin ] surface antigenic variation, and thymus-dependent t-lyn~phecytes in the induction of selfcure. athymio nu/nu mioe (zu. icr-strain) were reconstituted with immune peyer:~ patch lymphocytes obtained from self-healed littermat~ nu/+ mice. intestinal b-cells from these nu/+ mice @s well as from reconstituted athymic nude mice synthesized in vitro parasite-specific iga. this iga showed a predominant immunoblet reaction with the major surface antigen (a mr , polypep-tide) characterizing the giardia clone in question. nu/nu mice reconstituted wit~ immune cells acquired the potential to decrease significantly their intestinal parasite mass. thus the hypothesis on the causative role of intestinal iga in tl~ control of intestinal g. lamblia infection has found further experimental support. stephan jentsch, heinz tobler and fritz m er, institute of zoology, university of fribourg, p rolles, " fribourg the process of chromatin diminution in the parasitic nematode ascaris lumbricoides leads to the formation of somatic cells that contain less dna than the germ-line cells. during this process, which occurs between the third and the fifth embryonic cleavage divisions in five different blastomeres, the termini of the chromosomes are cut off and eventually degrade in the cytoplasm. to analyze this process at the molecular level we constructed a xzap somatic telomere library. the analysis of three cloned telomeres and their corresponding germ-line genomic regions revealed that chromosomal breakage takes always place within short specific chromosomal regions (cbrs) and is followed by the addition of the telomeric sequences (ttaggc)n to the breakage sites. the different cbrs do not crosshybridize to each other. a comparative nucleotide sequence analysis is now being performed to screen for the existence of conserved sequence motifs. in a parallel approach we analyze the chromatin structure of the cbrs and their flanking regions in developmental stages before and during the elimination process, putative recognition or regulation sequences important for the elimination process might be recognized as dnasel hypersensitive sites. once such sequences will be identified, it should be interesting to establish their role in the elimination process and to look for specific binding factors. chromatin diminution takes place in all presomatie ceils of the early embryo of ascaris lumbricoides and is followed by the addition of many repeats of the telomeric sequence ttaggc. chromosomal fragmentation is developmentally regulated and occurs within specific chromosomal regions (cbrs). one of these cbrs (cbr ) was analyzed in detail. agene located close to the cbr encodes a putative gtp-binding protein, whose promoter region is located within a distance of only kb from the telomeric ttaggc repeats of the corresponding somatic chromosome. a homologous gene was isolated from c. elegans. most interestingly, the c. elegans gtp-binding protein gene is also located at a telomeric position, namely at the end of chromosome v. in ascaris, transcripts of this gene are present in all developmental stages, though they seem to be stronger expressed in oocytes and early embryos than in later developmental stages and somatic tissues. a high degree of sequence conservation of these genes between the two nematode species and man (a corresponding partial cdna clone was identified in a human heart cdna library) suggests an important biological function. currently, we are using genetic methods to determine the possible function of this gene in c. elegans and to test whether its telomeric localization in both nematode species has any functional importance. it encodes a nuclear protein which is associated with she chromatin together with other probeins of the poiyoomb group. the mechanism of gene regulation caused by pelycomb seems to be similar to that of the modifier genes of the position variegation effect which also encode structural proteins of the heterochromatin. one of these proteins is hpi which shares with polycomb a region of similarity at the amino terminus called the ~omatin ~rganisation m~difier domain (chromo domain). the particular function of the chromo domain is not yet understood, but it seems to be important for the protein-protein interactions in chromatin associated complexes. furthermore, chromobox cdntai~ing genes were found in several species, suggesting that they are widespread in the animal and plant kingdoms. here we show chat ~. clemens contains at least one chromobox containing gene (cdna: cm h ). length and szrusture of the putative ~. elegs~ chromo domain protein are similar to those of the chromo domain containing prozeins of other species. the expression pattern of cm h is s:udied with antibodies raised against the putative chromo domain protein and by injecting a -ga! fusion construct inns the germ line of ~. eleman$. chromatin diminution in the parasitic nematode ascaris lumbricoides is a complex molecular mechanism, involving cleavage of the chromosomes at specific breakage regions, degradation of the eliminated chromatin and new telomere formation in all presomatic cells during the early embryonic development. the aim of the present project is to reproduce this dna elimination process in vitro, step by step or all in one, by using cell-free extracts of a. lumbricoides eggs. therefore, - cell extracts were established and their quality was assessed by the ability to transcribe s rrna genes (pol iii) and sl rna genes (pol li). faithful extracts were then tested for cleavage activity on dna substrates derived from different chromosomal breakage regions. finally, preliminary results revealed a possible non processiv rnase sensitive telomerase activity in the in vitro extracts, capable of adding specific nucleotide sequences to an oligonucleotide primer containing four repeats of the telomeric sequence traggc. as de novo synthesis of several kilobases of somatic telomere is likely to require a strong telomerase activity, we assume that this a. lumbricoides in vitro system is a good tool to isolate the telomerase protein and its corresponding gene or other implicated factors, s - university of berne, ch berne a common feature of all mycobacteria -whether pathogenic or not -is their richness and variety of lipids. among numerous lipids widely distributed, pathogenic mycobacteria exhibit a group of sulfated surface lipids thought to be determinants of virulence. therefore, we studied sulfolipid-bioslrnthesis in pathogenic and apathogenic (opportunistic) mycobacteria species by monitoring the kinetics of [ s]sulfate incorporation into cellular lipids, pathogenic mycobaeterla (two virulent m. tuberculosis strains) showed a marked sulfolipid-synthesis with highest rates during exponential growth, while apathogenic ones (an avirulent m. tuberculosis strain and an opportunistic species) manifested negligible incorporation of the label. the partial characterization of the [ s]-sulfated lipids of pathogenic mycobacteria by thin layer chromatographic separation, combined with autoradiographic detection, revealed the presence of several radiolabeled lipid components of different polarities and with altering expressionpatterns during growth. these results strengthen the hypothesis, that sulfolipids are involved in the pathogenesis of mycobacterial disease. maiada ( , ) . some features of human pf maiada are observed in mudne malaria, although the pattern of sequestration changes due to the different plasmodial species involved, in mice, we showed by immunohistocbemistry that the expression of vcam- and icam- is upregulated in brain, lung and heart of p/asmodium bergheiinfected baib/c and also in lps-pdmed cid mice. in $gid mice injected with labeled human erythrocytes (e), a higher rate of sequestration to brain was observed with pfe than with uninfected e. lps-pdming increased the retention of pfe in the brain and lungs significantly (t test), but decreased it in the spleen. we conclude that pfe express surface structures which allow them to adhere to the vasculature of the brain more efficiently than uninfected e. lps increases the number of adhesive molecules on the host endothelium. interestingly, sequestration in the cid mouse resembles that of pfe in man. f. roth, f. riniker, k. schopfer and t. burkart inst. med. microbiology, univ. of berne, ch berne it has recently been shown that bacterial lipids cancarry antigenic determinants. the study of antibody specificity to such lipid epitopes in vitro turns out to be difficult since hydrophobic antigens have to react with watersoluble molecules. we have developed a solide phase lipid antigen immunoassay (laia) that allows to quantify the antibody reactivities with an array of lipid antigens derived from mycobacterial cells. defined amounts of extracted lipids were immobilized as equidistant lines on a nitrocellulose sheet. small strips of nitrocellulose, each carrying the same sets of lipids were incubated with different human sera. bound antibodies were then reacted with [ -i]-iodine labelled antihuman antibodies and visualized by autoradiography. the reaction was quantified by densitometry. assay conditions were optimized for antigen and antibody concentrations and appropriate incubation times. in addition, the effects of different ingredients (salts, proteins, detergent) on antigen immobilization and epitope accessability were studied. the presented laia-system is rapid, sensitive and reproducible. it allows to compare the specificities of different bacterial lipid antigens and to quantify their reactivity with serumantibodies. the atomic force microscope is a very convenient tool for studying hard and flat samples with atomic resolution. biological objects, usually soft and rough, are sometimes difficult to image using this technique. in contrast bacteria, which are too small to be observed with high resolution using the optical microscope, are hard enough to be observed with an afm at a relative good level of magnification. this kind of microorganisms can be prepared for afm imaging using very rapid and simple techniques. this method could be a convenient tool to observe the effect of bioactive substances such antibiotics. we show in this poster an exemple with the action of penicillin on b. subtilis. within the family trypanosomatidae, leishmania is a protozoan pathogen producing a broad spectrum of human disease. its life cycle is divided in two stages. promastigote is the flagellated form which colonizes the gut of the sandfly vector, and amastigote is the non-flagellated form that is specialized for growth and survival in the vertebrate host macrophage. to understand gene regulation during the transition between the promasttgote and the amastigote stage, we were interested in the characterization of stageregulated genes. we isolated a gene, sw , from leishmania major whose mrna is more abundant in amastigote compared to promastigote. structural analysis of this gene showed that an additional polypeptide could be encoded by the opposite strand of sw . we confn'med the presence of an open reading frame on the opposite strand by in vitro translation of the sw antisense rna. in vivo, an amastigote sw antisense transcript and the corresponding polypeptide were also detected suggesting that the sw is transcribed in both directions and that stage specific transcripts and polypeptides could be produced. analysis of other gene segments tend to indicate that an intensive usage of the leishmania genome to produce various polypeptides could be a general phenomenon in trypanosomatidae. the four variants and / or posttranslational modifications of histone h of procyclic trypanosoma brucei brucei (protozoa, kinetoplastida) were purified by hplc reversed phase chromatograpy and characterized by their amino acid composition and partial amino acid sequence. differences in sequence of up to % as compared to histone h of higher eukaryotes exist. alkaline phospatase digestion and analysis of h by means of hpce was used to demonstrate the presence of phosphorylated modifications. the unique biochemical properties of h of t. b. brucei contribute to the structural differences seen in the chromatin of the parasite as compared to that of the higher eukaryote host. larvae of the parasitic wasp chelonus inanitus (braconidae, hymenoptera) develop inside embryonic and larval stages of the host spodoptera littoralis (noctuidae, lepidoptera). parasitism induces in the host a precocious onset of metamorphosis and developmental arrest in the precocious prepupa. the wasp injects, along with the egg, pdv and venom into the host egg. injection experiments with pdv and venom revealed that pdv play a crucial role in altering host development and in suppression of the host's immune system. the genome of the pdv of c. inanitus consists of at least i segments of double-stranded circular dna ranging in size from - kb. we analyzed the expression of viral genes in the course of parasitization with cdna made from mrna at various developmental stages of s.littoralis. we also investigated the localization of the expressed viral genes on the various segments. in addition, these bacteria were isolated and cultured from brain tissue. to identify the infectious agent we used, among other techniques, an atomic force microscope (afm). when the. isolation fluids derived from the cortex of three alzheimer's cases were examined with afm and scanning electron microscopes, we observed bacteria whose size and morphology fit to the order of spirochaetales. these images are presented and discussed on our poster. ~ottstein bruno, institute of parasitology, ch-berne. %lveolar echinococcosis ae is due to infection with the metacestode (larval stage) of echinococcus multilocularls. an immunodominant antigen em of e. multilocularis was characterized by mab-gii, respective studies revealed, that (a) antigen expression metacestode stage-sdeci-fic and (b} that expression was restricted to the laminated layer. the em -antigen i s a lectin-binding carbohydrate linked to a lipid residue. the "em positive" laminated layer proved to be a crucial factor in protecting the parasite from host immune-effector mechanisms: only e. multilocularis larval structures exp ressing em were able to induce secondary alveolar chinococcosis in rodents. subsequent experimental studies in resistant (c bl/ ) versus susceptible (c bl/ j and akr) mouse strains showed that resistance was as-sociated with synthesis of anti-em igg and igg i. sus-ceptibility of c bl/ j-mice was associated anti-em s and no igg . the parasite-specific in vitro lym-~hoproliferative i~une response remained stationary ~ith time after infection in c bl/ and decreased in r and aki< mice. day p.i. cd § -lymphoblast cells dominated in all meuse strains; day p.i. an in-creased nu/ ber of cd -/cd + and cd +/cd + cells were observed. only susceptible mice exhibited a significantly ~eereased response of lymph node cells to cona-stimula-~ien with time p.i. in conclusion, subtypes of parasitespecific cellular and humoral host immune responses seem leishmania species are protozoan parasites causing a spectrum of clinical manifestations in man ranging from cutaneous lesions to morbidity and death. the insect stage (promastigote) and intracellular stage (amastigote) differ in terms of morphology, physiology, antigenicity and gone expression. it is clear that the amastigote stage is uniquely adapted for survival within the inimical environment of the macrophage phagolysosome and that mechanisms regulating these adaptations must be called into play during the early stages of infection when the parasite undergoes transformation. in an effort to elucidate these regulatory mechanisms, we have constructed a "subtracted" cdna library which is specifically enriched for parasite transcripts expressed during the first hours of infection ("switch" genes). several clones have been characterized by sequence, northern, and southern analysis. three clones, sw , sw , and sw , are expressed at a several-fold greater level in the amastigote stage and potentially encode proteins which interact with nucleic acids. sw predicted protein is homologous with histone h proteins and sw and sw potentially encode ribosomal proteins. analyses of transcripts from this library are yielding clues not only to mechanisms underlying the regulation of parasite transformation, but also insights into post-transcriptional and translational control of gene expression in leishmania. $ - microbiology, university of geneva, medical school, c.m.u., avenue de champel, geneva . a viral rna representing the sequence of a defective interfering rna, naturally selected for efficient replication, i.e. only containing the replication promoter, was cloned under the control of the t rna polymerase promoter. the expressed transcript was then replicated in rive by supplying in trans the replication functions, equally expressed from plasmids. this system, completely accessible to genetic manipulations, allowed us to define a basic rule directing sendal virus replication, the "rule of six" (calain and roux, j.virol. : - , ig ). a second defective rna, representing a shorter version of the viral rna, in that it contains not only the replication but also the transcription promoters, was then cloned and replicated in the same system, although with lower efficiency. the replication of this "transcribing" viral rna was found to obey the "rule of six" as well. replicationitranscdpfion promoters were then achieved to define the cissequence requirements needed for efficient replication or for replication /transcdption. results obtained with these chimerical viral rnas will be presented and their contdbufion to the comprehension of the paramyxovirus replication and transcription processes bovine t-cells that become infected with the intracellular parasite theileria parva undergo lymphoblastoid transformation and acquire the ability to proliferate continuously. they cease to require specific antigenic stimulation, become independent of exogenous growth factors and cause tumors in nude mice. the il and il r genes are constitutively expressed and the transcriptional activator nfwd which regulates these two genes is continuously activated. these processes are all strictly dependent on the presence of the parasite in the host cell cytoplasm and cease upon the specific killing of the parasite by the theilericidal drug bw c. we have shown that the presence of the parasite results in the activation of the protein tyrosine kinases fyn and ick, which can participate in early t cell receptor signalling, suggesting that the parasite may use this signal transducti n pathway to induce continuous proliferation. cysteine proteinases of leishmania as targets for chemotherapy. jacques bouvier*t and judy sakanari*. *department of pathology, ucsf school of medicine, san francisco, ca, and tanimal health, ciba-geigy, ch st. aubin, switzerland. the overall goal of the study is to apply the structure-based approach to develop lead compounds and design novel enzyme inhibitors as potential therapeutic agents against leishmaniases. in this regard, we have identified and characterized the cysteine proteinases of leishmania major, and showed that they consist of excellent targets for drug design. we have purified a membrane-boand cysteine pmteinase not previously described in other trypanosomatids or protozoans and obtained amino acid sequence information from the purified enzyme. in addition, we have also characterized and purified the soluble cysteine proteinases of the parasite. consequently, we have isolated two genes encoding the soluble and membrane proteinases. the soluble cysteine proteinase gene occurs in multiple copies of . kb with flanking regions of . and . kb and is localized on one chromosome of approximately kb. the membranebound enzyme gene is . kb and occurs as a single copy on a chromosome of approximately kb. a three dimensional computer model of both genes wiu be generated and enable us to scan the vast chemical database for new lead componds. we akeady have identified several cysteine proteinase inhibitors that are effective compounds in killing both promastigote and amstigote stages of the parasite in the micromolar range in vitro without affecting the host cells. dollenn~ier, g., cassinotti, p. and weitz, m., institute for clinical microbiology and irananology, st .gallen/swit zerland hav is a ~r of the picornaviridae. its rna gencrne rf~y be divided into coding regions (pi, p and p ), qhe p region contains a protease cp ro that generates mature proteins by cis-cleavage of the initial polyprotein precursor. catalytic activity of cp r~ has been d ~aonstrated in a vacciniavirus/t ~qa-polymerase hybrid expression system. (balouter aided ali~ts of hav cp r~ sequence with that of other picornaviruses imply that amino acid residues his- and/or h) the complete hav open reading frame, hav specific expression products were identified by radioj_rmmnoprecipitation and lyy sds-page. the analysis with an antibody specific for putative nonstructural protein b revealed a , kda peptide. this is in contrast to its predicted size ( kda). however, analysis with anti-(putative) a antiserum confirmed an upstream location of the b n-terminus. the new bpeptide was also identified in lyeates of hav infected mrc- cells. hence, recombinantly expressed hav polyprotein underwent authentic processing and the predicted p organization has to be modified. we are interested in the development of animal models for one of the two major pathological changes found in the brains of patients with aizheirnar~s disease, the formation of fleuroitbrillary tangles, a main component of which is abnormally phosphorylaled tau-protaln. (tau itself is a protoin associated with mlcrotubuli.) two fundamental questions may be answered with our models: first of nit we woald like to reproduce the formation of tangles, and secondly we hope to answer the question, whether the formation of these pathological structures is sufficient for the bevalopment of senile dementia ot the alzheimer type (sdat). at present there are only a tow putative candidate genes known which may be in-voned in the hyperphosphor~lation ot tau in v/vo, one of which is the map-kinase erk . we have cloned this candidate kinase from rat txaln rna via an rt-pcr approach and expressed the cona under,the co.rat of tour different promoters in transgenis mice. these promoters were tested for maximal expression. a similar approach was chosen to express the human taut isofonn in the brain ot transganic mice. we have stated to analyze different tau an~ erk expressing lines, northern blot analysis has revealed high levels of expression for all transgenes (up to fnetoid {n comparison to endocjenous levels). the neuronal spectficry of the transgenio erk expression was c~nfirmsd by in situ hybridization analysis. ir~ addition several tauexpi'esalog tines were analyzed in wealernblots to determine the relative amount of htau= protein in the brain. sections of the brain were stained with tau-sp~c~c ardib~iies to identify the neurons expressing tau. from intemrosses of tau-with erk -expressing lines, we have already identified double-positive mice which we currer~y analyze by immunohistocbemistry and in western nots to examine the phosphorylaiton status of tau. we are well aware of the possibility that only animals trans~enic for both tau and erk might beveiop tang{es, whereas single transgenics mlsht not. pairs of individual neurons were recorded with sharp electrodes or in whole-cell configuratio~ in hippocampal slice cultures of rat. synaptic connections were studied between pre-and postsynaptie cells in ca and ca hippneampal fields by eliciting single aetinn potential in the presynaptic cell. monosynapfic excitatory connections were highly probable between ca and ca pyramidal ceils (p= . , n= ) with almost no feed-forward inhibition (p= . ). by contrast, disynaptic ipsps, blocked by cnqx or bieuculline were observed with a very high probability ( -- . ) between ca pyramidal cells. monosynaptic excitation was found ha % of cases between ca neurons (n= ) but only in % of cases (n=ll) between two ca cells. monosynaptie ipsps with very short lateneies (<lms) and blocked by bicuculline, but not by cnqx, were recorded within area ca ( / ) and within cai ( / ), but not between ca and cat fields ( / ). probability of transmitlzr release, studied in recordings where failures could be unambiguously distinguished from spontaneous or small psps, was found to be close to % at both excitatory and inhibitory synapses. specific inhibitors of presynaptie release at excitatory and hahibitory terminals (i.e. adenosine and opioid peptides, respectively) gradually reduced the anaplitude of the psps indicating that the release at excitatory and inhibitory unitary synapses is multiquantal. the cns of a neonatal opossum, monodelphis domestic& shows profuse growth of fibers across a lesion. as in other mammals, such repair is not seen in adults. experiments have been made to define the age of the animal and stage of development at which repair ceases to occur. the entire cns was removed from animals aged - days or - days after birth. the spinal cord was crushed and growth of fibers assessed days later by electrical recording of impulses conducted through the crush. repair after lesions to spinal cords of younger animals occurred in % of the trials ( animals, aged - days). in nervous systems from older animals ( - days after birth) the success rate was low (only out of preparations). similarly, the carbocyanine dye dil labeled numerous fibers in spinal cords of younger animals ( out of ), whereas only one preparation out of showed scant outgrowth into lesions in the older animals. our results suggest that there is a critical period for neuronal repair; at about days nerve fibers lose the ability to grow across lesions. it is an advantage that at this later stage the nervous system is still small enough to survive in vitro. features correlated with these changes are oligodendrocyte differentiation and myelin formation. with immunofluorescent staining as well as electron microscopy myelination becomes apparent at days after birth. we are now using time lapse-videomicroscopy to analyze molecular mechanisms that could play a part in promoting or preventing repair. supported by grants to j.g.n from the swiss nationalfonds ( - ) and from the internat. res. inst. for paraplegia. i. impulse conduction in axons has integrative functions. the dorsal root ganglion (drq) of the embryonic rat put into culture forms a monolayer and is thus accessible to eonventionalmieroelectrode technique, which allows testing the above hypothesis. we have chosen a dual approach by correlating ext~rimental el~ctrophysiological data from the chlture with results obtained from a compartrhental computer model. ii. the safety factor for conduction was found to be low. thus failures of ap invasion of the drg cell soma could occur at sites of impedance mismatch when a second stimulus felt into the relative refractory period of the first or when the axon was repetitively stimulated. the electrotonic resiuues (e.rs) of the failed aps had discrete amplitude levels, suggesting that the failures were always caused at the same site along the axon. these sites of low safety factor were found to be the branch point in the unipglar drg cell and the entrance of the stem piece into the soma in both cell types, the bipolar as well as the unipolar. a systematic comparison of oth cell types showed that the ap conduction through the latter is more reliable.'the length of this stem piece is inversely' correlated to the delay caused at the branch point, as tile electrical load of the soma is more efficiently masked by a long stem piece. the dendrites of motoneurons (and subsequently many other neurons) have been assumed to be electrically passive since the work of coombs, ecc]es and fatt in the 's. however, direct evidence has been impossible to come by until recently with the advent of appropriate dyes and equipment for measuring very small and very fast optical signals. we examined the propagation of action potentials, both spontaneous and evoked, in the dendritic trees of spinal cord neurons using a • array of photodiodes and a ccd camera as weft as conventional microelectrode techniques. organotypic slice cultures of embryonic rat spinal cord were stained with a voltage-sensitive dye (di- -anepps) or a calcium dye (fluo- ) and recordings were made from motoneurons identified by their location and morphology. the results indicate that there is in fact an increase in calcium in the dendrites that accompanies action potentials whether synaptically evoked or evoked by current injection at the soma. spikes in the dendrites up to i~m from the soma are much larger than could be explained by models assuming passive dendritic trees. work is currently underway to test the possibility that sodium conductances might contribute to the propagation of action potentials in the dendrites. cat auditory cortex contains multiple cochlear representations in both hemispheres that are interconnected through the corpus callosum (cc). the distribution of auditory callosal axons was studied on the mediosagittal level after injections of biocytin at electrophysiologically identified locations. single injections were done in ai ( cats), aaf, aii and paf; multiple injections in ai & aaf in one cat. an additional cat received injection in sii. the mediosagittal image of cc was subdivided into segments along a rostro-caudal axis. labelled axons crossing the midline were counted in coronal or horizontal sections. axons from auditory fields were found in the posterior / and those from the somatosensory cortex in the anterior third of cc. axons from a discrete injection (ca imm in diameter) spread over as much as i/ of cc. the rostrocaudal distribution of axons at the midline reflected roughly the rostrocaudal position of the field of origin, but apparently not the tonotopic order of a given field. the major efferent projections of substantia nigra pars reticulata (snr) convey information to the thalamus, to the tegmentum, to the superior colliculus, and to the spinal cord. such a range of targets suggests a highly organized activity within snr and this study investigates the temporal organization of spike trains. in the portion of snr between + . and + . mm interaural levels, we recorded single units along electrode tracks in young female wistar rats. most units (n= ) fired in a non-regular poisson process, although only a small number ( / ) was characterized by an average burst size with more than spikes. crosscorrelograrns were computed for pairs of units simultaneously recorded from the same electrode and for pairs simultaneously recorded from distinct electrodes. most crosscorrelograms ( / ) showed a symmetrical peak centered on time zero, which indicates a tendency to synchronous firing. when units were recorded from the same electrode tip we observed that / to / of their spikes were synchronous. about half of the significant interactions were characterized by a relatively long duration (> ms) and are likely to correspond to a different mechanism of synchronization. these results support the hypothesis that snr cells receive common afferences of two different types and their fine temporally organized activity yields coordinated activity between the target structures. the neuronal microtubule-associated protein map binds to microtubules via a domain containing or repeats of an amino acid sequence. we have previously shown that when cultured nonneuronal cells are transfected with map their microtubules become stiffer and are then capable of supporting process outgrowth when their cortical actin cytoskeleton is depolymerised. we hypothesise that this stiffening effect of map is caused by the amino acid sequences binding to neighbouring tubulin subunits in the walls of the microtubules thus reducing their freedom of movement relative to one another. to investigate this further we have created map mutants in which or repeats of the tubulin-binding motif were deleted. these were tested by transfection and expression in cultured cells. the results confirm that the number of repeats affects the stability, stiffness and the cytoplasmic arrangement of cellular microtubules. we were previously able to demonstrate the presence ot kinin-like immunoreactive structures in the mouse brain. in order to characterize them, we have now performed a donble-immunofluorescence study using both polyclonai auti-srudykinin (bk) and mouse monocloual anti-neeron-specificenolase (nse) antibodies. the latter is considered to be a specific neuronal marker. after fixation with bonin-hollande solution, the brains were removed, embedded in paraffin and serially sectioned at am. the sections were incubated with the antibodies, which were visualized by an indirect immunofluorescence technique using fitc for nse and by an avidin-biotin technique using texas red for bk. double-immunofluorescent cells were found in the thalamus ventralis, in the nucleus cechleuris ventralis and in the nucleus mesencephaficns nervi trigemini (v). bk-positive cells in the nucleus principalis v were nse-negative ih spite of their typical neuronal aspect. the other bk-positive structures (pia, ependyma, hnic~es) also were alwa~ nse-negatlve. most of the nsepbsifive bk-negative cells were seen in the cortex. the neuronal nature of many kinin-like immunoreacfive structures in the mouse brain is thus ascertained. in contrast to the amphibian optic nerve (on), on of adult mammals is unable to regenerate after injury. astrocytes disappear from and macrophages accumulate at the lesioned site (blaugrund et al., j. neurocytol, , , ) , we have followed the distribution of astrocytes and microglial cells in xenopus tadpole ons over d after mechanical crush. astrocytes were stained for cytokeratin and vimentin, and microglial cells for griffonia isolectin b . soon after crush, immunoreactivity for intermediate filament proteins was strongly increased. astroeytic processes extended into the lesion from both proximal and distal stump. at , d, astrocytes had crossed the injured region. in the distal stump, some of them contained vacuoles indicating phagocytic activity. while in the normal on microglial cells were ramified and scarce, . d after crush they were roundish and vacuolized, and scarce within the lesion but numerous distal to it. at d, their number was decreased and most of them had reacquired ramifications. conclusively, in the tadpole on, astrocytes are virtually never absent from the lesion site but rather extend rapidly into it. astrocytes may thus provide a guiding support for outgrowing axons, microglial cells that are frequent in the distal stump, do not accumulate at the lesion. the behaviour of the two cell types differs profoundly from that in mammals and is likely to be one of the factors that may contribute to successful neuronal regeneration of the amphibian on. ci mence, j. f. , ranieri, j. p. , aebischer, p. , and sigrist, h. , institute of biochemistry, university of berne, ch- berne; division autonome de recherche chirurgicale, chuv, ch- lausanne. small peptidic sequences of laminin (yigsr, ikvav) are known to promote attachment and/or neurite outgrowth of various neuronal cell lines (pc , nb , ng - ). new and established heterobifunctional photo crosslinkers were used to immobilize these peptides in a topically defined fashion onto biocompatible surfaces such as hydroxylated fluorinated ethylene propylene (fep-oh) and pely(vinymlcohol) (pva) . n-[m[( -trifluoromethyl) diazirine- -yl]phenyl]- -maleimido-butyramide (mad) was thermochemically linked to the synthetic peptide cdpgyigsr via its n-terminal cysteine, leaving the bioactive part (yigsr) free for cell receptor interaction. mad-cdpgyigsr was radioactively labeled by reductive methylation and purified by reversed phase hplc. patterned ( and #m) peptide immobilization was attained by photocoupling onto pva and fep-oh and visualised by autoradiography. light-structured biomaterial surfaces with molecularly oriented nerve growth promoting peptides were investigated for spatially controlled neuronal cell attachment and differentiation. it is striking to observe that these inhibitory or stimulatory effects were corroborated by binduced decrease or increase in -deoxyglucose uptake, respectively. this gives b a putative role in the limbic regulation. the sheet-like processes of oligodendrocytes wrap themselves spirally around central axons, thus formtng the myelin sheath. a single oligodendrocyte may donate internodal segments of myelin to each of - or more adjacent axons. it is not known, if one oligodendrocyte picks out axons randomly or if it prefers axons with a certain diameter or with a certain chemical specificity. we have studied this last possibility by combining intracellular injection of a fiuorochrome, and immunohistological detection of calciumbinding proteins (cabp's), markers for the axons ef certain subpopulafions of nerve ceils. lucifer yellow was injected into oligodendrocytes of the optic nerve, of living rat brain slices. the oligodendrocytes were identified by their electrophysiologieal properties. slices containing the iniected cells were immunostained /or parvalbumin or calretinin using second antibodies labelled with texas red. using co.focal laser-scanning microscopy combined with a three*dimensional reconstruction programm, the relationship between oligodendrocyte processes and axons positive for one of the cabp's was determined. for ultrastructural confirmation, single, lucifer-yellow injected, oligodendrocytes were photoconverted, and the cabp's-positive axor~ were revealed by gold-labelled antibodies. the few oligodendrocytes injected show the classic morphology, that is of a small cell body and few processes running parallel to the course of the axons. the proximity between glial processes and positive axons can be easily appreciated in confocal laser-scanning images. a preferential assodation of oligodendrocyte processes with parvatbumin-positive axons has not as yet been found. thus, the confirmation of the hypothesis that oligodendrocytes choose their axonal targets according to chemical cues awaits further work including the injection of a lar~er number of these cells. to initiate a molecular genetic analysis of brain development in insects, we are characterizing the mechanisms of embryonic brain development in the fly drosophila. early in embryogenesis, a small number of molecularly diverse brain neuroblasts generates the neurons of the brain. subsequently, pioneering brain neurons initiate axonal outgrowth along glialbound brain compartments. these pioneering neurons establish the primary brain tracts. the pioneering brain axons express the cell-cell adhesion molecule fasciclin i dynamically during outgrowth and initial fasciculation; a subset of the pioneering axons expresses fasciclin ii. the axonal framework of the embryonic brain tracts is used for outgrowth and fasciculation by subsequently differentiating axons and, thus, prefigures the tracts of the mature brain. a comparison of early brain development in drosophila and in vertebrates reveals common mechanisms for brain development. supported by the swiss nsf. institute ot histology, university of fribourg, p~rolles, ch- fribourg calretinin (cr), an ef-hand ca +-binding protein, is expressed in cajai-retzlus cells during development of the rat cerebral cortex. these cells are located in the marginal zone and are the ear{iest cortical neurons to be generated. they have been consldered as unusual on account of their morphology and their fate during corticogenesis. the functional significance of cajai-retzius cells and their destiny in adulthood is still controversial. in order to approach these questions we have applied organotypic culturing methods. slices of neocortex from brains of - days-old rat pups were cultured for - days applying the interphase technique (stoppini etal, j. neurosci. methods , ) or the roller-tube technique (g&hwiler, j. neurosci. methods , i ) . the fixed cultures were immunolabe ed with an antibody against cr. the obtained results showed that cr positive structures develop in vitro like in vivo. however, in organotyp[c cultures their distribution corresponded to a more mature stage than the situation on the day of the explantations. many cr-positive cells were seen in layer l they were horizontally oriented or had a pyriform shape. based on their morphology these ceils were identified as cajai-retzius cells. orpositive cells were numerous in layer i -l[i and showed mostly a bipolar morphology. some muripo[ar cells could also be observed. a fine net of crpositive fibers and puncta was found in layers [ and iv. the demonstration that cajai-retzius celts are detectable in these cultures will allow us to follow the fate of these cells dynamically and [nte~ene in their function by applying exogenous factors. cervical primary afferent input to vestibule-spinal neurones projecting to the dorsal horn: a double labelling study in the rat. wheat germ agglutinin-horseradish peroxidase (wga-hrp) was injected by pressure into cervical spinal ganglia c or c . in the same experiments fluorogold (fg) was iontophorefically injected into the ipsilateral dorsal horn of different cervical spinal cord segments. anterogradely labelled fibers (wga-hrp) were present mainly in the external cuneate nucleus, but also to a lesser extent in the caudal part of the medial and descending vestibular nuclei (mvn, dvn) . the fg injections, restricted to the cervical dorsal horn, revealed retrogradely labelled cells in the central part of the caudal mvn. a double exposure (fluorescent and polarisadon optics) under the microscope showed primary afferent fibers surrounding like baskets retrogradely labelled fg cells. the projection from cervical ganglia cells to the mvn represents a pathway for direct afferent information from neck receptors (in particular muscles) to vestibular nuclei and supplies the mvn with afferent information which could enable the vestibulospinal nenrones, projecting to the dorsal horn, to influence the local information processing. in the sciatic nerve, all myelinated and non-myelinated axons were snap-ir. in peripheral tissues, all nerve endings were positive. the preterminal schwann cells of motor axons were also snap- -ir. after sciatic nerve section, many myelinating sehwann cells also expressed snap- -ir. in conclusion, snap- is expressed by neurons in the pns. it seems to be also expressed by preterminal schwann cells and by myelinating schwann cells during regeneration. $ - repair of completely transected spinal cord of neonatal opossum in culture h.a. vischer, dept. pharmacology, iocenter, uni. of basel, switzerland woodward et al. (j. exp. biol. , have shown that fibers grow rapidly and profusely across a lesion made by crushing the spinal cord of the neonatal opossum monodelphis domestica. in such experiments careful controls must be made to establish that fibers were in fact broken by the lesion. tests have now been made to determine whether similar repair can occur after a more drastic lesion involving complete transection of the cord. after dissection of the entire gns from animals aged - days, the cord was cut into two with scissors at the c -c level. the two-ends were glued together with matrigel on a sylgard mold, which encompassed and held the cns. the rostral end was labeled with the fluorescent carbocyanine dye dil in order to visualize growing fibers. in preparations fibers grew over the cut into the separated part of the spinal cord. such growth could extend to ixm. fibers grew straight, branched and multidirectionally, or in fascicles. in another set of experiments cords were combined from different animals of the same or different ages. again, fibers could grow across the cut but they did so less frequently. these results set the stage for investigating fiber growth after rotating the two ends at a defined angle and for analyzing factors that influence the direction of growth. map la is a microtubule associated protein of kd. in rat brain two monoclonal antibodies, a and bw , recognized map la in neurons and their axonal and dendritic processes. in mouse cerebellum, monoclonal a recognized purkinje ceils and granule cells uniformly, as already described for rat brain. in contrast, monoclonal bw stained selectively some dendrites of purkinje cells, forming bands in the molecular layer. we compared this striation with that obtained with a monoclonal antibody, anti-zebrin ii, which recognized aldolase c. in the mouse vermis, zebrin stained in a striated pattern, but complementary to bw . these results demonstrate that map la is present in forms which are differentially distributed in the mouse cerebellum. these observations may be explained either by differences in metabolic states of neurons, or by differences in their regional functions, or by differences in the regional stability of microtubules. this work was supported by grant no - . from the swiss national science foundation. serum free aggregating brain cell cultures prepared from day old fetal rat telencephalon are used as a model to study brain development. in these cultures it is possible to distinguish ontogenetic events such as cell proliferation, differentiation and myelination, in the present study we examined synaptogenesis by analyzing the expression of synaptic proteins such as synaptophysin, snap- , synapsin i, and brain spectrin. different developmental stages were analyzed by western blotting. immunoreactivity for synaptic proteins was detectable already at day in culture, suggesting that the first synapses are already present at this early stage. the expression of synaptic proteins strongly increased between day and day in culture, reflecting a period of intense synaptogenesis. treatment of the culture with an elevated concentration of kci ( mm) increased the expression of synaptic proteins, suggesting a stimulation of synaptogenesis. these findings demonstrate the utility of this approach to study brain synaptogenesis, and possibly synaptic plasticity. drenhaus, u.i gunten, a.v., rager, g. ; institute of anatomy, university of ch- freiburg the retina of tupaia comprises three types of ganglion cells (rgcs) analogous to x-, y-, and w-cells found in other mammals. these cells differ in size and in their distribution pattern: large rgcs are frequent in temporal, small rgcs in nasal retina. as the fiber diameter correlates with the size of its respective rgc, it can serve as a parameter for the topographic representation of rgcs in the nerve. to study this we analyzed the optic nerves of three tupaia. using the electron microscope we recorded the density, diameter, and the position of fibers in the nerves. we found, that fibers with the largest diameter are located dorso-temporally and close to the center of the nerve. they are surrounded by zones of smaller fibers. the diameter decreases gradually towards the periphery and is smallest in the ventro-nasal region of the nerve. since the fiber diameter distribution corresponds to that of the size of rgc-types in the retina, we assume that the topographic relationships in the nerve and the retina are similar. (supported by s - stoppini luc, s. duport, l. parisl, c. oropes~, departement of pharmacology, cmu, geneva the micro environment of the central nervous system is important for neuronal function. in this context, the blood-brain (bbb) provides and maintains the extracellular medium that is compatible with normal neuronal and synaptic activity. due to the difficulties inherent using whole animal as an experimental model to study permeability and metabolic processes at the cellular level, major efforts have been engaged in recent years, in order to bring up suitable /n vitro models. we are presently developping an in vitro bbb by interfacing a coculture of endothelial cells monolayem and nervous organotypic cultures. the main idea of this study is based on the premise that the complex intercellular interactions between the different cell types pertaining to the nervous tissue and the neighbeurlng endothelial cell is of fundamental importance to promote a realistic bbb system. we expect that erganotypic culture of nervous tissue, combined to endothelial cell monolayers, will initiate and maintain a bbb phenomena/n vilro. we will test more specifically the hypothesis that neuronal activities [spontaneous or elicited) will influence gila] cell response which will in turn modify endothelial properties. preliminary results clearely show a good nervous tissue and endothelial cell survival. tight junction llke structures could be identified using freezefracture or conventional transmission electron microscopic thechniques. {work supported by chemodyne gent~ve ). the process of myelination is a prerequisite for the proper function of the brain since it enables rapid saltatory conduction of axons. in the central nervous system (cns) myelination is performed by oligodendrocytes. a differential screening approach was used to isolate new rat cdna clones that are expressed in this glial cell type. here we report the further characterization of two of these novel cdnas which appear to be expressed specifically in oligodendrocytes. the two characterized cdna clones (tentatively called cns and cop- . ) share an approximately bp region of sequence identity which suggests that they are dedved from the same gene by alternative splicing, within this area a putative open reading frame is located. we demonstrated by northern blot analysis and in situ hybridization that the two mrnas of the novel gene are expressed exclusively in oligodendrocytes. however, the mrnas show a different specific spatial localization within the cell. we compared mrna expression of myelin basic protein (mbp) and proteolipid protein (plp), with that of cns and cop- . in brain and optic nerve during development. it appeared that the mrnas of the novel gene were delayed significantly as compared to mbp and plp mrnas. streit. phyaiologisches institut, btihlplatz , bern in organotypic slice cultures of the embryonic spinal cord, rhythmic spontaneous activity arises when the inhibitory synaptie transmission is blocked. the rhythmic activity consists of bursts of regular oscillations of activity at - hz. this study investigates the origin of the regular oscillations. when stimulated electrically at hz, the synaptic transmission in the cultures showed strong depression by about % on average. the synaptie depression was highly variable among individual connections and tended do be less pronounced in frequently used connections. when random depression ratios with an average of % at hz were implemented in a computer model consisting of randomly connected excitatory cells, regular oscillations of activity did not arise, even in the presence of synchronous pacemaker cells. however, when individual depression ratios were adapted according to the rate of activity of individual connections, regular oscillations at - hz arose in the presence of few unsynchronized pacemakers. this finding suggests that the regular oscillations in the cultures originate in a network formed by the plasticity of synaptic depression. maier a., schlumpf m., beer h.f., sehubiger p.a. and lichtensteiger w., inst. of pharmacology, univ. of zurich, the ontogeny of expression of dopamine d receptor mrna in the rat brain and binding to the receptor was studied at time points from gestational day (gd) to postnatal day (pn) by quantitative receptor autoradiography and in situ hybridization.long evans rat pups and fetuses of time pregnant rats were frozen and sectioned coronally and sagitally at i um. d recepto~o~inding , determined with the selective antagonist ~ji-sch was first noted on gd in the developing striatum, basal ganglia and the olfactory tubercle, reaching adult levels at around pn . the expression of d i receptor ~na was studied by using a mixture of -~js-labelled oligonu-specific cleotides. on gd messages were noted in the developing stiatum, olfactory tubercle and retina. this study shows a good regional correlation between the development of receptor binding and mrna, however, mrna precedes detectable binding to the receptor by several days. earlier work had shown that q~ replicase forms complexes with q~, plus strand rna via interactions at two internal binding sites, the s-and the m-site, while binding of the '-end is mediated by host factor. in contrast, the '-end of the minus strand appeared to be directly accessible by replicase. we have prepared a series of plus and minus strand variant rnas containing either internal deletions or terminal deletions extending from the '-end. the template activities of the variants were determined by single-round replication assays. for the plus strand we find that while deletion of the s-site remains without effect (as expected from previous results), deletion of the m-site reduces template activity to % or less compared to wild-type rna; the residual activity shows a decreased dependence on the presence of host factor. the results agree with an important role of the m-site interaction both with replicase and with host factor for the formation of productive initiation complexes. the template activity of the minus strand was unexpectedly found to be strongly dependent on the presence of a segment between nt and from the '-end. this shows that recognition of the minus strand by rep(icase does not only involve interactions near the '-end but requires a previously unknown structural feature near the '-end of this template. $ - karsten graning and angela kr&mer d~partement de biologie cellulaire, universite de gen~ve, , quai emest-ansermet, gen~ve splicing of nuclear pre-mrnas takes place within multicomponent complexes (spliceosomes) that are assembled by interactions between the pre-mrna, snrnps and protein splicing factors. we have purified two splicing factors that function in the formation of the pre-splicing complex. sf a consist of three subunits of , and kda and corresponds to three proteins present in the functional s but not in the s u snrnp. it binds to the s u snrnp only in the presence of sf b, suggesting a two step assembly pathway of s u snrnp: binding of sf b to the s u snrnp generates a s intermediate particle that is converted to the active s u snrnp by the subsequent association of sf a. comparison of proteins enriched in the sf b fraction with polypeptides found in the purified s u snrnp suggests that sf b comprises at least five of the other s u snrnp specific proteins and together with sf a promotes the u snrnp/pre-mrna interaction. by edna cloning, two of the subunits of sf a have been identified as homologs of well characterized yeast proteins that function at the same stage of spliceosome assembly in yeast. splicing factor sf , a heat-stable -kd protein, functions early during spliceosome assembly. tryptic peptides of sf were sequenced and cdna libraries were screened with degenerate oligonucleotides. the information obtained by comparing the sequences of three overlapping cdnas suggests the existence of at least three mrnas that could be derived from a common pre-mrna by alternative splicing. in agreement with this assumption mrnas of the expected sizes that are differentially expressed depending on cell line or tissue are detected by northern blotting. in theory, three polypeptides could be generated that differ by the presence or absence of internal amino acids and in their ctermini. the deduced amino acid sequence is rich in prolines and contains several possible phosphorylation sites and a putative leucine zipper. proline-rich domains, which are also present in splicing factors psf and sap , as well as leucine zippers have been found in transcription factors and could mediate protein/protein contacts, suggesting that sf could function during splicing by interaction with other spliceesomal proteins. pre-mrna splicing is catalyzed by a multicomponent complex (the spliceosome) that consists of small nuclear ribonucleoprotein particles (snrnps) and non-snrnp protein factors. the spliceosome is assembled in a stepwise fashion on the pre-mrna. chromatographic fractionation of hela cell nuclear extracts and subsequent reconstitution of splicing in vitro has allowed the separation and isolation of snrnps and several protein factors. sf triggers the transition between splicing complex b, which contains all spliceosomal snrnps but is not competent for catalysis, and complex c, the active spliceosome. this transition involves a conformational change that leads to altered base pairing interactions between snrnas and pre-mrna. the purification of sf has been impeded by its low abundance; however, a correlation between sf activity and a polypeptide of kd could be established in the most purified fractions. we are currently investigating whether this protein represents sf . interestingly an atp-dependent rna-helicase cofractionates with sf through several purification steps. whether or not this activity is relevant for the splicing process is under investigation. selection of iron-responsive element rnas with high affinity for iron regulatory factor henderson, b.r., menotti, e., bonnard, c. , and kith_n, l.c., isrec, ch-i epalinges iron regulatory factor (irf) post-transcriptionally regulates iron homeostasis via binding to mrna iron-responsive elements (ires). the ire loop sequence ( '-cagugn- ') and "bulge" cytosine are phylogenetically conserved. we prepared a pool of , ire molecules randomized at these nucleotide positions, and employed in vitro selection to identify optimal sequences which bind human irf. we define two major classes of high affinity rna ligand, the optimal loop sequences of each are '-cagugn- ' (wild-type) and '-uaguan- '. this novel finding predicts base-pairing within the loop between positions i and . all nucleotide substitutions in the "bulge" or at loop positions , and decreased binding by % to %. in addition, binding specificity of rat irf differed with that of the related rat ire-binding protein, irf b. in vitro analysis of ire-like stem-loops identified by computer search has not yet revealed any new ire-containing genes. a $ - ' processing of histone pre-mrnas: a phylogenetic comparison reto kohler, petra duda and daniel schiimperli. zoologisehes institut der universtit/it bern, abteihing fiir entwicldungsbiologie, baltzerstr. , ch- bern, switzerland. we are using a phylogenetic approach to study the biochemical reaction by which animal histone pm-mrnas are processed at their ' ends. in mammals, the efficiency and specificity of this reaction is known to depend absolutely on the u snrna which interacts with conserved spacer sequences downstream of the proc~sing site. a highly conserved hairpin element which precedes the cleavage site serves as a binding site for an additional processing factor, but its importanea for efficient processing varies greatly between different historic genes and between extracts of different mammalian cell lines. to determine whether the relative importance of the hairpin and spacer dements have been conserved during evolution, we analysed the processing of histone genes from three different animal phyla in vitro, i.e. vertebrates (mouse), arthropods (drosophila melanogaster) and nematodes (c. elegans). in the mouse system, processing was strongly dependent on the presence of a mouse spacer element. in nuclear extracts of drosophila ke cells, processing occurred exclusively when a drosophila spacer element was present in the rna. processing efficiency was not reduced by foreign (mouse, c. elegans) hairpins. in whole cell extracts of ascaris lumbricoides embryos, an inverted situation was observed. ' processing products were only produced by rnas that included an upstream segment derived from a c. elegans histone gene, irrespective of the spacer sequences present. these surprising results correlate with certain unique features in the c. elegans upstream element. we are currently in the process of cloning ascaris lumbricoides histone genes, which will allow us to verify our results in a homologous system. in xenopus laevls ooeytes, wild type mouse u rna gets assembled into functional u snrnps, both when transcr~ed from an injected gene or when injected as/n vitro synthesized rna. if the sm binding site of u rna is converted into the canonical sm sequence (u sm opt), the rna assembles into a particle which accumulates more efficiently in the nucleus but wbach is nonfunctional. this u sm off particle inhibits the function of preformed endogenous u snrnps, most likely by nonproductive binding to histone pre-mrnas, histone pre-mrna processing can be demonstrated in c/s if u rna is placed ' of hlstone pre-mrna and injected into oocytes. only u sm wt sequences are active in this c/s processing. three proteins can be photoaffinity cross]inked to u sm wt rna, the common snrnp proteins g and b/b' and a u -specific protein of kd ( kd in mouse). these crosstinks map to closely spaced positions within the sm binding site with the u -specific crosslink being located most '. u sm opt rna does not become crosslinked to the u specific protein and is also more tightly associated with sm proteins. we now investigate the in vitro assembly of u snrnps using these characterized u rnas and a preparation of highly enriched snrnp proteins. u sm wt and u sm opt rnas associate with common sm proteins in this system, but the interaction with the u -speciflc protein could not be demonstrated. the double-stranded (ds)rna modifying enzyme, which can convert adenine to inosine, was first identified in xenopus laevis, but has since then been detected in different species and in mammalian cells. the enzyme is a specific adenine deaminase which uses dsrna as substrate and recently, it has been postulated to be responsible for a specific mrna editing reaction. we have purified this enzyme to homogeneity, approximately , fold from calf thymus whole cell extracts. the protein was purified primarily by ion exchange chromatography over six columns, with the final step being chromatography on a dsrna affinity column. the purified protein has a molecular weight of kd and is the only protein present when enzymatically active fractions are visualized on an sds-polyacrylamide gel stained with silver. the dsrna modifying enzyme is a very low abundant protein. we are in the process of further characterizing the purified enzyme and of cloning cdnas coding for it. detailed mutational analysis of histone rna ' end formation carmen spycher, andr furger, adrian streit, daniel albrecht, d. schiimperli, zoologlsehes iustitut der universtit/it bern, abteilung thr entwicklungsbiologie. baltzerstr. , ch- bern, switzerland histone rna ' ends are formed by cndonucleolytic cleavage resulting in poly(a)" mrnas with a highly conserved '-terminal hairpin loop structure. for the mouse h - gene major and minor processing sites are located and nucleotides downstream of the hairpin, respectively. for the cleavage reaction, the u snrnp interacts with the spacer element of histone pre-mrna by base-pairing through the ' end of its rna moiety, u rna. we have observed that this base-pairing potential extends further than previously recognised in either direction. we have made site-directed rnutagenesis in fltis potential hybrid region and around the processing site. rna processing and complex formation with the u surnp were analysed by incubation in nuclear extract from mouse k cells. our results indicate that base-pairing with u rna both in the classical spacer element and downstream of it are very important for histone rna processing. in contrast, sequences upstream of the spacer element do not seem to be involved in base-pairing with u rna, but mutations in this region may affect processing by other mechanisms. systematic mutations of the highly conserved four nucleofides immediately following the hairpin show no qualitative or quantitative effects in the processing reaction. alteration of nucleotides to around the minor processing site, however, result in a dramatic decrease in processing efficiency and alter the specificity of the cleavage site. in further experiments we will introduce specific nuclcetidc modifications at the two processing sites, which should allow us to characterize potential cleavage factors by chemical crosslinking. evolution callaerts p., glardon s., j.,oosli f., quiring r. and gehring w. cell biology, biozentmm, basel. the pax genes encode a family of dna binding factors which share the paired-box and play an important role in the control of development. they can be subdivided into four different classes which differ in the presence or absence of other conserved sequence motifs coding for a paired type homeobox or an octapeptide. the pax- gene, which contains a paired-box and a homeobox, has been cloned from humans, mice, rats and zebra fish. recently, we have cloned the pax- homologue of drosophila melanogaster. the five genes share a very high degree of sequence identity both in the paired-domain and the homeodomain. similar to its mammalian counterparts, the drosophila gene is expressed in the eye primordia and the nervous system. in addition, mutations in pax- affect eye development: aniridia in humans, small eye in mouse and rat, and probably eyeless in drosophila. this would imply that the pax- homologues share a conserved function in eye morphogeuesis in both vertebrates and invertebrates. furthermore, this suggests that pax- may have been present in a common ancestor. therefore, we are now trying to isolate pax- homologues from other, more primitive organisms by means of the polymerase chain reaction, and to determine whether they are implicated in eye development. putative candidates have been identified from a number of species and are being characterized. their sequence similarity and some evolutionary implications will be discussed. keegan liam and gehrin~ walter j., biozentrum, university of basel, klingelbergstr. , ch- basel, switzerland using an enhancer map screen we have identified two genes that may be direct targets of antennapedia involved in forming the adult leg. spalt major is repressed in the leg imaginal disc and tea, shirt is activated by antennapedia. we wish to determine whether the control of these genes by antennapedia is direct, spalt major is expressed in a ring in the antennal disc and is repressed by antennapedia in the leg disc. the antennal ring is repressed by ectopie antennapedia expression that transforms the antenna to a leg. we are defining an enhancer at spalt major that is required for antennal ring expression and repression by antennapedia. we are using various approaches to show that antennapedia binds to the antennal ring enhancer at spalt major. these include polytene chromosome banding studies using antibodies to antennapedia as well as in vitro dna-binding and genetic experiments. in search for novel regulators potentially involved in myogenesis, we identified a homeobox of the paired type in human muscle. subsequent cdna cloning revealed that we had cloned the full length coding region of human pax . our human sequence extends the known mouse edna both on the ' and the ' end. as a first step in order to test for a functional role of pax in myogenesis, we analyzed its expression pattern during chicken development. transcripts are present in the neural tube and in the dermamyotome of the developing somites. therefore, the pattern of expression of pax is conserved between mouse and chicken. next, we assessed the expression of pax in different mouse and human cell lines. we found pax transcripts specifically in myogenic cells and not in any other cell types. interestingly, pax is already present at the myoblast stage. moreover, ti/ fibroblasts converted to myoblasts by either transfection of myod or treatment with -azacytidine expressed pax , whereas the parental cells did not. while myod was able to activate pax in t / cells, expression of pax was not sufficient to induce the myogenic phenotype. nevertheless, our expression data are consistent with a role of pax during the mesodermal commitment to the myogenic lineage. the segmental organization of the drosophila head is achieved by a flow of positional information from maternal gene products to the zygotic gap genes. recent genetic analysis has identified three new gap genes involved in head development. one of these genes is the empty spiracles (ems) gene. mutations in eras cause severe defects in the head and the filzk~rper at the posterior end are missing. the eros gene encodes a putative transcription factor with a homeodomain. the eros rna is expressed in two phases of embryonic development. first expression is seen at the blastoderm stage in a single anterior band, correlating with its function as an anterior gap gene. later during embryogenesis eros is expressed in the posterior spiracles as well as lateral regions of each segment where the tracheal pits form and lateral neuroblasts originate. using g-gal fusions we could identify at least five different regulatory elements in the eros promotor region responsible for tissue specific expression of the gene. a bp element responsible for the early expression which is dependent on the maternal gene bicoid, the key gene of the anterior system, and the terminal gene tailless was identified and studied in detail. this element contains two bicoid and two tailless binding sites. the effects of muations in these binding sites on eros expression will be discussed.this analysis should allow us to elucidate the molecular mechanisms by which the morphogen bicoid regulates subordinate target genes like ems in the drosophila embryo. the pax- gene of the mouse encodes a transcription factor with both a paired-and a homeodomain. pax- is affected by several allelic mutations in the small eye (sey) gene. sey mutations cause a reduction of the eyes in heterozygotes, and homozygotes lack both eyes and nasal openings and die as newborns. the corresponding mutation aniridia in humans affects eye and cranlofacial development in a similar manner. we have isolated a pax- homolog from drosophila (dpax- ), which shares more than % sequence identity in the paired-and the homeodomain with the mammalian genes. also outside of these domains we find considerable sequence conservation arguing for a true pax- orthologue. the drosophila gene spans ~ kb and is expressed in the brain and the ventral nervous system of the embryo, and in the eye imagjnal discs and the brain of the larva. the gene was mapped to the eyeless (ey) locus. mutations at the ey locus cause either the partial or complete absence of the compound eyes or embryonic lethality. in one ey allele we have identified a transposable element in the first intron of dpax- , which affects dpax- transcription in the eye discs, suggesting that dpax- is encoded by the ey gene. this implies that pax- (sey) in the mouse is homologous to ey in drosophila. thus, despite of the different modes of eye morphogenesis, the same gene seems to control eye development in insects and vertebrates. the drosophila homolog of the vertebrate serum response factor (srf) was isolated by low stringency-hybridization. nucleotide sequence analysis revealed that the drosophila srf homolog (dsrf) codes for a protein which displays % of sequence identity with human srf in the mads domain, the region required for dna binding, dimerization and interaction with accessory factors. the dsrf gene is expressed during several phases of embryonic development. both the rna and the protein are maternally provided to the egg and slowly decrease in their levels during gastrulation. after germ band retraction, high levels of zygotic expression were observed in a distinct subset of peripheral tracheal cells distributed throughout the embryo. many of these cells are at the tip of tracheal branches and are in direct contact with the target tissues these branches tracheate. the dsrf gene was mapped to position c on the second chromosome, and overlapping deficiencies which remove the gene were identified. analysis of tracheal development in embryos carrying these deletions revealed a degeneration of most of the major branches of the tracheal system. although the initial migration of tracheal cells was not affected in those deficiency embryos, many tracheal cells appeared not to maintain their formerly correct position and continued to migrate. thus, the dsrf gene might play a role in the proper formation and maintenance of the major branches of the trachea. s - our experiments would be expected to provide insight into such problems as the evolution and function of transcription factors with multiple dna binding domains. ideally, we would be able to mimic "gene splitting" which occurred during evolution. the poan gene plays an important role during drosophila neurogenesis. it is expressed in specific subsets of sensory mother ceils (smcs) and neuroblasts. the smcs that express poxn differentiate into polyinnervated external sense (p-es) organs. in the absence ofpoxn, smcs differentiate into mono-innervated rather than poly-innervated external sense organs. as the poxn gene contains a paired-box, it probably encodes a transcription factor. since the function ofpoxn in the central nervous system is not known and no po~n mutants are available, an ems mutagenesis screen was initiated to isolate such mutants. based on the assumption thatpoxn null alleles are lethal, we screened for lethals uncovered by the deficiency df( r)wmg, which includes poxn, but survive over the deficiencies df( r)xte- and df( r)kl- flankingpoxn. in a further test, such lethals are screened for the absence of embryonic p-es organs. ifpoxn mutants are not lethal, they will escape this screen. therefore, we are inducing local hopping of p-elements that have inserted in the vicinity of poxn. the offspring of flies that display an altered eye phenotype are screened for p-element insertion into poxn. we hope that these approaches will generate mutants that will be crucial for future studies ofpoxn function in neurogenesis. chromatin structures and transcription of rdna in yeast saccharomyces eerevisiae reinhard dammaun, renzo lucchini, thee keller and jest m. sogo; institute of cell biology, eth-honggerberg, ch- ziirich the chromatin structure of yeast ribosomal dna was analyzed in rive by cross-linking intact cells with psoralen. we found that in exponentially growing cultures the regions coding for the s rrna precursor fall into two distinct classes. one class was highly accessible to psoralen and associated with nascent rnas, characteristic for transcriptionally active rrna genes devoid of nucleosomes, whereas the other class showed a cross-linking pattern indistinguishable from that of bulk chromatin and was interpreted to represent the inactive rrna gene copies. by cross-linking the same strain growing in complex or minimal medium, we have shown that yeast ceils can modulate the proportion of active (non-nueleosomal) and inactive (nucleosomal) rrna gene copies in response to variations in environmental conditions which suggests that yeast can regulate rrna synthesis by varying the number of active gene copies, in contrast to the vertebrate ceils studied so far. whereas intergenie spacers flanking inactive rrna gene copies are packaged in a regular uucleosomal array, spacers flanking active genes show an unusual cross-linking pattern suggesting a complex interaction of regulatory factors and histories with dna. $ - r. felleisen & b. gottstein; institute of parasitology, university bern, bern, switzerland most patients suffering from alveolar echinococcosis (ae) respond to infection with a marked igg synthesis directed against e.multilocularis metacestode antigen ii/ , which represents a novel member of the family of cytovillin related proteins. although the respective gene basically is also present and expressed in e.granulosus, most of the cystic echinococcosis (ce) patients do not recognize the antigen. this phenomenon was tackled by generating cdna derived from full length ii/ genes from both echinococcus species, performed by rt-pcr. sequence analysis revealed a very high degree of conservation of the primary sequence ( . % homology), cdna fragments were expressed as recombinant proteins and were comparatively assessed in elisa respective to antibody-binding characteristics. sera from patients suffering from ce were showing no significant differences in reactivity with the antigens derived from both species. therefore, parameters others than minor differences in the primary sequence seem to be responsible for the lack of antigen recognition with respect to ce. $ - patrick rigoni, sandro rusconi, institut f molekularbiologie h der universitat zarich, winterthurerstr. , z~rich, switzerland. trinuclcotide repeats are often found in the coding sequence of transcriptional regulators in both mammals and yeast, however, their function is yet unclear and data collected in our laboratory on the gheocorticoid receptor even suggest that they may not have any, and that their presence is probably the result of a selfish replicative behavior. nonetheless, we believe that these motifs can be used as tags to identify flexible protein domains typical of modular proteins such as transcription factors. another kind or repeat (coding for a poly-glutamine/alanine) has been discovered in the yeast regulators gall and ssn . from northern blots, we know that such caggcn repeats are present also in higher eukaryotes and we want to isolate them by constructing an enriched edna library using a ' race protocol. so far, no mammalian protein bearing the motif glrdala has been characterized. among the positive clones we hope to find the homologous or paralogous of gall and ssn that have escaped so far conventional cloning techniques. meanwhile, we have been testing the effect of coexpressed yeast gall on different reporter-activator combinations in mammalian cells. preliminary results show that galll but not the mutant form galllp can inhibit the activation properties of some (not all) gal chimeras. we are also producing antibodies directed against oligo-ala and oligo-gln and oligo-gln/ala motifs in order to better characterize natural proteins containing these repeats. we are interested in the early development of c. elegans, particularly in the contribution of genes whose homologues in vertebrates and d. melanogaster mediate positional infolzzations during development. therefore, we are working on ceh- which belongs to a cluster of at least homeobox containing genes. this cluster is considered to be equivalent to the homeotie cluster of drosophila and to the hox clusters in vertebrates. by generating ~-galactosidase transgenie lines, we have observed that ceh- is expressed very early during embryogenesis, embryonic expression appears to be restricted to the posterior half of the embryo, which is rather surprising, since the ceh- hemologues in drosophila and vertebrates are anterior-specific. during larval stages, ceh- is expressed in neuronal cells. these results are now in the process of being confirmed by immunolocalization of the ceh- protein product with a polyclonal antiserum. in order to clarify the function of ceh- in the c. elegans development, we have isolated a ceh- mutant from a tcl insertion library, in collaboration with r. h. a. plasterk from amsterdam. from this worm, which looks wt, we are now trying to obtain a deletion derivative. previous studies have shown that bovine herpesvirus (shv- ) infected cell pratein (bicp) acts -depending on the promoter -as a strong transactivator or as a repressor in transient expression assays. the cp polypeptide contains a cysteine-rich zinc finger domain (c hc ) which is conserved in a number of viral and cellular regulatory proteins including the icp homologs icr of herpes simplex virus type and protein of varicella zoster virus. this type of zinc finger (the so-called ring tinge was shown to bind zinc ions but functional requirement for zinc has not yet been demonstrated, a gap which we aimed to fill with the present study. transient expression assays were performed in oocytes which had been microinjected with thionein to chelate and deplete the intracellular pool of zinc. cp -induced cat activity of a promoter-cat construct was -fold higher than the basal cat activity of the reporter plasmid, in the presence of thionein, bicp -induced transaotivatlon was reduced old. with a set of control experiments we excluded that thionein might affect transcription and protein synthesis in general, to our knowledge this is the first demonstration of zinc-dependence for a member of the ring finger family. we have identified by pcr and edna cloning five pou genes expressed during early embryogenesis of zebrafislx four of these genes show extended homology to the brn- class of pou genes. preliminary evidence suggests that the brn- like pou genes overlap with most of their expression domains.the gene studied in most detail so far (zp- ) is first expressed on the ventral side of the future fore-and midbrain. slightly later, expression is also found in rhombomeres and in the hindbraln. these rbombomeres were identified by the specific expression of the krox- marker using a novel in situ hybridization protocol which allows the simultaneous detection of two different transcripts using different color substrates. in the hours old embryo a complex expression pattern is found involving a variety of brain structures and the spinal cord. the distribution of the transcript suggests that zp- is mostly expressed in glia cells. another pou gene we have identified (gp- ) defines a novel class of pou proteins. as a maternal mp, na it is initially ubiquitously present. after gastrnlation its transcript is found most notably in the developing hindbrain in rhombomeres and for which so far no good molecular markers were known. we are investigating the roles the identified pou genes may have in zebrafish hindbrain segmentation. we are currently attempting to manipulate gene expression in developing embryos by injection of rna or by the production of transgenic fish. we are also screening the zebrafish genome for new developmental marker genes. progress about these experiments will be presented. $ - willimann, t. and trueb, b. to gain insight into the regulatory mechanisms of collagen vi synthesis we have characterized the cis-acting elements of the chicken ctl(vl) collagen promoter. footprinting experiments with nuclear extracts from chicken embryos revealed three distinct elements, designated a, b and c, that were protected from dnase i digestion. the nuclear proteins that interact with the three sites were identified by gel retardation assays in combination with the use of various oligonucleotide competitiors as well as specific antibodies raised against well-characterized transcription factors. site a was found to be a target for transcriptional activator apf, whereas sites b and c were shown to be recognized each by two distinct nuclear proteins which belong to the spl multigene family. to address the question whether the three sites alone are able to direct transcription, a minipromoter construct was created in which the sequences of sites a, b and c were placed in front of a reporter gene. after transfection into chicken fibroblasts, this construct exhibited a high relative promoter activity when compared to a large genomic fragment containing the basic ctl(vi) collagen promoter. thus, the three sites are sufficient to induce transcription of this gene. octamer transcription factors (oct or otf) are a subset of the pou family of transcription factors which regulate expression of cellular and viral genes by binding to the octamer sequence atgcaaat. neurons and astroglial cells harbour, in addition to the ubiquitous oct-i factor, brain specific factors termed n-oct , , and . in the present study we determined the chromosomal localization of the gene encoding the n-oct transcription factor and characterized the structure of isolated n-oct genomie clones. the chromosomal mapping was performed by analysis of somatic cell hybrid panels and radioactive and fluorescence in situ hybridization of human metaphase chromosomes. it is interesting that the ' end of the n-oct coding sequence contains repetitive cag and ggc residues. several disorders have been discovered to be related to expansion of trinucleotide repeats. we are presently investigating if the n-oct cag or ggc clusters are hot spots for such mutation mechanisms and if so, which diseases could be associated with it. two dna regions upstream of the distal glucocorticoid receptor binding site interact with nuclear proteins that are tissue-specific (region a) or ubiquitous (region c). the characteristics of the dna-protein interactions have been studied in vitro. these sequences, in different combinations with the natural mmtv promoter, were tested in transfection assays of fibrcblast or mammary cell lines. we show that they are able to modulate the level of glucocorticoid-stimulated transcription. the proximal region of the mmtv promoter has binding sites for the transcription factors ctf/nf-i and oct- . p~asmids with a deletion or mutations in the oct- site were tested in stable transfections, that are most representative of the state of proviral dna with respect to both number of integrated dna templates and chromatin organization. we show that the oct- site is important for the basal level of promoter activity. the data further indicate that the functional outcome may depend beth on the relative ratio of factors to dna templates and on the relative affinity of binding sites, as determined by oligonucleotide competition footprinting. besides the fact that telomeres represent specialised structures important for chromosome stability, the particular significance of cloned c. elegans telomeres is that they will contribute to the completion of the physical mapping of chromosomes and that they provide one set of elements for the construction of artificial c. elegans chromosomes. the cloning of c. elegans telomeres, however, is impeded by the fact that tandem repeats of the sequence ttaggc are not only located at the ends of the c. elegans chromosomes, but also at many internal chromosomal sites. therefore, we have developed a special protocol for the construction of a c. elegans endlibrary in ~ zap. the resulting telomeric library was screened for recombinants containing ttaggc repeats and yielded about positive clones. so far, were analysed by partial sequencing and twelve of them satisfy all criteria required for telomeric clones. their ttaggc tandem repeats are located at the expected position, namely just adjacent to the blunt end cloning site in the polylinker. furthermore, the g-rich strand of the repeats is oriented ' to ' towards the end of the fragment, thus corresponding exactly to the defined orientation of eukaryotic telomeric sequences. finally, hybridisation data with one of these putative telomeric clones show that a subtelomeric fragment hybridises to bal -sensitive fragments on a southern blot with c. elegans dna, indicating that this clone represents a c. elegans telomere. $ - to investigate whether chromatin structure or transcription can interfere with replication, derivatives of the yeast trp ars minichromosome were constructed that contain either the ded or pet promoter firing against the origin of replication ars . while the pet constructs transformed yeast at high frequencies and were maintained as high copy number minichromosomes, the ded constructs transformed at low frequency and the constructs were integrated in the genome suggesting that ars function was impaired. insertion of the h -ars, a second origin of replication, rescued a ded construct as a minichromosome (yrpdh ).the chromatin structure mapped at low and high resolution of the ars region in yrpdh was indistinguishable to that of the pet construct yrpft . ananlysis of rna showed that transcription was going through ars in yrpdh and in yrpft , but the levels of transcripts in yrpdh were much higher. we conclude that transcription through are interferes with replication and prevents extrachromosomal maintenance. dna sequence of a repeated dna segment on circular and linear plasmids of borrelia burgdorferi w.r. z%ckert and j. meyer, abt. pzmom, zahn&rztliches institut der universit~t basel a plasmid-associated repeated dna segment of b. burgdorferi strain b was cloned. at least two copies of this segment appear to be present on the kb circular plasmid, whereas one copy is carried on the kb linear plasntid of this strain. dna sequence analysis revealed a region containing a novel bp putative open reading frame (orfl) on the kb linear plasmid, orfl displayed extensive sequence homology ( %) to putative open reading frames present on the clones obtained from the kb circular plasmid. heterogeneity is mainly caused by rd-base-wobbllng. flanking sequences share - % homology, including the ' end of an additional putative open reading frame irmaediately downstream of orfl. whether orfl and/or the related sequences are being transcribed and yield, in the case of orfl, an approximately . kd, lysine-rich polypeptide, is yet unknown. the repeated sequence seems to be specific for b. burgdorferi sensu lato and therefore may be useful in nucleic acidbased diagnostics of lyme disease. $ - similar to type ib oculocutaneous albinism in humans, overall production of pigment is greatly reduced in dark eyed albino and only obvious in eyes. we have studied the molecular basis of the c h mutation and show that expression of the tyrosinese gene is not affected. after sequencing tyrosinase cdna isolated from c h/c h homozygotes we uncovered a single base alteration from wild type leading to a serine to isoleucine exchange. the importance of this mutation was demonstrated by generating transgenic mice containing a mutated tyrosinase minigene. this showed that the single base change is sufficient to severely depress pigment production in transgenic mice. we therefore conclude that the point mutation is responsible and sufficient to generate the dark eyed albino phenotype. members of the pou-family of transcription factors are involved in developmental and differentiation processes. using a pcr-based cloning strategy with degenerated oligonucleotides we identified several pougenes from the lactating and involuting mouse mammary gland. three of these cdna clones which contained as yet unknown sequences were chosen for further investigations: clone . which has a high homology to pit-l, clone . which is related to the brn- gene and clone . which belongs to the class iii of pou-proteins. the expression levels were analyzed in different mouse organs and in several developmental stages of the mammary gland, including the postlactational process of involution. because of the low abundance of the specific transcripts we used a semiquantitative, reverse transcriptase-mediated pcr assay to investigate the mrna levels of the three novel pou-family members. distinct and specific expression patterns for all three members were obtained in the different investigated organs. interestingly, the expression of the pit- related cdna clone , was elevated in all developmental stages of the lactogenic-hormone dependent mouse mammary gland and in sceletal muscle, whereas its expression was low in other organs. repetitive sequences in dna can allow ectopic (outof-register) homologous recombination, which is often undesirable and can even lead to disease in humans. to prevent this, long homologies should often be interrupted during evolution. accelerated mutation of repetitive sequences by so-called ripping may be one of the mechanisms used to accomplish this in fungi. we are investigating if introns in vertebrate genes have an undiscovered role as interrupters of homology within and/or between genes, in addition to their established role in exon shuffling. by inserting homology-poor introns in an otherwise homology-rich region the genome could prevent ectopic homologous recombination. such a mechanism could be especially useful where there is an advantage in encoding highly repetitive protein sequences. it appears that within the collagen gene family there is indeed a correlation between repetitiveness and intron density. the influence of prenatal diazepam exposume (i, mg/kg/d, s.c.) from gestational day to on the development of the ~-opioid binding sites in striaturn, nucleus accumbens and midbrain of pn , pn and week old male and female rats was studied. week old prenatally diazepam exposed male rats showed a significant decrease of k-opioid binding sites in the nucleus accumbens. a sex-dependent difference in k-opioid binding site densities could also be detected between pn prenatally diazepam exposed male and female rats. we now investigate by in situ hybridisation whether changes in the level of mrna encoding fo~ prodynorphin, the precttrsor for various endogenous ~-opioid agonists, could be responsible for the decrease of ~-opioid binding sites in the nucleus accumbens of week old prenatally diazepam exposed male ~ats. $ - distribution and morphology of nitric oxide-positive neurons in the marmoset cerebral cortex during pre-and postnatal development j.p. hornung* and j. schultz** *institute of anatomy, university of lausanne, lausanne, and **university of fdbourg, fribourg nitric oxide, synthesized by the enzyme nitdc oxide synthase (nos), is a neuroactive substance and a limited number of neurons were shown to express this enzyme either by histochemistry or by immunocytochemistry. both techniques were performed on brains of marmosets with ages ranging from weeks before birth to adult. the morphology and distribution of nos-positive neurons were analyzed in all lobes of the cerebral cortex. the same pattern was revealed by histochemistry or immunocytochemistry. three populations of nos-positive neurons were revealed. first, a transient population of neurons in the molecular layer of the embryonic cortex, many having the morphology of the retzjus-cajal cells. a second population was made of large multipolar neurons in the deep layers of the cortex and the underlying white matter, which persisted from embryonic to adult life. a third, permanent, population was made of small, weakly reactive neurons in the supragranular layers appearing postnatally, this study demonstrates that no can exert its actions in the cerebral cortex through several pathways at different developmental stages. trimethyltin (tmt), a well-known neurotoxicant, was used to study the sensitivity of the different brain cell types (i.e. neurons, astrocytes, oligodendrocytes and microglial cells) to a neurotoxic insult. aggregating brain cell cultures of fetal rat telencephalon were treated during days with tmt at different concentrations, ranging from - m to - m. microglia were found to be the most sensitive cell type, since already at - m of tmt an increased number and clustering of griffonia simplicifolia-positive ceils could be observed. at - m of tmt astrocytes showed increased staining for glial fibrillary acidic protein, characteristic of gliosis. neurons and oligodendrocytes appeared to be the least sensitive cells, since a decrease in the activity of cell type-specific enzymes was observed only at - m of tmt. these results show that microglial cells and astrocytes respond to a toxic insult before any neuronal changes could be detected, and that the microglial reaction may be the first target of tmt. this reaction could then trigger the astrocytic response. vif is widely distributed in brain with suggested functions related to development. vip expression was studied during rat brain development using hybridization histochemistry with a met probe. vip mrna was found in thalamic nuclei on el , later recognized as the ventrolateral and reticular nuclei after further maturation during prenatal period, vip mrna was found in hypothalamus, especially suprachiasmatic nucleus on el and its expression matured over the next days. few cortical neurons contained vip mrna on e , they continuously increased in number and signal intensity over the perinatal period. vip gradually matured in the first three postnatal weeks and adult-like patterns were found on p , when cerebral cortex, ventrolateral and reticular thalamic nuclei, hypothalamus, esp. suprachiasmatic nucleus, were the regions with most prominent vip expression. these results demonstrate the relatively late appearance of vip gene expression in the rat forebrain, as compared with peptides like srif and cck, not suggesting a major role in earlv brain maturation. in primary cultures of mouse cerebral cortical astrocytes, a rapid glycogenolysis followed by a massive glycogen resynthesis ( six-to ten-fold over basal levels after hr) are induced by vasoactive intestinal peptide (vip) or noradrenaline (na). both actions of these neurotransmitters are mediated by camp. since the induction of glycogen resynthesis triggered by vip or na is abolished by inhibition of transcription and translation, we applied the -d gel electrophoresis technique to search for astrocytic proteins induced by vip or na. the comparison of s-labeled proteins from primary astrocyte cultures treated or not with vip ~tm revealed two newly synthesized proteins: a kda protein (pi= ) and a / kda protein (pi= ). only the latter is visible on a silver stained -d gel, which is a prerequisite to consider its purification and microsequencing. induction of the / kda protein by vip was time-dependent with a maximum at - hr. preliminary results indicate a similar induction by na and isoproterenol. in humans, the central analgesic effect of tramadol (t) is only weakly reversed by the ~t opioid antagonist naloxone (nx) (br j clin pharmacol ; : p). t analgesia may thus result from an action on monoaminergic pathways as suggested by in vitro and animal data. we therefore investigated the effect of ct -adrenoeeptor antagonism by yohimbine (y) on t analgesia. according to a randomised double-blind crossover and placebo (p) controlled design, healthy volunteers (n= ) received t ( mg orally), followed (+ h) by y ( . mg/kg intravenously), and y + nx ( . mg intravenously). analgesia was assessed over h by subjective pain threshold (numerical scale -ns-) and objective pain threshold (rill nociceptive reflex -riii-) monitoring. peak analgesic effect was observed at ca. . h (riii + . +semi. ma and ns + . + . ) vs p (rih - . + . and ns + . + . , p< . ) and lasted ca. h. y reversed t analgesic effects during ca. h (r/ii - . + . , p< . and ns + . + . , p< . ), whereas y + nx abolished t effects thoughout the study period (rill - . - . and ns + . + . , p< . ), y alone tended to lower pain thresholds (rill - . + . and ns - . + . , p> . anova). yohimbine, an r antagonist, reverses tramadol effects, thus pointing to the major role of monoaminergic modulation in tramadol anfinociception. a monoclonal antibody (in-l)was raised against neurite growth inhibitory proteins present in rat cns myelin (caroniand schwab, neuron : , ) . in- neutralizes the inhibitory ettect of cns tissue in vitro and in vivo, thus permitting regeneration of certain lesioned cns fiber systems. we have used this antibody to immunostain cryostat sections of the nervous system of the rat. we found that in- stains white matter and myehnated fiber tracts in the cns. the staining pattern corresponds to that of the known myelin specific antigens mbp (myelin basic protein), mag (myelin associated glycoprotein) and mog (myelin oligodendrocyte glycoprotein). the staining of in- in the cns is found in regions of the adult nervous system which have been shown to be inhibitory for axonal growth. interestingly, the regions which show a high abundance of in- antigens are low in staining for gap- , a marker for growth and plasticity in the developing and adult brain. in contrast, gap- is strongly expressed in unmyelinated fiber tracts or nuclei. prevention of oligodendrocyte development in rat spinal cord resulted in suppression of in- expression and elevated gap- levels. this suggests that inhibitory myelin proteins may negatively influence plasticity in the rat cns. to date, the existence of anp and npy in the adrenal medulla has been shown only for a few mammalian and anuran species. thus the present immunohistochemical investigation attempts to localize anp-like and npy-like peptides n the adrena gland of representative mammals, birds, reptiles, amphibia and bony fish by the use of antisera specific for mammalian anp and npy. furthermore, the catecholamine-containing cells were characterized by antisera against enzymes of catecholamine synthesis. anp-and npy-immunoreactivies were detected in adrenal chromaffin cells of all vertebrates studied while no immunoreactivities were observed in the adrenal cortex or in its homolog, the interrenal. the representatives of the different vertebrate classes exhibited marked differences in the proportions of anp-immunoreactive and npy-immunoreactive cells, in the presence of anp-and npy-immunoreactivities in noradrenalineand/or adrenaline-containing cells and in the amount of of cells containing both anp-and npy-immunoreactivities. our results give evidence for a long phylogenetie history of adrenal anp-and npy-like peptides. thus, they stress the physiological impact of these peptides as adrenal paracrine and/or endocrine hormones. human neuronal growth inhibitory factor (gie) is involved in the regulation of neuronal growth and is deficient in the brains of alzheimer's disease victims. this brain-specific metalloprotein consists of amino acids out of which are cys and has been found to contain copper and zinc. proteins with similar primary structure were isolated from bovine and equine brain. the amino acid sequence of these proteins shows about % homology to those of mammalian metallothioneins (mt), with two conserved inserts of thr and a glutamic acid rich hexapeptide in the n-and c-terminal regions, respectively. in contrast to mts, which usually contain only zn(i ), the presence of cu(i) and zn(ii) ( - moles total) in all gif isolations suggests the functional importance of both metals ions. to spectroscopically probe the native zn-binding sites, the zn(ii) ions were replaced by cd(ii). both bovine and equine cd/cu-gif derivatives exhibit similar absorption and cd spectra with features characteristic of cd-thiolate clusters. the release of h-arachidonic acid evoked by glutamate was characterized in cerebral cortical neurons in primary culture grown under conditions that prevent glial cell proliferation. in these cultures, % of the total ceils are immunocytochemicauy characterized as being positive for specific neuronal markers such as neuron-specific enolase and neurofilament. specific markers for astrocytes, oligodendrocytes or microglia were not detected. glutamate induces a concentration-dependent release of h-arachidonic acid with a ec of pm. the pharmacological profile of this glutamate response shows the involvement of two receptor types: the nmda and the ampa ionotropic receptors. the glutamate-evoked release of h-arachidonic acid is phsensitive. when the incubation buffer is alkalinized from . to . , both basal and glutamate evoked release of h-arachidonic acid are enhanced. the glutamate response is also enhanced as intracellular ph is alkalinized by pharmacological treatments: such as inhibition of c -/hco -antiport by dids. these results further stress the role of intracellular ph in glutamate-mediated neurotransmission. $ - multiceli culture system for the study of drug kinetics wechsler d., *schittny j.c. and honegger u. e., depts. of pharmacology and *anatomy, university of bern, ch- bern a cell culture system was developed for the investigation of cellular drug kinetics in cultures with up to cell types. it was used for the study of uptake, competitive uptake, release and redistribution of drugs. individual cell types (human skin fibroblasts, rat astrocytoma cells (c ) and rat hybfidoma ceils (oligodendrocyte x astrocytoma, roc)) were separately grown to confluency on glass circle sectors. in the same petri dish sectors in various combinations were exposed to media containing radiolabelled chlorpromazine (cpz). single and repetitive uptake and release of cpz were measured in each cell type after individual exposure or exposure in any combination of cell types: in hour competitive uptake studies fibreblasts reached . and . times the concentrations of c -and roc-cells, :respectively. in redistribution experiments the exchange of cpz from preloaded to unloaded cells was tested. the exchange rate was dependent on cell type and loading period. it decreased with increasing time of exposure suggesting the formation of more stable cpz stores. we have previously demonstrated that certain neurotransmitters such as vip, noradrenaline (na) and adenosine promote glycogenolysis in mouse cerebral cortical astrocyte cultures (brain res. : - , ) . the question that we then addressed was the nature of the metabolic substrate released by astrocytes following glycogenolysis. we therefore determined the presence of various metabolic substrated released from astrocytes under static conditions and in the superfusate of a laminar flow system developed in our laboratory. neither glucose nor any intermediate of the tficarboxylic acid cycle could account for the decrease in glycogen stores; astrocytes however were shown to release great quantities of pyruvate and lactate at a rate of nmol/mg protein/minute. noradrenaline but not vip promotes a significant increase ( %) in lactate release. the effect of noradrenaline is mimicked by isoproterenol and inhibited by propranolol, suggesting a i~adrenergic mediation. when astrocytes are incubated in an isotonic solution containing no energy suhstrate, the glycogen stores are rapidly depleted and lactate, but not glucose, is released in the medium. in conclusion, lactate appears to play a major role in cerebral energy metabolism homeostasis, as substrate released by astrocytes to prey!de energy fuel for neurones and other neighboring cells. it is known so far that calcium-binding proteins (cabps: calbindin, pan, albumin and ca/retinin) are expressed in some cells of the pineal gland of laboratory mammals. the exact phenotype of these cells is unknown. nevertheless, according to recent studies, it seems that some of them are ganglion cells. therefore we studied the pineal gland of different species (gerbils, rats, goats, cows and humans) using calretiain (cr) antibody which is considered as marker for neurons (andressen & al, cell & tissue rss, t: , ) , the cr-expressing cells were found in all oar species: in gerbils, rats, cows and humans they were scattered throughout the parenchyrna, whereas in goats they lined mostly the pericapillary spaces. according to our findings, it seems that most of the reactive ceils are rather neuron-like elements since morphological criteria for true neurons (axosomatic synapses, nissl bodies, bundles of neurofilaments) are missing. however one cannot exclude that among these cells some are intrapineal ganglion cells, because of ranvier's nodes found along the processes of some crpositive cells. thus, it is possible that these cells are involved in the modulatory control mechanism of the pineal neurohumoral activity and/er by accumulation of intracellular calcium in the formation of pincal calcifications. besides the excitatory amino acid transmitter glutamate, its sulfur containing analogue homocystcic acid (hca) could play a role in excitatory processes in the central nervous system. hca is present in and released from nervous tissue and is a potent neuronal excitant, predominantly activating n-methyl-d-aspartate receptors (do et al., ) . these properties are suggestive of a classic synaptic neurotrausmitter role. on the other hand, hca seems to be localized not in neurones but in glial cells (grandes et al., ; tschopp et al. ) . the efflux of hca is temporally delayed following activation of specific pathways (klancnik et at., ) ; these latter observations are not in accordance with the classic concepts of synaptically-mediated neurotransmission. a laminar flow superfnsian system of monolayer cultures was developed and the released materials from mouse cortical astrocytes, previously preincubated with [ s]-methioulne, were investigated. during application of either noradrenaline or the b-adrenergie agonlst isoproterenol, the extracellalar level of [ s]-methianine was decreased and that of labelled hca was increased. this effect was not observed with the -adrenergic agonist methoxamine, and could be blocked by the ll-adrenergic antagonist propanolol these results, combined with the delayed hca release, the glial localisatian of hca and its neuroactivity, strongly suggest a potential role of hca as a gliotransmitter, modulated by noradrenaline inputs. vasoactive intestinal peptide (vip) induces long lasting metabolic effects in the mouse cns. we have investigated a shorter neuromodulatory action of vip on the synaptic responses in coronal slices of adult mouse cingulate cortex by means of intracelhilar current clamp recordings. electrical stimulation of the underlying white matter evoked a multiphasic synaptic potential consisting of early epsp~ early ipsp, late epsp and late ipsp mediated by non-nmda, nmda, gaba a and gaba b receptors, respectively. vip ( . ~d~l) superfused for rain. had no effect, whereas concentrations over . ixm selectively and reversibly depressed the nmda-medlated responses in / cells. in the presence of cnqx ( [tm) and biencuuine ( ~tm), the isolated nmda-mediated late epsp was still attenuated by . ~m vip in / cells (mean:- % al~er rain.). in other cells vip induced a spreading depression (sd) and a strong, reversals inhibition of the late epsp (mean:- % after min.).these results indicate that in the absence of gabaergic inhibition vip can induce two types of effects in the mouse cingulate cortex, a prolonged decrease of the nmdamediated syaaptic response and a striking sd. interaction between vip and glutamate on c-fos mrna expression. |.-l. martin, d. gasser and p.] . magistretti. institut de physiologie, universit de lausanne, lausanne, switzerland. the regulation of c-los mrna expression by vip and glutamate was examined in primary cultures of neurons originating from the mouse cerebral cortex. in primary cultures of cortical neurons, vip increases camp levels and stimulates c-los mrna expression. interestingly vip stimulation is completely blocked by mk- , an nmda receptor antagonist but not by cnqx or ap , which antagonize ampa/kainate and metabotropic receptors respectively, c-los expression stimulated by glutamate shares the same pharmacology. these results suggest an involvement of endogenously released glutamate in the stimulation of c-fos mrna expression evoked by vip. furthermore, when added together, vip and glutamate interact synergistically to increase cfos mrna levels. consistent with the pharmacology of vip and glutamate, the synergistic interaction between vii' and glutamate on c-fos mrna expression is also inhibited by mk- . interestingly, this synergism is completely inhibited by h- , a protein kinase a inhibitor, whereas staurosporin and kn- which block protein kinases c and ca++/calmodulin dependent respectively exhibit smaller inhibitory effects. presynaptic proteins involved in neurotransmitter release (i.e. synapsin, b- ) and some postsynaptie gaba a receptor subunits possess phosphorylation sites for camp-dependent protein kinase (pka) and/or protein kinase c (pkc). the functional consequences of phosphorylation by these kinases is not known. we have studied the action of specific activators of pkc and pka, phorbol ester (pdbu) and forskolin, respectively, on gabaergic synaptic transmission in area ca of hippocampal slice cultures. pdbu significantly enhanced the amplitude of evoked monosynaptic ipscs (in cnqx and ap ), and both pdbu and forskolin increased the frequency of spontaneous miniature ipscs (m[pscs) in the presence of cnqx, ap and ttx. this effect by pdbu was antagonized by staurosporin, a protein kinase inhibitor. pdbu and forskolin did not change the amplitude or decay of mipscs, suggesting that only presynaptic kinases are involved. furthermore, pdbu enhanced mlpsc frequency by a comparable amount in the presence of the ca + channel blocker cd +, indicating that pdbu did not enhance gaba release from presynaptic endings by facilitating ca + influx into axon terminals. valiunas, v., sc~ilinsky fluri, g. and weingart, r. arachidonic acid (aa) reversibly impairs the gap junction conductance (g~) in cardiac cells. now, we examined whether aaacts directly or via its catabolites. experiments were performed on pairs of neonatal rat myocytes using a dual voltage-clamp method. we used blockers which interfere with enzymes of various catabolic pathways. pretreatment with i ~mpoca (blocks carnitine acyltransferase i) did not prevent the decline in g by i ~m aa; i.e. intermediates of p-oxidation are not involved. similarly, preexposure to i ~m indomethacin (blocks the cyclooxygenase pathway) did not prevent aa from uncoupling; this rules out an involvement of prostaglandins and thromboxanes. exposure to ~mndga (blocks the - ipoxygenase pathway) per se produced a decrease in gj. likewise, exposure to i ~m etya (blocks the - ipoxygenase pathway) also impaired gj. these effects cannot result from accumulation of endogenous aa because preexposure to ~m bpb (blocks phospholipase a ) did not prevent ndga-or etya-induced uncoupling. exposure to ~m skf -a (blocks the epoxygenase pathway) also produced a decrease in gj. hence, ndga, etya and skf -a, compounds with different biochemical actions and of different chemical structure, act on g~ either directly or via an unknown mechanism. $ - potential changes associated with electrical activity in nonmyelinated nerve fibres a. robert and p. jirounek d~partement de pharmacologie, cmu, gen~ve simuheneous measurements of the extracellular concentration of k + ([k+]e), and of the concomitant changes in the membrane potential (era) were measured in the rabbit vagus nerve during and after a period of activity. during a hz stimulation there was a large increase in the [k+]e , accompagned by a change in era, which roughly followed the depolarization expected from the increase in [k+] e . at the end of the stimulation, although the [k+]e was still elevated, the nerve developed a posttetanic hyperpolarization (pth). the pth was generated by the electrogenicity of the na+-k + pump, but its amplitude and time-course were strongly affected by two depolarizing currents: (i) an inward ba +-sensitive current of k + and (ii) an outward current of ci-. we hypothesize that during activity and during the early phase of recovery there was a ba +-sensitive uptake of potassium by the schwann cells. the ci" current, by short-circuiting the na+-k § pump, contributes to the control of the e m, and by this way to the driving force for the inward k § current. overdose of ifosfamide has ooo/red in a canc~c patient ( g i.v. iy / hours with g as ~utectarfe). urir~ collece{on was obtaj/ ed in hour ~ fcr t~o days. c~ was measured ~ a gas liquid d~romatogr~ method of the u ~ ~ (s~@pl. v, pag. - g) ~sir~ a ~e~s ar~ ni~, sensitive ths~mi(~ ic d~. ~ u~s p~mt ~ ~ ~ ~ ~ ~ m~/ h and r~/ h at day one ard day res~ec~vely. ~ _h=~=mmtly ~ has also bsm foa~ in mti~s ~ mgh e~e ~ ~ (~/o~e). w~n ~ ~s am/nist~ to rats (i~ m~/~ ~c m~f~ intraperit~neally), no ~ omild be detected in urine within ht~r~ after drug administl- (cicn. from these ~ data we conclude that c~ is extensi~_ly metabolized in rats and ~ mscbard_sm of ~ el ~mlnaticn in pati~rfc t=cj_ne m~cjlr[c reflect a flmicticrsl der~,~t of c~ m~t~ olisn in man to ifo~=~de ~tion. there are numerous reports of improved memory functions in rats following a dietary supplementation with choline. in some cases, this improvement can be related to enhanced cholinergic transmission (mack et al., behav neurosci., , - , ). but it is not clear whether there is a general improvement in memory or whether specific aspects of learning and memory are affected. this work was aimed at analysing the effects of perinatal cholinergic supplementation on the development of spatial abilities and upon adult performance. choline supplementation ( . g./l. in . m saccharine solution in tap water) was maintained during two weeks before birth. additional supplementation was maintained from the th to the oth week postnatal. spatial learning capacities were studied at the ages of , or days in a cimular swimming pool (morris place navigation task) and at the age of of months on a homing board. adult and aged rats were tested on a radial maze. selective aspects of the performance were markedly improved. this treatment had no specific effect upon spatial memory per se, but, instead, affected learning abilities by promoting efficient behavioural strategies hypothetically based on active representation and anticipation processes. the effects of the treatment remained evident up to months following the supplementation. barcelona, e- bellaterra females of both lines (n=b/grp) were injected sc daily between days - of pregnancy with vehicle (v), i(di) or (d ) mg/kg diazepam, or not(c). in these studies(a,b) their - mo old offspring were subjected to a -run session in a shuttlebox(a: males, females of each line/condition) or a min session in a hexagonal tunnel maze with strategically-placed barriers and a lit central arena(b_: - males of each line/condition). a: the freezing behavior of ld rats was reduced ~s lc, this behavior reverting more to escapes in males & avoidances in females, although v had an effect in the latter also. no within-line differences in inter-trial responses were seen, but pre-session activity was increased in female lvs and ldis, returning to lc levels for ld s. no differences existed among the h groups. b: whereas h maze activity exceeded that of l, no wtthin-line differences appeared. hd s entered the lit arena less than hcs or hvs, indicating an increased timidity after pnd, and a trend in the same direction existed for ldi/ld vs lv. two groups of abstinent female smokers, performing either a fixed rate or a subject paced version of a rapid information processing task were compared. both groups participated in two test sessions (in which the task was performed twice) to compare two different types of cigarettes which were smoked before and during the second task trial: a) the subject's habitual cigarette with a nicotine yield of at least . mg/cigarette and b) a nearly nicotine free test cigarette with a tar yield comparable to that of the habitual cigarette. reaction times to correct detections (series of three odd or even digits in a pseudorandom sequence of single digits) were longer with the subject paced version and not affected by the type of cigarette but shorter with the fixed rate version and even more decreased with the smoking of the habitual cigarette. on the other hand, the pre-to posttreatment increase in the subject paced processing rate was greater with the habitual than the test cigarette. it was concluded that the two task versions assess different cognitive functions. whereas the fixed rate version primarily assesses vigilance performance and seems to be more suitable to measure changes in reaction time, the subject paced version is more sensitive to changes in speed capabilities in rapid information processing. heart rate, physical activity, and cigarette consumption were continuously recorded under field conditions, whereas subjective craving was assessed six times per day and saliva cotinine was measured once daily. abstinence, habitual cigarettes (with a nicotine yield of at least . mg/cigarette), and nearly nicotine free test cigarettes but with a tar yield comparable to that of the habitual cigarettes were compare~) in a sample of twelve female smokers for two days each. whereas physical activity was similar for the three conditions, heart rate was highest with the habitual cigarettes, lowest on abstinence days and in between with the test cigarettes. cigarette consumption was similar for both types of cigarettes and subjective craving was higher on abstinence than on smoking days but no differentiation between the two cigarette types was obtained. saliva cotinine values were highest on the days with the habitual cigarettes but lower and similar with the test cigarettes and on abstinence days. it was concluded that heart rate and saliva cotinine depended only on the amount of nicotine absorbed, whereas subjective craving was reduced by smoking independently of the actual nicotine yield of the cigarette. (ptps) is the rate limiting enzyme in the synthesis of bh, in man, a cofactor for several hydroxylases involved in catecholamine and serotonin biosynthesis. the human and rat liver cdnas encoding the kda subunit of ptps were expressed and the recombinant enzymes purified to homogeneity. the apparent k~ for the substrate, pi and heat stability of the re-combinant enzymes were similar to the native enzymes. the rat enzyme was crystallized and the x-ray structure showed a hexameric native form arranged as a sandwich of two trimers. three histidine, a glutamic acid, and -also shown by site-directed mutagenesis -a cysteine residue characterize the active center of the enzyme thought to be mg +-dependent. according to the proposed ligands we replaced mg * by fe § or mn ~+ and observed an enhanced activity up to %. m. neerman-arbez, v. cirnlli and p. halban. laboratoires/eantet. centre m dical universitaire, gen~ve . pci( ) and pc , members of the mammalian family of pro-protein convertases homologous to the yeast kex , are both expressed in pancreatic islets of langerhans and are thought to be responsible for the conversion of proinsulin to insulin and c-peptide in beta cells. however, the insulin secreting beta ceils are not the only ceils present in these complex microorgans, prompting us to evaluate the expression of pc and pc in islet beta and non-beta cells. rat islet cells were sorted by autofluorescence activated flow cytometry to separate beta cells from non-beta ceils and conversion cndoprotease levels were analysed by western blotting. in beta cells, pc levels were higher than pc (pc /pc = . + . ) whereas the opposite was found for non-beta cells (pc /fc = . • confirming that pc is important for proinsulin conversion while suggesting a role for pc in the conversion of proglucagon, prosomatostatin and propancreatic polypeptide. transformed murine cell lines (insulin-producing beta-tc and glucagonproducing alpha-tc) were found to faithfully reflect the primary rat cells in terms of their pc /pc ratios. finally, post-translational modification of the convertases themselves was found to differ between cell-types, in particular, a precursor kd form of pc accumulated in beta cells whereas only the fully processed kd form was detected in the non-beta cells. the kringle ( + ) domains (k + ) were successfully expressed in e. coil a n-terminal hexahistidine tag was fused to insure the isolation of k + by affinity chromatography on a ni- +-ntaagarose-column. to be able to remove the hexahistidine tag a factor xa cleavage site was introduced between the histidine residues and the n-terminus of the kringle structures. the correct arrangement of the disulfid bonds was determined by amino acid-and sequence analysis. the lysine binding site was proven by affinity chromatography on iysine-bio-gel, as previously shown for k . the dissociation constant of k + was measured by intrinsic fluorescence titration with -aminohexanoic acid. a replication protein a copurifying dna helicsse from calf thymus anthl georgakl, narendra tuteja, blrglt sturzenegger and ulrich hobscher department of veterinary biochemistry university of z rich-lrchel winterthurerstrasse f ch- z rich, switzerland a dna helicase from calf thymus copurified with replication protein a through several steps of purification including deae-sephacel, hydroxyapatite and single stranded dna cellulose. it is finally separated from replication protein a on fplc mono q where the dna helicase elutes after replication protein a. we named this new calf thymus enzyme dna helicase f. characterization of the helicase f by affinity labeling with [a p]atp indicated that the enzyme has a catalytic subunit of kda. gel filtration experiments suggested that dna helicase f can exist both in a monomeric and an oligomedc form. the enzyme unwinds in the ' -> ' direction in relation to the dna strand it binds. all eight deoxyribonucleoside-and ribonucleosidetriphosphates could serve as an energy source. testing a variety of dnndna substrates indicated that the dna heitcase f preferentially unwinds very short substrates and is slightly stimulated by a single stranded '-tail replication protein a allowed the dna helicase f to unwind longer dna substrates up to bases suggesting that copurification of replication protein a with the dna helicase might be of functional relevance. , , , ', ', '-hexachlorobiphenyl ( -cb) shows limited elimination and extensive storage in adipose tissue in vivo (lipophilie unmetabolizable compound) while chlorpromazine (cpz) is known for its lysosomal storage. uptake and release of -cb and cpz were studied in t -preadipocytes (p) and t -adipocytes (a). t -cells were cultivated in dulbecco's minimal essential medium supplement with % fetal calf serum and grown to eonfluency. differentiation was stimulated by dexamethason and bezafibrate exposure for h. radiolabelled -cb ( ~tm) or cpz ( ~tm) was added to the medium. following a single dose - % of cpz were taken up within an hour into p and a. in contrast, -cb reached an intracellular plateau of - % of the dose after lh in p whereas dependent on the triglyceride content a accumulated up to % of the dose. -cb uptake into a was temperature dependent and not saturable with repetitive daily doses up to week. -cb uptake into p was fully reversible while the release from a was limited to % of the accumulated drug. in contrast, the release of cpz was higher from p than from a as a consequence of the different lysosomal activities of the respective cells. the use of t cells allowed to show a remarkable difference in cellular handling of neutral and basic lipophilic drugs and may be helpful to further elucidate basic mechanisms involved. chronic granulomatous disease: an attempt to reconstitute the function of the x-linked form of the disease. chronic granulomatous disease (cgd) is a group of congenital immunodeficiencies that predispose patients to recurrent severe bacterial and fungal infections. the cause of the disease is lack or impairment of -production in phagocytic cells due to mutations in any of the four components of the superoxide producing system. approximately % of all cgd patients suffer from an x-linked form of the disease where the phagocyte oxidase gp phox is affected. this project concentrates on the reconstitution of the deficient gene in phagocytic patient cells. expression of recombinant gp was assayed in vitro and in vivo. attempts to "tag" gp with a foreign epitope were unfortunately so far unsuccessful. the target cells for gene reconstitution are non-dividing monocytes. thus, we have chosen an adenovirus over other viral systems because of: a) its ability to infect nondividing cells, b) its genetic stability and, c) lack of human adenovirus related malignancies or side-effects. we constructed two recombinant adenoviruses: one recombinant expresses a lacz gene (control), another the gp lphox gene. we are trying the expression of recombinants adeno/lacz and adeno/gp in normal and patient-derived cells (for example ebvtransformed normal and patient b-cell lines), as well as the reconstitution of nadph oxidase function in peripheral blood monocytes of cgd patients. kinetics of molecular chaperone action schmid, d., gehring, h., *baici, a. and christen, p., biochemisches institut der universit&t z~rich, ch- zorich; *rheumaklinik, universit&tsspital, ch- z%rich molecular chaperones of the hsp type transiently sequester unfolded segments of proteins and promote their correct folding. target peptides labelled with an environmentally sensitive fluorophore allowed to follow their binding to the molecular chaperone dnak of escherichia coli in real-time. the two-step process is characterized by relaxation times z~ = s and z = s at ~m dnak and .i ~m ligand at ~ in the presence of atp, the formation of the complex is greatly accelerated and follows a single-exponential process with z : . s. the rate of dissociation of the complex was even more increased than that of association resulting in a decreased net affinity for ligands in the presence of atp. the binding-release cycle of dnak thus occurs in the time range of polypeptide chain elongation and folding and is too fast to be stoichiometrically coupled to the atpase activity of the chaperone (ko~ ~ = . min i at "c). supported by the olga mayenfisch stiftung, z%rich, and the hartmann m~ller-stiftung, z~rich. comparison of the activities of native bovine seminal ribonuclease and that secreted from e. coil schein, ch* (siat, technopark, pfingstweidstr. , ch ziirich) and haugg, m (lab. org. chemistry, e.tm., . seminal ribonuclease (bs-rnase) differs from the pancreatic rnase a in that it forms a cysteine-linked dimer and has a relatively higher specific activity in the cleavage of double-stranded (ds-) rna substrates. we expressed bs-rn in a secretion system similar to that described previously (biochem. j. : - , ) using the signal sequence of routine pancreatic ribonuelease. about i mg bs-rn was isolated per liter culture supernatant. human and bovine recombinant interfcron-~ (ifn-r) inhibit the degradation of ss-and ds-rna by bovine pancreatic rnase while they activate the activity of bs-rn on the same substrates (febs letters, : - , ) . recombinant bovine or routine pancreatic rnase (produced in our secretion system) are inhibited by ifn-y, while the recombinant bs-rn or a cysteine-linked dimer of rnase a is also activated by ifn-y. ifn-y activates bs-rn even in the presence of inhibitory concentrations ( . - ram) of mononueleotides. thus the mode of activation cannot be attributed to alleviation of product inhibition. kinetic studies using bp ds-rna as substrate suggest that ifn-~ interacts directly with the bs-rn to increase the rate of highmolecular weight producffsubstrate release, as the apparent ks increases proportionately to the increase in kcat. significantly smaller movements were observed in the simulation with the liganded enzyme. the structural differences between the protein in the crystal and the ensuing calculated structures might arise from the absence of lattice forces in solution. evolutionary relationships among pyridoxal- '-p-dependent amino acid decarboxylases sandmeier, e., hale, t.i. and christen, p. biochemisches institut der universit~t zgrich, z~rich. the pyridoxal- '-p (plp)-dependent amino acid decarboxylases (de) can be subdivided into four apparently unrelated groups. group i is represented by glycine dc, group ii comprises glutamate, histidine, tyrosine and aromatic-l-amino acid dc, group iii procaryotic ornithine and lysine dc as well as the procaryotic biodegradative type of arginine dc, group iv eucaryotic ornithine and arginine dc as well as the procaryotic biosynthetic type of arginine dc and diaminopimelate dc. (n-l) profile analysis, a more stringent application of profile analysis [gribskov et al. ( ) methods enzymol. , - ] established the homology among the enzymes of each group. a search with the profile of group ii indicated a distant relationship with aminotransferases and thus with the ~ family of plp-dependent enzymes. no evidence was obtained that groups i, iii and iv are related with other plp-dependent enzymes or any other protein in the database. apparently, the amino acid dc are of multiple evolutionary origin, in some cases even if they have the same substrate specificity. a. lo russo, a.c. passaquin, u.t. r~eg~, ecole de pharmacie, %hiv. lausanne, c~- lallsaraqe drug-induced local vasoccmstricticn appears to be respmnsible for the hypertensive side effect of the imn/nosti~pressant cyclosporin a (csa). we have therefore ex~rnir~ the [ca +] i increase caused by the vasoccr~trictor hormcme [ar~]vascpressin (avp) in vascular ameoth ~uscle cells (vsmc) treated with csa. [ca +] i levels were m~nitored either with a fluoresc~qce analyser using fura or by ca + efflux. pretreatment of v~mc with csa increased the avp induced rise in [ca +]i. a leftward and upwsxd shift of the ccncentraticm-respmnse curve of the avp-induced rise in ca + efflux was also observed after csa treatxr~%t. ilzg/cticn of csa dote~tiaticn w-as detectable after rain, took to h to become fully established and was reversed after washout. csa potentiaticm was cc~centraticn-deperdent with a threshold at - m. %]]is potentiating effect of csa may be the underlyin~ cause for csa-ind/ced hypert~ sicn. ) barja, f. and turian, g. i, ilab. gen. microbiology, dept. biochemistry, station exp. zoology, university of geneva, quai ernest-ansermet, geneva the actin has been found from the simplest forms of cell to the highly evolved ones. in higher organisms it is transcribed by multigene families but in yeast and several filamentous fungi there appears to be only one actin gene. it was therefore of interest to study the expression of actin gene in n. crassa in which three actin isoforms have been characterized (barja et al., , fems left. : - ) . after isolating genemic dna from wild type n. crassa a pcr was performed with primers corresponding to actin consensus sequences and an amplified fragment of the gene (verified by sequencing) was obtained. this fragment was 'used as a probe in a southern to assess the number of aetin gene(s). our results suggest that n. crassa has alsc only one actin gene. the blocking effect of the n-terminal decapeptide of msmooth muscle actin (sma) (ac.eeedstalvc) on the monoclonal antibody anti-asm- was compared with that of synthetic peptides modified by changing the n-terminal acetyl group or by substituting a single amino acid in position to . using immunofluorescence or immunoblotting techniques, anti-c~sm- activity was abolished after incubation with the native peptide and peptides modified in position (only when e~a) or . on immunoblots running bsa crosslinked peptides on denatured gels, only the natural peptide and peptides with substitution in position or were detected by the antibody. our results indicate that ac.(e)eed is the epifope for anti-c~sm-l. binding of anti-~sm- to sma increased actin polymedzation by decreasing the critical concentration. as control this action was not exerted on skeletal actin. this is the first example of the role of a precise n-terminal sequence in the polymerization of a single actin isoform. double immunofluorescence for msma and total actin on cultured aortic sm cells microinjected with the native peptide showed a selective disappearance of msma staining suggesting that this peptide traps a protein involved in msma polymerization. work is in progress to search for the putative actin-binding protein(s) physiologically interacting with the n-terminal sequence of o~-sma. ( within mitochondria, the mechanism of selective protein degradation is poorly understood. while the bulk of mitochondrial proteins are long-lived, some are degraded rapidly. the selective degradation of mitochondrial proteins is likely to be mediated by proteases similar to those found in bacteria; this is based on the hypothesis that mitochondria have evolved from bacterial endosymbionts, in conjunction with the finding that mammalian mitochondria contain an atp-dependent proteolytic activity similar to that in bacteria. one atp-dependent protease from bacteria, lon, is of particular interest because it is involved in regulated protein turnover. degenerate oligonucleotide primers corresponding to two regions of the bacterial lon gene were used to pcr amplify a kb fragment from yeast dna that encoded an open reading frame with striking similarity to the bacterial lon protease. by screening a yeast cdna library, we isolated a . kb clone potentially encoding a protein of amino acids. haploids bearing a disruptedlon gene were unable to respire or to grow on ethanol/glycerol. fractionation of rnjtochondrial matrix from wild-type cells revealed a peak of aip-dependent proteolytic activity which was absent in the disruptant. furthermore, we have identified matrix proteins that are degraded with a half-time of ~ min in intact wild-type ceils but are stable in the ion disruptants. electron microscopy of lon disruptants revealed the presence of many electron dense bodies in the mitochondrial matrix which are not found in lon + cells. hydrolysis of high molekular weight kin nogen by plasma kallikrein benner, s. and duffer, h., laboratory for organic chemistry, eth h nggerberg, ch- z/inch high molecular weight kininogen (hmwk) is cleaved by the serineprotease plasma kailikrein to generate the hypotensive peptide bradykinin in human blood. we established a measuring system for the time course of the hydrolysis of hmwk in its native and deglycosylated form and in the presence or absence of c~-inhibitor. analysis of the bradykinin release and the yielded protein fragments lead to the three following results: . the hydrolysis did not follow plane michaelis-menten kinetics due to a hmwk-fragment operating as a competitive inhibitor. . addition of the ci-inhibitor stopped the hmwk-hydrolysis immediately, which is in contrast to the physiological role of plasma kallikrein in blood. . so far we have no evidence that the high content of o-glycosylated side-chains of the hmwk-light chain has an effect on the kinin-liberation as proposed in literature. ( tetrahydrobiopterin (bh ) is the essential cofactor for the hepatic phenylalaoine hydroxylase, but also for tyrosine and tryptophan hydroxylases that are responsible for the production of nettrofransmitter precttrsors. furthermore, bh is required for the deavage of giyceryl ethers aztd is i~vo[ved in the biosynthesis of nitric oxide by the nitric oxide synthase. a vazia~t type of hyperphenylalani~emia is caused by a deficiency of bh . the most frequent form of this cofactor deficiency is due to lack of -pyruvoyl-tetrahydropterin synthase (ptps) activity. the human cdna for ptps was isolated, and the recombinant protein was found to be active when expressed in e. coil, thus allowing us to perform hmctional t e, ting of mutant proteins. primary skin fibroblasts derived from ~everal ptps deficient patieilts were investigated for the molecular nature of this autosomal recessive disorder. all fibroblasts had f "ps mrna expressed, and subsequent cdna sequence analysis revealed different mutatkons in the coding region of ptps (codon replacemenls, deletions, or nonseme codons) responsible for a lowered or no enzyme activity. in order to potentially correct ptps activity (and restore bi-i biosynthesis) we are establishing a recombhtant rel~oviral-mediated gene transfer system to efficiently introduce the wild-type human ptps-cdna in these fibroblasts. the thylakoid membrane (tm) contains i molecular species of phosphatidylglycerol (pg). the relative amount of these species depends on the plant species and growth conditions. using phospholipase a (pla ) at ~ to digest preferentially pg in the outer monolayer of spinach tm, we have shown previously that this monolayer was enriched in pg (ca ~). the purpose of this investigation was to determine the transmembrane distribution of each pg molecular species. to this aim, we have studied the hydrolysis kinetics of each species in the presence of pla in spinach and squash tm containing different relative proportion of molecular species. the hydrolysis rate and extent of the molecular species depended on the unsaturation degree of the fatty acid at the sn-i position as well as on the presence or absence of : ( t) at the sn- position. for instance, the hydrolysis rate of pg ig: / :l( t) was greater than that of pg : / : . these results will be discussed in terms of the thylakoid transmembrane distribution of each pg molecular species. (supported by the snsf - . ). from previous electrophysiological evidence, we concluded that metal ions (hgz+,cd +,ag +) at low concentrations (i - m) increase rapidly ( - s) cation (na + , k + ) conductance at the apical membrane of epithelial cells. we investigated here the effects of these metals on [ca +]i in mdck-i cultured cells, for a potential correlation between functional membrane changes and [ca +]i . metals ( - - - m) were added at the apical side of the epithelium, and [ca +]i was measured with fura- . hg + induced a rapid (peak - s) and marked increase in [ca +]i, whereas cd + entailed a slower (peak - min) and marked response. the time-course of effects for both metals was similar to the electrophysiological changes. in contrast, the pattern of effects of ag + on [ca +]i differed markedly from that on apical membrane conductance. thus, the effects of metals on [ca +]i are conspicuous but not tightly associated with changes in membrane conductance. c. bulliard and l-l. dreyer, institut de biochimie, universit de fribourg, ch- fribourg trans-plasma membrane oxydoreductases (pmo) play a significant role in energy transduction and post-receptor signal transmission, but the enzymes have not been characterized so far. we have achieved the purification of pmo from rat brain synaptic vesicles and from synaptic plasma membranes. the membrane enzymes were extracted by % lubrol xl tm, precipitated with ammonium sulfate (between and % saturation) and purified by means of hydrophobic chromatography on butyl-agarose and gel f'fltration on superose- , followed by page under native conditions. specific enzymatic staining of native page gels yields two homogenous diaphoraselike activities, pmo-i and pmo-ii . sds-page of the enzymes showed that pmo-i is made of two subunits of kda and kda whereas pmo-ii consists of a single sub-unit of kda. the ki)a subunits from pmo-i and pmo-[i are probably identical from their respective properties. partial n-terminal sequencing ( aa) of the kda subunit from pmo-i showed % homology with rat brain fructose-l, -bisphophate aldolase. these data indicate that pmo is closely associated with the glycolytic enzyme that co-purifies in all steps under suitable conditions. the biochemical functions of pmo's remain to be established. ferredoxin:thioredoxin reductase (ftr) is the key enzyme in the ferredoxin/thioredoxin system, the light-dependent regulatory system in oxygenic photosynthesis. it is a nucleus encoded ironsulfur protein, composed of two dissimilar subunits, one of which is the catalytic subunit containing a redox-active disulfide bridge functional in the reduction of thioredoxins and a fe- s cluster. using pcr we have isolated and characterized a cdna clone of bp from spinach and a clone of bp from corn coding for the catalytic subunits including the transit peptides. the first (spinach) resp. (corn) amino acids after the start exhibit typical features of chloroplast transit peptides. following the transit peptides are (spinach) or (corn) ami[~o acids representing the catalytic subunits. the primary structures are highly conserved between these two species with % similarity and % homology. seven of the eight cys residues found in the spinach subunit and important for the catalytic activity are at conserved positions. (snf - . and - . ) $ - r. zurbriggen and j.-l. dreyer, institut de biochimie. univarsit de fribourg, ch- fribourg most mammalian cells display transplasma membrane oxidoreductase activity (pmo). the enzymes use intracellniar reduced pyridine nucleoticle to reduce an unspecific extracellular electron accepter as substrate. in order to set up a methodology for purifying, characterizing and quantifying pmo's, the plasma membrane from a neuroblastoma cell line, nb a , has been biotinylated. subsequently the biotinylated proteins were purified by immanoprecipitation with avidin, antiavidin-antibodies and insoluble protein a. the protein recovery of an immunopurified membrane preparation was < . % of the protein content in the cell extract. specific pmo activity was increased to fold compared to the activity in whole cells. this approach demonstrates the presence o ' pmo within the cell plasma membrane. this activity accounts for about one third of the total cellular diaphorase activity and cannot be attributed to an increased perraeabilization of the plasma membrane induced upon biotinylation nor to intracelluiar activity from lysed cells. pmo also promotes cell growth at low substrate concentrations ( . -ll.tm). native gel electrophoresis of iarinobiotinylated and affinity purified plasma membrane extracts displays two diaphorase-positive bands, indicating that a homogeneous cell population may express several pmo activities at the plasma membrane. fluvastatin (fs) is a new hmg-coa reductase inhibitor. its affinity for major human drug metabolizing p monooxygenases (cyp c , cyp d and cyp a ) was determined in liver microsomes. fs showed low affinity (ki > ~tm) for cyp d and cyp a whereas cyp c was selectively and competitively inhibited (ki = . ~tm). the potential for in vivo fs interactions with cyp c substrates was therefore investigated in volunteers using dicinfenac (df) as a probe ( mg orally) on days , and . df and '-ho-df kinetics were determined by hplc/uv. df cmax increased over time ( . [sd . ], . [ . ] and . [ . ] mg/l on day , and , resp.). oral clearance was reduced on days and ( % and %, resp.). a time dependent decrease in urinary metabolic ratio ( '-ho-df/df) was noted ( . [ . ], . [ , ] and . [ . ] on day , and , resp. (p < . )) only over the first hours. fluvastatin therefore exhibits a two-phase interaction in vivo: an early transient and a late cumulative inhibition. the transient phase matches inhibition profiles predicted by quantitative models integrating in rive pharmacokinetics and in vitro inhibition data (q-dips). simple competitive inhibition by fs cannot explain the late phase. other mechanisms (i.e. fs or metabolite cumulation, mi complexation) are hypothesized. in some situations fs is expected to cause interactions with cyp c substrates, which are partially predicted by quantitative modeling of in vitro data (snsf project n o - . ). gen~ve, switzerland. previously, a parvalbumin mutant, pvf~ w, was constructed with a unique reporter trp in the middle of the hydrophobie center. in the present study three new parvalbumin mutants, derived from pvft~w and containing alterations essential for ca +-binding in either one, pv.c• and pv_m, or both, pv-co~, of the two ca +-binding sites, were analyzed in order to study the metal binding properties of individual ca~+-binding domains and their contributions to the structure and stability of the protein. both, pv_ct, and pv.~, bind i ca ~ § with affinity constants, kc,, of . , and . - m "~ in the absence of mg +. mg + shifts the ca~+-binding isotherms of pv.co to higher free [ca z+] according to the competition equation with km~ = m t. pv.m~ is much less effected by mg z* (kmg = m "t) and can be considered as possessing a ca +-specific site. nevertheless, in the absence of ca § both, pv.co and pv~, bind i mole of mg + with much higher affinity than predicted from the competition studies. pv_cd;~ binds neither ca . nor mg +. trp-fluorimetry and uv-difference spectroscopy revealed that the ca~*-loaded conformations of pv_co, pv.ef and pvr~o~w are similar, with the trp- deeply buried in the hydrophobie core. however, striking differences between the mutants are found in the metal-free and mg~+-loaded forms. the conformation of pv~t~;~ is not affected by ca ~ or mg z*. our results indicate that destroying the ca +-binding of the ef loop, but not of the cd loop, strongly destabilizes the protein in the absence of ca z § . biochemlsches institut der unlversitfit zfirich, switzerland, ch- . the sea urchin, strongylocentrotus purpuratus, metallothionein gene, mta, was constructed and expressed under the control of a heat shock promoter in a protease-deficient strain of e, coll. the nascent recombinant protein was stabilised by the addition of exogenous cd. the cd-containing protein was precipitated with ethanol and subsequently purified to homogeneity by gel permeation and ionexchange chromatography. the purified protein was identified as the desired gene product by tryptic peptide map analysis, sequence analysis and mass spectroscopy. typical yields of protein were mg per litre of culture. the presence of cd ions per molecule was confirmed by the sh/cd ratio and by the existence of h cd nmr resonances. the apparent association constants of sea urchin mt with cd, which has a charge of - , were found to be approximately times weaker than for rabbit mt, at ph . (mt) are small metalloproteins displaying as their main feature clusters of d i~ metal ions bound to the thiolate ligands of the abundantly occurring cysteine residues (cys). from a set of over sequences a general multialignment of class i mt has been obtained on the basis of the needleman & wunsch algorithm. all cys are conserved in the man%mals and over % of them in other vertebrates and invertebrates. on the basis of similarity/identity scores we can isolate groups of sequences. evolutionary relationships between species and groups have been calculated with the maximum parsimony method of felsenstein and with the distance matrix method of fitch and margoliash. the divergences observed among the mammalian mts indicate a partition into four distinct subforms which all may have arisen before the separation of the species in evolution and which in part may differ in function. $ - bourque, a., singh, a., dykeman, a., macmahon*, a., and foster*, w. atlantic veterinary college, charlottetown, canada, and *reproductive toxicology section, environmental health centre, tunney's pasture, ottawa, canada. hexachlorobenzene (hcb) is a known environmental pollutant that is produced as an industrial by-product of certain chemicals. when administered in high dosage, hcb induces alterations in the rhesus monkey ovary. a purpose of the study was to determine a no observable adverse effect level (noael) for the compound. twenty, cynomolgus monkeys, - years old were placed in five groups. hcb was given orally in concentrations of . , . , . , and mg/kg b.w. in gelatin capsules, daily, for weeks. one group of animals fed glucose only, served as the control. ovary specimens fixed in % buffered glutaraldehyde were prepared by conventional methods for transmission electron microscopy. ultrastructural alterations were revealed in the developing ova and follicular cells in all the ovaries from hcb-treated animals in a dose-related manner. clinical chemistry was unaffected by hcb. a noael for this reproductive toxicant is yet to be determined. a.b. was a recipient of the nserc undergraduate student award. an acidic haem-and zinc-containing protein has been isolated from bovine brain. native and sds-polyacrylamide-gel-electrophoresis reveal a monomeric protein with an apparent molecular weight of , kda. the absence of co-staining with tetramethylbenzidine implies that the haem group is non-covalently bound. the electronic absorption spectrum, with a soret-band absorption maximum at nm and c~-and i~-bands at and nm, respectively, is similar to that of fe(ll)-myoglobin, consistent with the presence of haemiron in the ferrous oxidation state. in air the protein was easily oxidized to the fe(lll) form with a subsequent shift of the soretband maximum to nm. atomic absorption measurements indicate approximately mole of zinc and mole of iron per mole of protein. the amino acid composition is similar to that of indoleamine- , -dioxygenase, although no such enzymic activity has been detected. in this study, we looked for this substance and its metabolites in young and old bovine lenses, because of their possible role in cataract formation. the substances were detected by hplc analysis. the kynurenine aminotransferase (kat) activity was determinated by the method of tobes (methods enzymol ~ : ) . the fluorescent substance formed from -hk was characterized by thin layer chromatography followed by reaction with rtinhydrin, uv and fluorescence spectrometry, and atom bombardment for molecular mass determination. -hk at concentrations of . -+ . , . _+ . , and _+ . btg/g of tissue was detected in iris/ciliary body, retina, and calf lens respectively. this substance is deaminated by kat. the activity of this enzyme was . _+ . , . _+ . , and . +_ ~tmol/g of tissue/hour in retina, iris/ciliary body, and lens respectively of old bovine eyes, but it was not found in calf eyes. the deamination of -hk resulted in the formation of a fluorescent substance which was identified as oxo-xanthurenic acid (oxa) with a molecular mass of daltons. the accumulation of oxa acid possibly interacting with lens proteins could induce cataract formation. snf - . , emdo, and hartmann-mtiller. the goal of covalent immobilization of oligonucleotide capture probes to solid supports has been accomplished by light-dependent, photolinker polymer mediated immobilization strategies. synthetic oligonucleotides were immobilized by facile layer coating procedures. the procedure does not require functionalization of the oligonucleotide probe or the matedal surface. oligonucleotides were linked to polystyrene with a biopolymer which was multiply substituted by photoactivatable diazirines. capture probe photoimmobilization ( nm irradiation) was strictly light and diazirine dependent. using radiolabeled oligonucleotides as capture probes and polystyrene as material surface, the light dependent coupling efficiency was %. nonspecific oligonucleotide binding was below %. photojmmobilized oligonucleotides retained the ability to form stable complexes with complementary dna strands, and target oligonueleotides of varying length were captured as well as dna fragments produced by pcr techniques. indoleamine , -dioxygenase (ido), a superoxide radical scavenger, is present in transparent human lenses and produces -hydroxykynurenine, a uv-b filter. the synthesis of -hydroxykynurenine by ido and its degradation by kynurenine aminotransferase (kat) were measured in transparent lenses and cataracts. post-mortem eyes of elderly persons between and years of age with transparent lenses were obtained from the eye-bank of the department. fresh cataracts were obtained from cataract surgery patients. ido activity was measured by the method of yamazaki et al (biochem j ; : ) adapted for hplc. kat activity was measured by the method of tobes (methods enzymol ; : ) . ido activity found in post-mortem transparent lenses (cortex and nucleus) was . _+ . nmol/mg protein/hour. in cataracts a low do activity of . _+ . nmol/mg protein/hour was found in the cortex but not in the nucleus. kat activity, found in the eight cataracts examined, was . _+ . nmol/mg protein/hour. inhibition of ido activity and induction of kat activity seems to be involved in human cataract formation snf - . , emdo, and hartmarm-m er photolinker polymer mediated covalent immobilization of antibodies and f(ab') fragments has been achieved by newly established lightdependent coupling procedures. anti alpha-l-fetoprotein monoclonal antibodies and f(ab') fragments derived from anti prostate specific antigen monoclonal antibodies were covalently linked to microplates. diazirine derivatized bovine serum albumin served as multifunctional light activatable linking agent. both immunoreagents, monoclonal antibodies and f(ab') fragments remained immunologically active after nm irradiation (irradiance . mw cm - for minutes). covalency of antibody binding was inferred from i) the photoreagent dependence, ii) the light dependence of the immobilization process, and iii) the reversibility of immunocomplexation after acid treatment. $ - two ~ of extracelluiar elecirical resistance in v~kwricular myocardil~ fleischhauer j.c., kl~ber a.g., dept. of physiol. bern in myocardial tissue, the electrical resistive properties of the extracellular space are important for local current flow during e~citation and therefore influence impulse conduction and the magnitude of the extracellular electrical field. to assess the r~tlti~t nature of the resistance of the extracellular space, electrical cable analysis was applied on an arterially perf~-sed rabbit papillary mascle. ~he resistivity of the intravascular space was changed ( to ~i~n) by variation of hematocrit frcrn to % in the perfusate. in order to change the voltm~ and hence the electrical resistance of the interstitial space, colloidosmotic pressure in the perfusate was varied by changing dextran {m r ) concentration (i to g/l). altering the interstitial space volume had a marked influence on the extracellular resistance (ro) , conduction velocity and the magnitude of the extracellular field. variations of the resistive properties of the intravascular space had no significant influence on ro, conduction velocity and the magnitude of the extraeellular electrical field. our results suggest that the microvascular tree is insulated from the interstitial space and local electrical current flow during excitation in heart muscle is confined to the narrow interstitial clefts. s - , s - s - , s - s - , s - bchini hooft van huijsduijnen s - s - s - s - s - , s - s - s - , $ - s - nook, ek s - , s - s - , s - s - , s - , s - s - , s - , $ - s - , $ - s - s - s - s - , s - , s - s - lrmler s - , s - s - , s - , s - s - lipp, p. s - so - s - , s - , $ - , $ - , $ - , $ - $ - ,$ - s - s - , s - s - , s - molar s - s - , s - e s - , s - s - , s - , s - e. s - s - , s - , s - s - s - s - s - , s - , s - , s - , $ - s - , s - s - , s - s - , s - stalder h. s - s - s - , s - , s - s - teheesen s - , s - , s - s - van dillewyn s - , s - , s - s - , s - s - , s - huber d., ojha m. and turian g. laboratory of general microbiology, university of geneva, sciences iii, quai ernest-ansermet, geneva , switzerland. ca +-dependent protease in allomyces is predominantly localized in the apical region of the exponentially growing hyphae (huber and ojha, submitted) . many cytoskeletal proteins like actin and tubulin are also mainly localized in this growing region (heath, l~ ). it is known that the ca +-dependent protease proteolyzes a number of cytoskele~ ton proteins in order to regulate the plasticity of the cell (mellgren, ) . we have studied the proteolysis of some cytoskeletal proteins by this protease purified from the aquatic fungus allomyces erbuscula. actin, tubulin and desmin, were found to be rapidly proteolyzed in vitro at a high substrate to enzyme ratio. immunofluorescence studies of proteolyais made in situ in exponentially growing hyphae showed modification of apieally localized cytoskeletal proteins. this colocalization of the ca +-dependent protease and cytoskeletal proteins in the same apical region is inferred in relation to hyphal elongation. jbiological databases grow exponentially. in order to provide access, as much data las needed and as little volume as possible shall be returned upon a query. the icurrentiy available dna and protein sequence databases are updated in a sophisticated network of procedures and expose a considerable amount of redundancy. research is performed on optimizing the creation of non-redundant data sets. the resulting data are disttibuted in switzerland, or made accessible to researchers who cannot utilize local resources. transparent access to the swiss resources, as well as paneuropean services, is provided with a job scheduling system which was developed in basel, the hierarchical access system for sequence libraries in europe ("hassle"), and various other computer programs which allow comprehensive browsing such as "gopher" and "www". training courses to use the resource effectively are running throughout the year. key: cord- -a t u authors: nan title: alphabetic listing of diseases and conditions date: - - journal: handbook of autopsy practice doi: . / - - - - _ sha: doc_id: cord_uid: a t u part ii begins with a list of special histologic stains, their for use and their corresponding references. at the end of this list is a procedure for removal of formalin precipitate from tissue sections. diseases. there may also be a list of possible associated conditions. these entities are generally linked pathogenetically to the main disease entry. any asterisk after a related disease indicates that that disorder is also listed as a disease entry. many disease entries will be followed by a three-column table that provides the reader with a listing of the pathologic findings to be expected with the disease as well as the prosection and dissection procedures necessary to demonstrate those findings. it is expected that routine hematoxylin-eosin stains will be done on all sections submitted for histologic examination. special stains will be recommended in the procedures column of the tables, when indicated. any table immediately following the two columns of disease entries always refers to the disease in the right column. prepare smears of undiluted blood. obtain blood for molecular studies for preservation of small intestinal mucosa and for preparation for study under dissecting microscope, see part i, chapter . submit sample for histologic study. submit stool for chemical analysis. record weight and submit sample for histologic study. freeze liver for molecular studies record appearance of spine (see also chest roentgenogram). for removal and specimen preparation, see chapter . request luxol fast blue stain. for removal and specimen preparation, see chapter . below-normal weight in infants. kyphoscoliosis. very low concentrations of cholesterol and decreased triglycerides; serum~-lipoprotein or absent; a.-lipoproteins present. acanthocytosis (spiny red cells). gene mutations ( ) . abnormal shape of villi; vacuolation of epithelial cells. fatty stools fatty changes. gene mutations ( ) systemic manifestations of malabsorption syndrome* and of vitamin a deficiency. * kyphoscoliosis. axonal degeneration of the spinocerebellar tracts; demyelination of the fasciculus cuneatus and gracilis ( ) . possible involvement of posterior columns, pyramidal tracts, and peripheral nerves. atypical retinitis pigmentosa ( ) with involvement of macula. angioid streaks ( ) . synonym: cerebral abscess. note: for microbiologic study of tissues and abscesses, see part i, chapter . include samples for anaerobic culture. it is best to study the brain after fixation but if specimen is examined fresh, aspirate and prepare smears of abscess content. photograph surface and coronal slices of brain. request giemsa stain, gram stain, pas stain, and grocott's methenamine silver stain for fungi. external examination if there is evidence of trauma, see also under "injury, head." prepare roentgenograms of chest and skull. submit for microbiologic study. for removal and specimen preparation, see chapter . for microbiologic study, photography, and special stains, see under "note." for exposure of venous sinuses, see chapter . sample walls of sinuses for histologic study. for exposure of paranasal sinuses, mastoid cells, and middle ears, see chapter . for removal and specimen preparation, see chapter . procedures depend on suspected lesions as listed in right-hand column. skin infections in upper half of face. edema of forehead, eyelids, and base of nose, proptosis, and chemosis indicate cerebral venous sinus thrombosis. * trauma; craniotomy wounds. skull fracture and other traumatic lesions. for possible intrathoracic lesions, see below under "other organs." the national transportation safety board (ntsb)* has authority over aircraft wreckage and the legal authority to investigate and to determine the cause of air crashes. ( ) the dead are the responsibility of the medical examiner or coroner. local police will seal off the area of the crash. other than for the purpose of determining that death has occurred, no one should be allowed to approach the bodies or any objects until the identification teams and the medical examiner or coroner have taken charge. the sudden influx of bodies after a commercial air carrier accident and the request for speedy identification of the victims would overburden almost any institution. managing such a disaster is eased by writing a contingency plan beforehand. temporary morgue facilities may have to be established near the scene of the crash. refrigerated trucks may serve as storage space. a practical approach is to deal first with those bodies that seem to be the easiest to identify, in order to narrow the field for the more difficult cases. if bodies are scattered, their locations can be referenced to stakes in the ground or spray paint on pavement; only then should these bodies (or parts) and personal effects be collected. for large-scale crashes a locations can be referenced to a string-line grid benchmarked to gps coordinates. records and diagrams of the relative positions of victims are prepared during this phase. if bodies are still within the airplane, their positions are recorded, and photographed. the personnel of the medical examiner or coroner can augmented by d-mort team staffed by forensic pathologists, anthropologists, dentists, morgue technicians, and investigators supplied by the national disaster medical system. ** the airline will provide a list of the passengers and the federal bureau of investigation (fbi) disaster team will make itself available to take and identify fingerprints and aid in the acquisition of other identifying data such as age, race, weight, height, and hair color and style. if dental records can be obtained, this provides one of the most certain methods of identification. a medical history indicating amputations, internal prostheses, or other characteristic surgical interventions or the presence of nephrolithiasis, gallstones, and the like will be helpful. fingerprints (and footprints of babies) should be taken in all instances. wallets with identification cards,jewelry, name tags in clothing, or other personal belongings may provide the fastest tentative identification. the medical examiner may elect to autopsy only the flight crew but not the passengers of an aircraft crash. however, the grossly identifiable fatal injuries should be described, photographed, and x-rayed. this may reveal identifying body changes. if comparison of somatic radiographs, dental records, fingerprints, or photographs do not identify the victim, dna comparison must be considered. burned or fragmented bodies of passengers and the bodies ofcrew members, and particularly the pilots, must have a complete autopsy, including roentgenographic and toxicologic examinations, which must always include alcohol and carbon monoxide determinations. internal examination might reveal a coronary occlusion, or roentgenograms may disclose a bullet as evidence that violence preceded the crash. in some airplane crashes, particularly in light airplane accidents, suicide must be considered. in such cases police investigation is required to determine if the pilot exhibited suicidal ideation in the recent past.. when resources permit, autopsies should be performed on all deceased occupants of aircraft crashes, including passengers, in order to distinguish among blunt impact trauma, smoke inhalation, and flash fires as causes ofdeath, and to answer future questions concerning pain and suffering, intoxication, and sequence of survivorship. after a crash victim has been identified, the coroner or medical examiner will issue a death certificate. if remains of a decedent cannot be found, a judge can, upon petition, declare a passenger dead and sign a death certificate prepared by a medical examiner. *phone # ofntsb command center: - - **phone # of dmort: - - . entry should be followed. usually, the circumstances that led to drowning are not apparent from the autopsy findings but can be reconstructed from reports of witnesses and the police. because the reflex drive to seek air is triggered by hypercarbia, not hypoxia, loss of consciousness and drowning can ensue after hyperventilation and breath-holding by experienced swimmers who then drown without a struggle. there are no specific autopsy findings. a search for trauma, including a posterior neck dissection, should be made in all instances. head and cervical injuries may be responsible for loss of consciousness and drowning, usually in individuals diving into shallow water. toxicologic examination as described below for scuba diving accidents is always indicated. with scuba diving fatalities, investigation of the equipment and circumstances is usually more important than the autopsy. scuba fatalities should be studied by or with the aid of diving experts-for instance, members of a diving club or shop (not the one providing the gear used by the decedent) or the u.s. navy. ( ) careful investigation of the scene and study of reports of witnesses and the police are essential. the investigation should ascertain the site of diving (currents and other underwater hazards), the estimated depth, the water temperature (exposure to cold), and a description of water clarity. electrocution should be considered if the site has electric underwater cables (see "injury, electric"). cerebral concussion should be considered if explosives were used in the vicinity. knowledge of the method of recovery of the body and the type of resuscitation efforts can aid in the interpretation of apparent wounds. the medical history of the diving victim should be sought, as it may lead to a diagnosis for which the autopsy is typically silent, such as seizure disorder, or may reveal asthma, emphysema, or chronic bronchitis, all of which increase the risk of air trapping and arterial air embolism. although drowning may be the terminal event in some scuba deaths, the investigation should be focused on the adverse environmental and equipment factors that place a capable swimmer at risk of drowning (see "embolism, air" and "sickness, decompression"). because scuba divers risk arterial air embolism if they ascend with a closed glottis, on can attempt to document gas bubbles at autopsy, but their interpretation is problematic: bodies recovered immediately are subjected to resuscitation efforts, which can by themselves produce extra-alveolar air artifacts, and bodies not recovered immediately tend to be found in a putrefied condition, full of postmortem gas. in the remaining cases, the pathologist must consider the potential of introducing artifactual gas bubbles by the forcible retraction of the chest plate and by sawing the calvarium. the following procedures apply primarily to scuba diving accidents. interrogation of witnesses is important; the behavior and complaints of the decedent, if any, might help distinguish between a natural death by heart disease and an unnatural death by air embolism. external examination eyes and ears head (skull and brain) chest blood (from heart and peripheral vessels) heart tracheobronchial tree and lungs a procedures photograph victim as recovered and after removal of wet suit and other diving gear. record condition of clothing and gear. impound all diving equipment for study by experts, particularly scuba tank, breathing hoses, and regulators. residual air in tank should be analyzed. record color of skin (including face, back, soles, palms, and scalp). palpate skin and record presence or absence of crepitation. record extent and character of wounds. prepare histologic specimens. record appearance of face (including oral and nasal cavities) and of ears. prepare roentgenograms. if air embolism must be expected, as in the presence of pneumomediastinum, follow procedures described under "embolism, air." for evaluation of findings, see also above under "note." if decompression sickness (caisson disease) is suspected, also prepare roentgenograms of the elbows, hips, and knees. otoscopic examination. funduscopic examination. save vitreous for possible toxicologic and other studies. for removal of brain, see chapter . record contents of arteries of the circle of willis and its major branches and basilar artery. strip dura from base of skull and from calvarium. for removal and specimen preparation, see chapter . for demonstration of pneumothorax, see under "pneumothorax". if gas is visible in coronary arteries, photograph. photograph and aspirate gas in heart chambers. submit samples of heart blood and peripheral blood for toxicologic study and drug screen. examine lungs in situ. save bronchial washings for analysis of debris. fresh dissection is recommended. if decompression sickness is suspected, prepare sudan stains from fresh-frozen lung sections. complete toxicologic sampling should be carried out (see chapter ). record nature of gastric contents. remove neck organs toward end of autopsy. for posterior neck dissection, see chapter . incise tongue. for removal, see chapter . for removal, see chapter . for removal, prosthetic repair, and specimen preparation, see chapter . consult roentgenograms. in decompression sickness, fatty change of liver, and ischemic infarctions of many organs. interstitial emphysema. aspiration (see above). trauma to cervical spine. mottled pallor of tongue after air embolism. contusion of tongue after convulsive chewing. nitrogen bubbles in spinal cord arteries may occur after rapid ascent. air embolism;' cerebral edema in decompression sickness. aseptic necroses (infarcts, "dysbaric osteonecrosis"), most often in head of femur, distal femur, and proximal tibia. infarcts indicate repeated hyperbaric exposures. nitrogen bubbles in and about joints and in periosteal vessels ("bends") occur during rapid ascent. related terms: automobile accident; motorcycle accident. note: a visit to the scene can make the interpretation of the autopsy findings easier. the vehicle can also be inspected in a more leisurely fashion at the impound lot. this is particularly useful for correlating patterned injuries with objects in the vehicle. most vehicular crashes occur as intersection crashes or because a vehicle with excessive speed left a curved road. the medical examiner or coroner should gain a basic understanding of the crash mechanism so that informed descriptions can be rendered, e.g., "impact to the b pillar of the decedent's automobile by the front of a pickup truck which failed to stop for a stop sign at an intersection, resulting in a -feet intrusion into the cabin; restraint belts not employed; air bag deployed; extrication required which took minutes." police are responsible for determining mechanical and environmental risk factors for the crash and for determining some human risk factors such as suicidal or homicidal intent. the pathologist determines other risk factors for crashes such as heart disease, a history of epilepsy, and intoxication by carbon monoxide, drugs, and alcohol. suicide as a manner of death should be considered when a single-occupant vehicle strikes a bridge abutment or a large tree head-on, with no evidence of evasive action or braking. in such a situation, the standard police traffic investigation should be supplemented of interviews of the victim's family and friends. the ambulance run sheet is an invaluable source of observations that often are not available from the police. this document should be acquired in all instances, even if the paramedics determined that death occurred and did not transport. the basic autopsy procedures are listed below. most traffic victims who die at the scene or who are dead on arrival at the hospital died from neurogenic shock caused by wounds of the head or vertebral column, or from exsanguination from a tom vessel or heart. as such, they have little lividity, and little blood is found in the vehicles. presence ofintense lividity may indicate suffocation or heart disease as a cause of death. if postural asphyxia is suspected, the first responders to the scene should be interviewed to determine the position of the decedent in the vehicle, and the vital signs, ifany, ofthe decedent from the time of the crash to the time of extrication. posterior neck dissection is indicated in these instances. if manifestations of heart disease, intense lividity, and absence oflethal wounds suggest that a crash occurred because the driver was dead, other drivers on the road may have observed that the victim was slumped at the wheel before the crash. the determination of heart attack at the wheel is usually simple, because most such victims realize that something is wrong, and bring the vehicle to a stop at the side of the road, or coast gently into a fixed object. in such instances, damage to the vehicle is minor, and wounds to the decedent are usually trivial. while pattemed wounds can often be matched to objects (see below), patternless wounds usually cannot be visually matched to specific objects, although an opinion can sometimes be given as to what object was struck, based on the direction of motion and position ofthe body with respect to the vehicle. impacts with the a-pillar produce narrow vertical zones of facial laceration and fractures extending from forehead to jaw. tempered glass shatters into small cubes on impact, and leaves so-called "dicing" wounds, which are abraded cuts arranged in a somewhat rectilinear pattern. windshield glass leaves shallow, abraded, vertically oriented cuts on the face or scalp. with pedestrians, the lower extremities are of particular forensic interest, to determine the height and direction of impact from vehicles that left the scene. scalp hair and blood should be collected from such "hit and run" victims and from occupants of a suspect car if police have a question as to which occupant was the driver; these exemplars can be compared to fibers and tissue recovered from the vehicle in question. likewise, foreign material in wounds can sometimes be matched to suspect vehicles, and should be sought and retained as evidence. for pedestrians, the distance between the impact point on the lower extremities and the soles of the feet should be recorded. the legs should be opened to inspect tibial fractures; cortical fractures initiate propagation opposite to the side of impact, where they usually have a pulled-apart appearance, and then splinter the cortex at the side of impact. abrasions are better impact markers than contusions, because subcutaneous blood extravasation can be caused not only by impact to the skin, but also from blood extravasating from underlying fractures. if no cutaneous abrasions or fractures of the leg bones are found, the skin of the legs should be incised to expose contusions. fracture descriptions should include location in the bone (e.g., proximal metaphysis or shaft), whether the fracture is complete or incomplete, and whether the fracture is displaced or distracted. lacerations of intervertebral disks, facet joint capsules, and ligamenta flava should not be loosely termed "fractures." the presence or absence of blood extravasation in soft tissue adjacent to the fractures should be recorded, and its volume estimated if it appears severe enough. venous air embolism from tom dural sinuses cannot be diagnosed without a pre-autopsy chest radiograph or an in situ bubble test. if an x-ray machine is readily available, an anterior-posterior chest radiograph should be obtained in every traffic victim who dies at the scene or after a failed resuscitation attempt. if a hemothorax is suspected, the rib cuts should be placed further lateral and the chest plate reflected so that the internal mammary vessels can be inspected before the chest plate is removed. after measuring and removing the bloody effusion, the underlying serosal surfaces should be inspected for defects. lacerations of the heart and aorta will be obvious. tamponaded lacerations of the aorta, around which the adventitia still holds, must be noted as such. if no lacerations are found at the usual sites, lacerations of the azygous veins must be considered, especially in association with fracture dislocations of the thoracic vertebral column; other sites are the internal mammary arteries, especially with fractures of ribs i and or of the sternum, and intercostal arteries with displaced rib fractures. only after the serosal defect is identified should the organs be removed, because that procedure creates many more holes in the serosa. for that reason, as much information as possible should be gained by in situ observation. the only evidence of concussion of the heart may be a cardiac contusion or a sternal fracture. the usual clinical history suggests cardiovascular instability that is not associated with craniocerebral trauma and which does not respond to the infusion of intravenous volume agents. the autopsy assistant may saw but should not retract the skull cap and remove the brain. the pathologist should observe in situ whether shallow lacerations of the pontomedullary junction with stretching of the midbrain are present. these lesions cannot be distinguished from artifact by examining the brain later. thus, only after appropriate in situ inspection should the pathologist remove the brain. a posterior neck dissection is required if no lethal craniocerebral or cardiovascular trauma is found, or if suffocation is suspected; neck trauma must be ruled out to diagnose suffocation in a traffic fatality. sudden death in a patient with seemingly trivial wounds may be caused by undiagnosed trauma of the craniocervical articulation. a posterior neck dissection is required in these instances. the diagnosis of diffuse axonal injury of the brain in victims with no appreciable survival interval requires that suffocation be ruled out and that no resuscitation from a cardiac arrest has been attempted. clinicians are quick to apply the label "closed head injury" when a victim of a traffic crash has cerebral edema on a computerized axial tomogram of the head, even if no cerebral contusions, scalp contusions, or skull fractures are evident. this may be a misinterpretation, because cerebral edema can be caused by hypoxic encephalopathy made evident after resuscitation from a cardiac arrest, or from hypoxia caused by suffocation. procedures possible or expected findings record presence of lividity. photograph all external wounds; measure all lacerations and any abrasions or contusions with a pattern. collect scalp hair and blood (see below) from victims of hit and run accidents. collect foreign material in wounds. intense lividity and absence of lethal wounds may indicate that the crash occurred because the driver was dead from heart disease or suffocation. wound documentation. patterned injuries often sometimes be matched to objects in or about the vehicle (the most common patterned wound is that from tempered glass; see above under "note"). impact patterns in pedestrians may help to reconstruct the accident. hair and blood of the victim may be matched to transfer evidence on a vehicle suspected of having left the scene. part ii / diseases and conditions internal examination of body cavities heart and great vessels abdomen skull and brain; neck soft tissue compartments at any location prepare roentgenograms of chest is cases with head impact and skull fractures. collect samples for toxicologic study from all victims, including passengers. create pleural window to detect pneumothorax. if blood is seen, examine internal mammary vessels (see under "note"). measure volume of blood in cavity bleeds, and note whether chambers of heart and great vessels are collapsed or filled. record evidence of cardiac contusion, sprain of intracardiac inferior vena cava, laceration of pericardial sac, and fracture of sternum. laceration of heart or great vessels (measure volume of blood). follow routine procedures for dissection of heart and great vessels (see chapter ) . in situ bubble test to confirm venous air embolism. record evidence of trauma and volume of blood in peritoneal cavity; estimated volume of blood in retroperitoneal soft tissues. autopsy assistant may saw the skull but pathologist should inspect brain in situ and remove it personally. for removal and specimen preparation of brain, see chapter . record brain weight. posterior neck dissection is indicated if there is no craniocerebral or cardio-vascular trauma, or if suffocation is suspected. record evidence of trauma and estimate volume of blood. venous air embolism.' evidence of alcohol or drug intoxication. pneumothorax, hemothorax, e.g., after laceration of internal mammary vessels. evidence of significant hemorrhage. indirect evidence of cardiac concussion. evidence of exsanguinating wounds. evidence of cardiovascular disease that may have felled the driver before the crash. in european countries, the concentration is expressed in promille (grams per liter). in the united states, it has become customary to refer to concentration by percentage (grams per deciliter), and values in these units have been written into legislation and included in the uniform vehicle codes. unless qualified, the use of promille or percentage does not indicate whether the result of the analysis is weight/weight, weight/ volume, orvolume/volume. another common way ofexpressing concentration, milligrams per deciliter, has also been used to indicate alcohol concentrations. the method ofexpressing concentration must be clearly specified whenever the alcohol level is mentioned. the desired expression canbe derived from the toxicologic report by using the following equation: i, ~g/ml = mg/dl = . g/dl = . mmolll = . promille = . % what is the legal interpretation of alcohol (ethanol) intoxication? objective impairment of driving ability is observed at threshold blood alcohol concentrations of . -. g/dl. as of august all states and the district of columbia have adopted laws that make it criminal offense for a driver to operate a motor vehicle with a blood alcohol concentration of . g/ dl or greater. many states have an enhanced penalty for high concentrations such as . g/dl or above. several states have zero tolerance laws, under which drivers who are minors are legally operating only if their blood alcohol concentration is . g/dl or less, and in some states, not detectable at all. blood alcohol concentrations obtained at autopsy are valid until putrefaction begins. specimen tubes with sodium fluoride should be used, and the specimen should be stored in the refrigerator. if the air space above the blood samples in the container is large, alcohol can evaporate and a falsely low blood alcohol level can result. putrefactive changes before autopsy or during storage may cause a falsely high blood alcohol concentration. ethanol can be produced in the specimen container; this is more likely in the absence of a preservative. because fluoride inhibits bacteria far more than fungi, higher fluoride concentrations are required for the inhibition of fungal growth ( ) . although there is no major difference in the alcohol concentrations ofblood samples from the intact heart chambers and the femoral vessels ( ), autopsy samples from pooled blood in the pericardial sac or pleural cavity are unsatisfactory. we therefore recommend that blood be withdrawn from peripheral vessels. is there normal "endogenous" blood alcohol (ethanol) in a living person? blood alcohol concentrations are generally believed to be negligible in the absence of ingested alcohol. "endogenous" ethanol in human blood exists at a concentration of about . g/dl, which is below the limit of detection for most methods ( ) . first in such a list would be postural asphyxia, for example, in drunks who fall asleep face down. also, depressant drugs in the tricyclic, analgesic, barbiturate, and benzodiazepine classes all potentiate the effect of alcohol ( ) . also included in such a list would be infancy and childhood; ischemic heart disease;' chronic bronchitis and emphysema;' other chronic debilitating diseases; poisoning with carbon tetrachloride' or carbon monoxide;' and other causes of hypoxia.' how can one estimate blood alcohol (ethanol) concentrations from vitreous, urine, or tissue alcohol levels and from alcohol in stomach contents? the ratio of serum, plasma, urine, vitreous, and various tissues has been compiled by garriot ( ) . the values may vary considerably. for vitreous, the ratios varied from . - . . these variations may depend on whether blood alcohol concentrations were increasing or decreasing at the time of death. most other body fluids and tissues showed ranges closer to . most urine values were above the blood alcohol concentrations. in another study ( ) , the blood/vitreous (bn) ratio in the early absorption phase was . (range, . - . ; sd . ) and in the late absorption and elimination phase, the bn ratio was . (range, . - . ; sd . ). blood ethanol concentrations probably can be estimated using b = . v for early absorption and b = . v for later phases. a urinelblood ethanol ratio of . or less indicates that the deceased was in the early absorption phase. how can one use alcohol (ethanol) concentrations in postmortem specimens to estimate the blood alcohol concentration at various times before death? with certain limitations, one can base calculations of this kind on the assumption that the blood alcohol level decreases from its peak at a fairly constant rate of . -q. g/dl/h until death ( ) . if blood is not available, conversion factors (see above) must be used. alcoholics have been reported to metabolize at a rate of up to . g/dl/h ( ) . example: the driver of an automobile drinks at a party until midnight. he leaves his host at about : a.m. and is involved in a head-on collision at : a.m. he dies in the emergency room at : a.m. there are multiple injuries and the patient exsanguinates. the autopsy is done at : p.m. although this appears quite unlikely, let us assume that no satisfactory blood sample was obtained before death and that no blood or plasma expanders were given. if under such circumstances the alcohol concentration in the vitreous was found to be . g/dl, what was the alcohol concentration in the blood at the time of the accident? vitreous and blood alcohol concentrations may be assumed to have remained unchanged after death. therefore, the blood alcohol level at the time of death must have been approx . (vitreous humor alcohol) x . (conversion factor, see above) = . g/dl. the time interval between the accident ( : a.m.) and death ( : a.m.) is hand min or / h. if we assume that the decedent was not an alcoholic and that the blood alcohol concentration was decreasing from its peak at a constant rate of . g/dl/h, then the concentration at the time ofthe accident is estimated to have been . (concentration at time of death) + ( / x . ) = . + . = . g/dl or . %. the blood alcohol concentration at the time of the accident could have been lower if the victim stopped drinking later than h or / h before the accident. in the latter case, the peak alcohol level would have occurred after the accident, reflecting the time to absorb the latest drink. the blood alcohol concentration at the time of the accident could have been lower or higher if the time when the patient stopped drinking, the time of the accident, or the time of the death is uncertain. the blood alcohol concentration at the time of the accident could have been higher if the victim was a chronic alcoholic. the elimination rate in such persons may be as high as . mg/dl, which would change the figures in our example above to . + ( / x . ) = . + . = . g/d or . %. only rough estimates are possible. first, the peak blood alcohol level must be determined or calculated, as described in the previous paragraphs. tables (see below) are available that relate blood alcohol level to the minimal amounts of whiskey, wine, or beer that must have been consumed ( ) . however, tables of this type are often based on the minimum amount of alcohol circulating in the body after specific numbers of drinks; such tables do not yield reliable results if used conversely. furthermore, inasmuch as drinking and elimination of alcohol may take place concomitantly, over a longer period the total amount of alcohol consumed may have been much greater than the tables would indicate. it cannot be lower. according to these tables, pints of ordinary beer or fl oz of whiskey would be the minimal amounts needed to produce a blood alcohol level of about mg/dl in a person weighing - pounds. the total body alcohol can be calculated from the blood alcohol level by using widmark's formula: average concentration of alcohol in entire body = . concentration of alcohol in the blood in a person weighing kg, the blood alcohol concentration would be increased mg/dl ( . %) by the absorption of oz of ethanol ( z of -proof whiskey). strength of alcohol is measured in "proof'; absolute alcohol is proof. therefore, in the united states, alcohol content as volume percent is half the proof (for example, -proof whiskey contains % alcohol by volume). the alcohol content of various beverages is shown in the following table. approximate alcohol content in various beverages t toata from glaister, rentoul e. medical jurisprudence and toxicology, th ed. e & s livingstone, edinburgh, with permission. twithin h after consumption of diluted alcohol (approx %) on an empty stomach, assuming body weight of - pounds ( . - . kg) reproduced from ( ) with permission. *one ounce (about ml) of whiskey or z (about ml) of beer. what is the toxicity of alcohol other than ethanol? in general, the toxicity increases as the number of carbon atoms in the alcohol increases. thus, butyl alcohol is two times as toxic as ethyl alcohol: but isopropyl alcohol is only twothirds as toxic as isobutyl alcohol and one-half as toxic as amyl alcohol. primary alcohols are more toxic than the corresponding secondary isomers ( ) . anemia, hemolytic synonyms and related terms: acquired hemolytic anemia; extracorpuscular hemolytic anemia; hereditary hemolytic anemia (hereditary elliptocytosis, pyropoikilocytosis, stomatocytosis. spherocytosis); immunohemolytic anemia; intracor-puscular hemolytic anemia; microangiopathic hemolytic anemia; spur cell anemia. possible associated conditions: disseminated intravascular coagulation;* eclampsia;* glucose- -phosphatase deficiency (g pd); hemolytic uremic syndrome;* malignant hypertension; lymphoma* and other malignancies; paroxysmal nocturnal hemo-globinuria; sickle cell disease;*thalassemia;* thrombotic thrombocytopenic purpura.* (see also below under "note.") note: hemolysis also may be caused by conditions such as poisoning with chemicals or drugs, heat injury, snake bite,* or infections or may develop as a transfusion reaction* or be secondary to adenocarcinoma, heart valve prostheses (see below), liver disease (see below), renal disease, or congenital erythropoietic porphyria. * procedures prepare skeletal roentgenograms. jaundice; skin ulcers over malleoli. in young patients: thickening of frontal and parietal bones with loss of outer table ("hairon-end" appearance); paravertebral masses caused by extramedullary hematopoiesis; deformities of metacarpals, metatarsals, and phalanges. osteonecrosis* of femoral heads. remove and place in fixative as early as possible in order to minimize autolysis (alternatively, formalin can be injected in situ; see below). samples should include oxyntic corpus and fundus mucosa. record weights. submit tissue samples for histologic study. record weight of thyroid gland. for removal and specimen preparation, see chapter . request luxol fast blue stain. for removal and specimen preparation, see chapter . if there is a clinical diagnosis of anemia-related amblyopia, follow procedures described under "amblyopia, nutritional." jaundice. manifestations of malnutrition. * stomatitis with cheilosis and perianal ulcerations due to folic acid deficiency. chronic exfoliative skin disorders. vitiligo. macrocytosis; poikilocytosis; macroovalocytes; hypersegmentation of leukocytes; abnormal platelets. atrophic glossitis with ulcers. pharyngoesophagitis (folic acid deficiency). previous total or subtotal gastrectomy. carcinoma of stomach. autoimmune gastritis (diffuse corporal atrophic gastritis) with intestinal metaplasia. crohn's disease;* sprue;* other chronic inflammatory disorders; jejunal diverticula; intestinal malignancies; fish tapeworm infestation; previous intestinal resection or blind intestinal loop; enteric fistulas. hepatosplenomegaly. alcoholic liver disease. * giant epithelial cells. hyperthyroid goiter; thyroiditis. demyelination of cerebral white matter (in advanced cases). demyelination in posterior and lateral columns of spinal cord, most frequently in thoracic and cervical segments. demyelination of peripheral nerves. retinal hemorrhages; demyelination of optic nerves. hypercellular; megaloblastic. myeloproliferative disorder. brain other organs if mycotic aneurysms are expected and microbiologic studies are intended, follow procedures described below under "aneurysm, mycotic aortic." request verhoeff-van gieson, gram, and grocott's methenamine silver stains. for cerebral arteriography, see chapter . if arteriography cannot be carried out, rinse fresh blood gently from base of brain until aneurysm can be identified. record site of rupture and estimated amount of extravascular blood. for paraffin embedding of aneurysms, careful positioning is required. expected findings depend on type of aneurysm. mycotic aneurysms are often multiple and deep in brain substance. berry aneurysms are the most frequent types and often are multiple. most frequent sites are the bifurcations and trifurcations of the circle of willis. saccular atherosclerotic aneurysms are more common than dissecting aneurysms, which are very rare. with congenital cerebral artery aneurysm: coarctation of aorta;* manifestations of hypertension;* and polycystic renal disease. with mycotic aneurysm: infective endocarditis;* pulmonary suppurative processes; and pyemia. aneurysm, dissecting aortic (see "dissection, aortic.") aneurysm, membranous septum of heart note: for general dissection techniques, see chapter . most aneurysms ofthe membranous septum probably repre-sent spontaneous closure of a membranous ventricular septal defect by the septalleafiet of the tricuspid valve. aneurysm, mycotic aortic note: (i) collect all tissues that appear to be infected. ( ) request aerobic, anaerobic, and fungal cultures. ( ) request gram and grocott methenamine silver stains. ( ) no special precautions are indicated. ( ) no serologic studies are available. ( ) this is not a reportable disease. chest and abdominal organs aorta other organs submit blood samples for bacterial culture. en masse removal of adjacent organs is recommended. photograph all grossly identifiable lesions. aspirate material from aneurysm or para-aortic abscess and submit for culture. prepare sections and smears of wall of aneurysm and of aorta distant from aneurysm. request verhoeffvan gieson and gram stains. septicemia and infective endocarditis. * streptococcus, staphylococcus, spirochetes, and salmonella can be found in mycotic aneurysm. para-aortic abscess. septic emboli with infarction or abscess formation. aneurysm, syphilitic aortic part ii / diseases and conditions heart and aorta other organs en masse removal of organs is recommended. for coronary arteriography, see chapter . request verhoeff-van gieson stain from sections at different levels of aorta, adjacent great vessels, and coronary arteries. see also under "syphilis." aneurysm usually in ascending aorta. may erode adjacent bone (sternum). syphilitic aortitis may cause intimal wrinkling, narrowing of coronary ostia, and shortening of aortic cusps. disruption of medial elastic fibrils. aortic valvulitis and insufficiency;* syphilitic coronary arteritis; syphilitic myocarditis. external examination aorta prepare chest and abdominal roentgenograms. open aorta along line of blood flow, or bisect into anterior and posterior halves. photograph tear(s). measure bloody effusions in body cavities. measure or estimate amount of blood in mediastinum. request verhoeff-van gieson stain. cutaneous impact trauma. mediastinum widened by hemorrhage in case of tarnponaded dissection. a bleed into a body cavity of less-thanexsanguinating volume should point to an alternate mechanism of death such as neurogenic shock or lethal concussion; a posterior neck dissection may be required in such instances. microscopy may show transmural rupture, false aneurysm, or localized dissection. angiitis (see "arteritis, all types or type unspecified.") angina pectoris note: see under "disease, ischemic heart" and chapter . angiokeratoma corporis dittusum (see "disease, fabry's.") angiomatosis, encephalotrigeminal (see "disease, sturge-weber-dimitri.") angiopathy, congophilic cerebral synonyms and related terms: beta amyloid angiopathy due to~-amyloid peptide deposition (~a ) (associated with alzheimer's disease; hereditary cerebral hemorrhage with amyloid angiopathy of dutch type; or sporadic beta amyloid angiopathy); hereditary cerebral amyloid angiopathy, due to deposition of other amyloidogenic proteins such as cystatin c (icelandic type) and others (e.g., transthyretin, gelsolin) ( ). procedures possible or expected findings request stains for amyloid, particularly congo red, and thioflavine s (examine with polarized and ultraviolet light, respectively). request immunostain for~a . some tissue should be kept frozen for biochemical studies. multiple recent cerebral cortical infarctions or small cortical hemorrhages, or both, or massive hemispheric hemorrhages, both recent and old. amyloid deposition in leptomeninges and cortical blood vessels. senile plaques are usually present. in some cases, angiopathy is part of alzheimer's disease. * other organs a prepare material for electron microscopy. electron microscopic study permits definite confirmation of diagnosis. organs and tissues may be minimally affected by amyloidosis. anomaly, coronary artery possible associated conditions: with double outlet right ventricle; persistent truncal artery; tetralogy of fallot;* and transposition of the great arteries.* note: coronary artery between aorta and pulmonary artery, often with flap-valve angulated coronary ostium. coronary artery may communicate with cardiac chamber, coronary sinus, or other cardiac veins, or with mediastinal vessel through pericardial vessel. saccular aneurysm of coronary artery with abnor-mal flow, infective endarteritis of arteriovenous fistula, and myocardial infarction may be present. ifone or both coronary arteries originate from pulmonary trunk, myocardial infarction may be present. heart perform coronary angiography. if infective endarteritis is suspected, submit blood sample for microbiologic study. ectopic origin of coronary arteries or single coronary artery. sudden death. for a detailed description of possible additional findings, see above under "note." anomaly, ebstein's (see "malformation, ebstein's") anorexia nervosa note: sudden death from tachyarrhythmias may occur in advanced cases and thus, autopsy findings may not reveal the immediate cause of death. external examination all organs record height and weight, and prepare photographs to show cachectic features. record abnormalities as listed in righthand column. follow procedures described under "starvation." record weight of endocrine organs and submit samples for histologic study. cachexia, often with preserved breast tissue; hirsutism; dry, scaly, and yellow skin (carotenemia). mild edema may be present. parotid glands may be enlarged. manifestations of starvation.* ovaries tend to be atrophic; other endocrine organs should not show abnormalities. synonyms: cutaneous anthrax; gastrointestinal anthrax; pulmonary (inhalational) anthrax. note: ( ) collect all tissues that appear to be infected. this is a reportable disease. bioterrorism must be considered in current cases. external examination and skin blood photograph cutaneous papules, vesicles, and pustules. prepare smears and histologic sections. submit samples for bacteriologic study. submit sample for serologic study. disseminated anthrax infection may occur without skin lesions. edema of neck and anterior chest in nasopharyngeal anthrax. anthrax septicemia. see above under "note." part ii i diseases and conditions lungs gastrointestinal tracts and mesentery neck organs record character and volume of effusions. after sampling for bacteriologic study (see above under "note") perfuse one or both lungs with formalin. extensive sampling for histologic study is indicated. extensive sampling for histologic study is indicated. photograph meningeal hemorrhage in situ. pleural effusions;* hemorrhagic mediastinitis; anthrax pneumonia (inhalational anthrax; woolsorter's disease). histologic sections reveal hemorrhagic necrosis, often with minimal inflammation and gram-positive, spore-forming, encapsulated bacilli. gastrointestinal anthrax with mucosal edema and ulcerations. hemorrhagic mesenteric lymphadenitis. tongue, nasopharynx, and tonsils may be involved. hemorrhagic meningitis (hemorrhage tends to predominate). external examination distal colon and rectum photograph perineum. measure depth of anal pit, if any. dissect distal colon, rectum, and perirectal pelvic organs in situ (as much as possible). search for opening of fistulous tracts from lumen. use roentgenologic study or dissection, or both, to determine course of tract. absence of normally located anus; anal dimple. abnormal termination of the bowel into the trigone of the urinary bladder, the urethra distal to the verumontanum, the posterior wall of the vagina, the vulva, or the perineum. aortitis note: see also under "arteritis" and "aneurysm, ascending aortic." heart and aorta other organs and tissues remove heart with whole length of aorta and adjacent major arteries. record width and circumference of aorta at different levels. describe and photograph appearance of intima and of orifices of coronary arteries and other aortic branches. submit multiple samples for histologic study and request verhoeff-van gieson stain. procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. secondary aortic atherosclerosis or intimal fibroplasia. widening of aorta; syphilitic aneurysm. * giant cell aortitis; rheumatoid aortitis; syphilitic aortitis; takayasu's arteritis.* manifestations of rheumatoid arthritis, * syphilis,* systemic sclerosis,* hodgkin's lymphoma, and many other diseases associated with vasculitis. external examination brain spine and spinal cord other organs prepare roentgenogram of spine. for removal and specimen preparation, see chapter . for removal of spinal cord and specimen preparation, see chapter . expose nerve roots. record appearance and photograph spinal cord in situ. submit samples of spinal cord and inflamed tissue for histologic study. request gram, gomori's iron, and grocott's methenamine silver stains. procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. signs of previous spinal surgery or lumbar puncture (myelography). evidence of previous trauma or previous myelography. cerebral arachnoiditis. fibrous arachnoidal adhesions and loculated cysts. tuberculosis;* syphilis;* fungal or parasitic infection. systemic infection (see above). ascending urinary infection or other manifestations of paraplegia. arch, aortic, interrupted synonym: severe coarctation. note: the basic anomaly is a discrete imperforate region in the aortic arch, with a patent ductal artery joining the descending thoracic aorta. type a interruption is between the left subclavian and ductal arteries; type b between the left subclavian and left common carotid arteries; and type c (rare) between the left common carotid and brachiocephalic (innominate) arteries. for general dissection techniques, see part i, chapter . possible associated conditions: bicuspid aortic valve (with type a); di george syndrome* with thymic and parathyroid aplasia (with type b); hypoplasia of ascending aorta (with all types); persistent truncal artery (truncus arteriosus); ventricular septal defect. arrhythmia, cardiac note: see also under "death, sudden cardiac." toxicologic studies may be indicated, for instance, if digitalis toxicity (see "poisoning, digitalis") is suspected. if a cardiac pacemaker had been implanted, the instrument should be tested for malfunction. arteriosclerosis (see "atherosclerosis.") arteritis, all types or type unspecified synonyms and related terms: allergic angiitis and granulomatosis (churg-strauss);* allergic vasculitis; anaphylactoid purpura* and its synonyms; angiitis; buerger's disease;* cranial arteritis; giant cell arteritis;* granulomatous arteritis (angiitis); hypersensitivity angiitis; infectious angiitis; necrotizing arteritis; polyarteritis nodosa;* rheumatic arteritis; rheumatoid arteritis, syphilitic arteritis; takayasu's arteritis;* temporal arteritis; thromboangiitis obliterans; and others (see also below under "note"). note: autopsy procedures depend on ( ) the expected type of arteritis, such as giant cell arteritis,* polyarteritis nodosa,* or thromboangiitis obliterans (buerger's disease*); and ( ) the nature of suspected associated or underlying disease, such as aortic arch syndrome,* beh~et's syndrome,* cogan's syndrome, degos' disease,* dermatomyositis,* erythema nodosum and multiforme,* goodpasture's syndrome,* polymyositis, rheumatic fever, * rheumatoid arthritis,* syphilis,* and other nonspecific infectious diseases, systemic lupus erythematosus,* systemic sclerosis (scleroderma),* or takayasu's disease. for histologic study of blood vessels, verhoeff-van gieson stain or a similar stain is recommended. temporal and ophthalmic arteritis. arteritis of ciliary and retinal vessels. clinically, polymyalgia. anemia. arteritis, takayasu's synonyms: aortic arch syndrome; pulseless disease. external examination heart, aorta, and adjacent great vessels kidney eyes and optic nerve brain for in situ aortography, clamp distal descending thoracic aorta and neck vessels as distal as possible from takeoff at aortic arch. remove heart together with aorta and long sleeves of neck vessels. for coronary arteriography, see chapter (method designed to show coronary ostia). test competence of aortic valve. open aortic arch anteriorly and measure (with calipers) lumen at origin of great neck vessels. photograph aorta and neck vessels and submit samples for histologic study. request verhoeffvan gieson stain. submit tissue for histologic examination. for removal and specimen preparation, see chapter . for removal and specimen preparation, see chapter . facial muscular atrophy and pigmentation. narrowing at origin of brachiocephalic arteries. dilated ascending aorta. narrowing of coronary arteries at origins. myocardial infarction. aortic insufficiency. * aortic atherosclerosis. thromboses of brachiocephalic arteries. giant cell arteritis. * diffuse mesangial proliferative glomeulonephritis ( ) . atrophy of optic nerve, retina, and iris; cataracts; retinal pigmentation. ischemic lesions. artery, patent ductal synonym: patent ductus arteriosus. note: the basic anomaly is persistent postnatal patency of the ductal artery, usually as an isolated finding (in % of cases in infants, and in % in adults). it is more common in premature than full-term infants and at high altitudes than at sea level. possible complications in unoperated cases include congestive heart failure, * plexogenic pulmonary hypertension,* ductal artery aneurysm or rupture, fatal pulmonary embolism,* or sudden death. in some conditions, such as aortic atresia* or transposition with an intact ventricular septum,* ductal patency may be necessary for survival. possible associated conditions: atrial or ventricular septal defect;* coarctation ofthe aorta;* conotruncal anomalies; necrotizing enterocolitis in premature infants; postrubella syndrome; and valvular or vascular obstructions. artery, persistent truncal synonym and related terms: type i, pulmonary arteries arise from single pulmonary trunk (in %); type , pulmonary arteries arise separately but close-by (in %); type , pulmonary arteries arise separately but distal from one another (in %). note: the basic anomaly is a common truncal artery, with truncal valve, giving rise to aorta, pulmonary arteries, and coronary arteries, usually with a ventricular septal defect. interventions include complete rastelli-type repair, with closure of ventricular septal defect, and insertion of valved extracardiac conduit between right ventricle and detached pulmonary arteries. possible associated conditions: absent pulmonary artery (in %); atrial septal defect (in %); absent ductal artery (in %); coronary ostial anomalies (in %); di george syndrome;* double aortic arch; extracardiac anomalies (in %); interrupted aortic arch* (in %); right aortic arch (in %); truncal valve insufficiency (uncommon) or stenosis (rare); trun-cal valve with three (in %), four (in %), or two (in %) cusps. heart and great vessels if infective endocarditis is suspected, follow culture procedures for endocardial vegetation described in chapter . request verhoeff-van gieson stain. infective endocarditis,* usually of truncal valve. late postoperative conduit obstruction. postoperative late progressive truncal artery dilation with truncal valve insufficiency. hypertensive pulmonary vascular disease. cerebral abscess,* if right-to-ieft-shunt was present. arthritis, all types or type unspecified note: for extra-articular changes, see under the name of the suspected underlying conditions. infectious diseases that may be associated with arthritis include bacillary dysentery, * brucellosis, * gonorrhea, rubella,* syphilis, * tuberculosis, * typhoid fever, * and varicella. * noninfectious diseases in this category include acromegaly,* beh<;et's syndrome,* felty's syndrome,* gout,* rheumatoid arthritis,* and many others, too numerous to mention. remove synovial fluid and prepare smears. submit synovial fluid for microbiologic and chemical study. for removal of joints, prosthetic repair, and specimen preparation, see chapter . for removal and specimen preparation, see chapter . in the polyarticular variant, facial asymmetry may be noted. rheumatoid factor positive in some cases. pericarditis.* interstitial pneumonitis; pleuritis. (see also under "arthritis, rheumatoid.") lymphadenopathy. splenomegaly. monarthritis or severe, erosive polyarthritis; see also under "arthritis, rheumatoid" and above under "externalexamination and skin." ankylosing spondylitis* may be present. chronic iridocyclitis. see "arthritis, rheumatoid." arthritis, rheumatoid synonyms and related terms: ankylosing spondylitis;* felty's syndrome;* juvenile rheumatoid arthritis* (still's disease); rheumatoid disease; and others. possible associated conditions: amyloidosis;* polymyositis (dermatomyositis*); psoriasis;* sjogren's syndrome;* systemic lupus erythematosus;* systemic vasculitis, and others. subcutaneous rheumatoid nodules on elbows, back, areas overlying ischial and femoral tuberosities, heads of phalangeal and metacarpal bones, and occiput. deformities and subluxation of peripheral joints (see also below under "joints"). subaxial dislocation of cervical spine may be cause of sudden death. pneumothorax;* pleural empyema.* t-cell abnormalities ( ) . bacteremia. positive rheumatoid factor. rheumatoid granulomas in myocardium (septum), pericardium, and at base of aortic and mitral valves; constrictive pericarditis;* aortic stenosis;* coronary arteritis. systemic vasculitis (arteritis*). rheumatoid granulomas in pleura and lung (with pneumoconiosis*); bronchopleural fistula; rheumatoid pneumonia with interstitial pulmonary fibrosis and honeycombing; bronchiectasis;* bronchiolitis with cystic changes; pulmonary arteritis. pneumoconiosis* in caplan arthrogryposis ( ) may be a primary muscle disease, or it may involve abnormalities of the brain, spinal cord, and/or peripheral nerves. etiologies are numerous, as are the modes of inheritance. critical to making the appropriate diagnosis is the collection of muscles from various sites for routine histology, muscle histochemistry, and electron microscopy. portions of peripheral motor nerves must also be prepared for histology and electron microscopy. abdominal cavity intra-abdominal lymphatic system puncture abdominal cavity and submit fluid for microbiologic study. record volume of exudate or transudate and submit sample for determination of fat and cholesterol content. prior to routine dissection, lymphangiography (see below) may be indicated. possible associated conditions: with pulmonary aspergillosis-bronchiectasis; * bronchocentric granulomatosis;* sarcoidosis;* tuberculosis. * with systemic aspergillosisleukemia;* lymphoma;* and other conditions complicated by immunosuppression (l, ) . other organs a carefully make multiple parasagittal sections through the unperfused lungs. culture areas of consolidation. if diagnosis was confirmed, perfuse lungs with formalin. prepare histologic sections from walls of cavities, cavity contents, and pneumonic infiltrates. procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. assault note: all procedures described under "homicide" must be followed. asthma note: spray death* may occur in asthma sufferers from pressurized aerosol bronchodilators. record thickness and position. perfuse one lung with formalin. because mucous plugs may block bronchial tree, attach perfusion apparatus to pulmonary artery or to bronchus and pulmonary artery. monitor perfusion to ensure proper inflation. prepare photograph of fixed cut section. submit samples of pulmonary parenchyma and bronchi for histologic study. request azure-eosin and verhoeff-van gieson stains. record weight and thickness of walls. leave attached to stomach. photograph and submit samples for histologic study. eczema. conjunctival hemorrhages and subcutaneous emphysema may be present after fatal attack. pneumothorax;* mediastinal emphysema. low diaphragm (see below). increased igeconcentrations in fatal asthma; postmortem tryptase determination is of doubtful value in this regard ( ) . hypertrophy. low position of diaphragm. hyperinflated lungs. thick-walled bronchi with prominent viscid mucous plugs. typical microscopic inflammatory changes ( ) . asthmatic bronchitis with eosinophilic infiltrates. bronchocentric granulomatosis.* pulmonary atherosclerosis with breakup of elastic fibers. paucity of ecosinophils in mucous ( ) . cor pulmonale. refl ux esophagitis ( ) . peptic ulcer. * pneumatosis of small intestine; emphysema of colon. centrilobular congestion and necrosis. petechial hemorrhages in hypothalamus; necrosis of cerebellar folia; anoxic changes in cortex, globus pallidus, thalamus, sommer's sector of hippocampus, and purkinje cells of cerebellum. suspected changes in anterior hom cells of spinal cord in patients with asthma-associated poliomyelitis-like illness (hopkins syndrome) ( ). allergic polyps and other allergic inflammatory changes ( ) . increased erythropoiesis. atresia, aortic valvular synonym: aortic atresia; aortic atresia with intact ventricular septum; hypoplastic left heart syndrome. note: the basic anomaly is an imperforate aortic valve, with secondary hypoplasia ofleft-sided chambers and ascending aorta. for possible surgical interventions, see two-stage norwood and modified fontan procedures in chapter . possible associated conditions: atrial septal defect* (or patent foramen ovale, usually restrictive); dilatation of myocardial sinusoids thatcommunicate with coronary vessels; dilatation of right atrium, right ventricle, and pulmonary trunk; fibroelastosis ofleft atrial and left ventricular endocardium; hypertrophy of ventricular and atrial walls; hypoplastic left atrium, mitral valve, left ventricle, and ascending aorta; mitral atresia* with minute left ventricle; patent ductal artery (ductus arteriosus); small left ventricle with hypertrophic wall; tubular hypoplasia of aortic arch, with or without discrete coarctation. synonyms and related terms: congenital biliary atresia; extrahepatic biliary atresia; infantile obstructive cholangio-pathy; syndromic (alagille's syndrome) or nonsyndromic paucity of intrahepatic bile ducts ("intrahepatic" biliary atresia). possible associated conditions: alpha]-antitrypsin deficiency;* choledochal cyst;* congenital rubella syndrome;* polysplenia syndrome* ( ); small bowel atresia; trisomy - ; trisomy ; turner's syndrome;* viral infections (cytomegalovirus infection;* rubella*). dissect extrahepatic bile ducts in situ or leave hepatoduodenalligament intact for later fixation and sectioning (see below). record appearance and contents of gallbladder and course of cystic duct. in postoperative cases, submit sample of anastomosed hepatic hilar tissue for demonstration of microscopic bile ducts. remove liver with hepatoduodenalligament. prepare horizontal sections through ligament and submit for histologic identification of ducts or duct remnants. prepare frontal slices of liver and sample for histologic study. request pas stain with diastase digestion. procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. jaundice. congenital rubella and other viral infections. alpha]-antitrypsin deficiency;* defects in bile acid synthesis. chromosomal abnormalities. in atresia of the hepatic duct, the gallbladder will be empty. in isolated atresia of the common bile duct, the gallbladder contains bile but it cannot be squeezed into the duodenum. atresia or hypoplasia of bile duct(s); choledochal cyst(s). biliary drainage created by kasai operation. obliterative cholangiopathy ( ) . intrahepatic cholelithiasis; postoperative ascending cholangitis; secondary biliary cirrhosis; giant cell transformation; paucity of intrahepatic bile ducts. pas-positive inclusions in alphal-antitrypsin deficiency.* polysplenia syndrome* ( ) with malrotation, situs inversus, preduodenal portal vein, absent inferior vena cava, anomalous hepatic artery supply, and cardiac defects. for other abnormalities outside the biliary tree, see under "possible associated conditions"). nephromegaly ( ) . atresia, cardiac valves (see "atresia, aortic valvular," "atresia, mitral valvular," "atresia pulmonary valvular, with intact ventricular septum," "atresia, pulmonary valvular, with ventricular septal defect," and "atresia, tricuspid valvular.") atresia, duodenal possible associated conditions: with membranous obstruction of the duodenum-annular pancreas; atresia of esophagus* with tracheoesophageal fistula; congenital heart disease; cystic fibrosis;* down's syndrome;* hirschsprung's disease; imperforate anus* or other congenital obstructions of the intestinal tract ( ); intestinal malrotation; lumbosacral, rib-, and digitllimb anomalies; single umbilical artery; spinal defects; undescended testis ( ). see also under "atresia, small intestinal." the basic anomaly is an imperforate pulmonary valve, with a hypoplastic right ventricle. in unoperated cases, ductal closure is the most common cause of death. for possible surgical interventions, see modified blalock-taussig shunt, mod-ified fontan procedure, and pulmonary valvulotomy in chapter . for general dissection techniques, see chapter . possible associated conditions: dilated myocardial sinusoids that may communicate with epicardial coronary arteries or veins; patent ductal artery (ductus arteriosus); patent oval foramen (foramen orale); tricuspid atresia with minute right ven-tricle; tricuspid stenosis with hypoplastic right ventricle (in %); tricuspid insufficiency with dilated right ventricle (in %). synonym: tetralogy of fallot with pulmonary atresia. note: the basic anomaly is atresia of the pulmonary valve and ofvariable length ofpulmonary artery, and ventricular septal defect (membranous or outlet type), with overriding aorta, and with pulmonary blood supply from ductal or systemic collateral arteries. for possible surgical interventions, see rastelli-type repair and unifocalization of multiple collateral arteries in chapter . possible associated conditions: right ventricular outflow tract a short blind-ended pouch ( %) or absent ( %); atresia of pulmonary artery bifurcation, with nonconfluent pulmonary arteries; right aortic arch ( %); atrial septal defect ( %); persistent left superior vena cava; anomalous pulmonary venous connection; tricuspid stenosis or atresia; complete atrioventricular septal defect; transposed great arteries; double inlet left ventricle; asplenia, polysplenia, or velocardiofacial syndromes; dilated ascending aorta, with aortic insufficiency. related term: jejuno-ileal atresia. possible associated findings: esophageal atresia* with tracheoesophageal fistula; lumbosacral, rib-, or digit/limb anom -alies; undescended testes (l) . note: see also under "atresia, duodena ." fascia lata, blood, or liver these specimens should be collected using aseptic technique for tissue culture for chromosome analysis (see chapter ) . intestinal tract for mesenteric angiography, see chapter . leave mesentery attached to small bowel, particularly to the atretic portion. trisomy . multiple atresias; proximal dilatation; volvulus; malrotation; meconium impaction; other evidence of cystic fibrosis. anorectal malformation (l) . annular pancreas ( ). atresia, tricuspid valvular note: the basic anomaly is an absent right atrioventricular connection ( %) or imperforate tricuspid valve ( %), with a hypoplastic right ventricle ( %), muscular ventricular septal defect ( %) that is restrictive ( %), and a patent oval atresia, urethral foramen ( %) or secundum atrial septal defect ( %). for possible surgical interventions, see modified fontan or glenn procedures in chapter . for general dissection techniques, see chapter . possible associated conditions: juxtaposed atrial appendages; large left ventricular valvular orifice; large left ventricular chamber; persistent left superior vena cava; pulmonary atresia; transposition of the great arteries ( %), with aortic co-arctation ( % of those); anomalies of musculoskeletal or digestive systems ( %); down's,* asplenia, or other syndromes. heart aorta and cervical arteries brain if infective endocarditis* is suspected, culture using the method described in chapter . for dissection of carotid and vertebral arteries, see chapter . for removal and specimen preparation, and cerebral anteriography, see chapter . if a foreign body is discovered during a medicolegal autopsy or if the discovery of a foreign body may have medicolegal impli-cations (e.g., presence of a surgical instrument in the abdominal cavity), the rules of the chain of custody apply. for the handling of bullets or bullet fragments, see "injury, firearm." if analysis offoreign material is required, commercial laboratories may be helpful. bolus (see "obstruction, acute airway!') burns note: fatal bums should be reported to the medical examiner's or coroner's office. the questions to be answered by the pathologist depend on whether the incident was accidental, sui-cidal, or homicidal, and whether the victim survivied to be treated in the hospital. a pending death certificate should be issued if the fire and police investigators are not sure of the circumstances at the time of the autopsy. for electrical bums, see under "injury, electrical." for victims who were treated at the hospital, autopsy procedures should be directed toward the discovery or confirmation of the mechanism of death, such as sepsis or pulmonary embolism.* death can be caused primarily by heart disease, with other-wise minor bums and smoke inhalation serving as the trigger that leads to lethal ventricular arrhythmia. because carbon monoxide concentrations are halved approx every min with % oxygen therapy, the pathologist must obtain the first clinical laboratory test results for co-hemoglobin. soot can be detected with the naked eye or d after inhalation of smoke. ambulance records should be examined to determine whether a persistent coma might have been caused by hypoxic encephalopathy following resuscitation from cardiac arrest at the scene. admission blood samples should be acquired to test for cohemoglobin and alcohol. this may not have been done in the emergency room. persons suffering from chronic alcoholism succumb to fire deaths more often than persons who do not drink. a very high initial serum alcohol concentration suggests a risk factor for the fire and presence of chronic alcoholism. patients with chronic alcoholism typically are deprived of alcohol when they are in the bum unit and this can cause sudden, presumably cardiac, death,just as it occurs under similarcircum-stances, not complicated by bums. under these circumstances, the heart fails to show major abnormalities. this mode of dying seems to have no relationship to the presence or absence of liver disease. if the body is found dead and charred at the scene, prepare whole body roentgenograms, before and after removal of remanants of clothing. see also under "identification of the body" and "external examination" in chapter ). one or two fingerpads may yield sufficient ridge detail for identification. if this is not possible, ante-and postmortem somatic and dental radiographs must be compared for identification, or dna comparison must be used. external examination, heart and lungs abdominal cavity and liver see below under "cardiomyopathy, dilated." record volume of ascites. record actual and expected weight of liver. request iron stain. see below under "cardiomyopathy, dilated." alcoholic cirrhosis and alcoholic cardiomyopathy rarely coexist. however, in genetic hemochromatosis,* cirrhosis and heart failure are common findings. cardiomyopathy, dilated (idiopathic, familial, and secondary types) note: for general dissection techniques, see chapter . external examination heart other organs and tissues record actual and expected weights. record ventricular thicknesses and valvular circumferences. evaluate relative atrial and ventricular chamber sizes. procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. note: huntington's disease maps to the short arm of chromosome . the gene is widely expressed but of unknown function; it contains a cag repeat sequence, which is expanded (range, to ) in patients with huntington's disease. a sensitive diag-nostic test is based on the determination of this cag sequence, which can be done on fresh-frozen tissue or blood ( ) . in the absence of genetic confirmation, sampling of organs and tissues cannot be excessive because a complex differential diagnosis must be resolved. note: disseminated intravascular coagulation (dic) often is a complication of obstetrical mishaps such as abruptio placentae or amniotic fluid embolism,* or it complicates malignancies (such as adenocarcinomas or leukemia*) or bacterial, viral, and other infections. other conditions such as aortic aneurysm* or hemolytic uremic syndrome* are known causes also. ifthe nature of the underlying disease is known, follow the procedures under the appropriate heading also. note: this is a cause of diarrhea. microscopic colitis is associated with older age; collagenous colitis is associated with female sex ( ). the colon is grossly normal but microscopically, increased lymphocytes in the lamina propria and a subepithelial band of collagen is found. if only the lymphocytic infiltrate is found, the term "lymphocytic colitis" or "microscopic colitis" should be applied. a trichrome stain should be ordered in all instances, because the collagen band may be difficult to see without the special stain. i death, anaphylactic synonym: generalized anaphylaxis. note: autopsy should be done as soon as possible after death. neck organs should be removed before embalming. if death is believed to be caused by drug anaphylaxis, inquire about type of drug(s), drug dose, and route of administration (intravenous, intramuscular, and oral or other). this will determine proper sampling procedures-for instance, after penicillin anaphylaxis. allergy to bee stings, wasp stings, fire ants, and certain plants may also be responsible for anaphylaxis. however, envenomation also can be fatal in the absence of anaphylaxis. external examination search for injection sites or sting marks. if such lesions are present, photograph and excise with -cm margin. freeze excised tissue at - °c for possible analysis. prepare chest roentgenogram. foam in front of mouth and nostrils. swelling of involved tissue. antigen-antibody reaction in involved tissues. antibodies against suspected antigen. laryngeal edema may recede soon after death. foamy edema in trachea and bronchi; diffuse or focal pulmonary distention ("acute emphysema") alternating with collapse; pulmonary edema and congestion; accumulation of eosinophilic leukocytes. eosinophilic leukocytes in red pulp. death, anesthesia-associated. note: there are many possible causes of anesthesiaassociated death that are not drug-related, such as acute airway obstruction* by external compression, aspiration, arrhythmia of a heart not previously known to be diseased, tumor, or an inflammatory process. some ofthe complications are characteristically linked to a specific phase of the anesthesia, and many are not revealed by customary morphologic techniques. the task for the pathologist charged with investigating an anesthesia-associated death is to reconstruct the chain of physiologic events culminating in cessation of vital signs. autopsy morphology plays a supporting role; the main investigations center around the record left by the anesthesiologist, testing of anesthesia equipment, and toxicological testing. a consulting anesthesiologist can divine much more information from the anesthesia and recovery room records than can the pathologist, and can suggest avenues of further investigation. therefore, the most important step in these autopsies is to obtain the anesthesiaassociated records and to secure the consulting services of an independent anesthesiologist. the changes in the vital signs during and after anesthesia will help to focus the investigation toward a cardiac mechanism ofdeath or depression ofbrainstem function as a terminal mechanism. when information is gathered about drugs and chemical agents that have been administered or to which the victim may have had access, the pathologist must keep in mind that some non-medical chemicals and many drugs are known to affect anesthesia. drugs and their metabolic products, additives, stabilizers, impurities, and deterioration products (one of which can be carbon monoxide) may be present and can be identified in postmortem tissues. therefore, all appropriate body fluids and solid tissue should be submitted for toxicological examination. if the anesthetic agent was injected into or near the spinal canal, spinal fluid should be withdrawn from above the injected site into a standard toxicologist's collection tube with fluoride preservative. if the anesthetic agent was injected locally, tissue should be excised around the needle puncture marks at a radius of - em. serial postmortem analysis of specimens may permit extrapolation to tissue concentration at the time of death. the time interval between drug administration and death sometimes can be calculated from the distribution and ratio ofadministered drugs and their metabolic products. for a review of anesthetic death investigation, see ref. ( ) . halothane anesthesia and some other anesthetic agents may cause fulminant hepatitis and hepatic failure. the autopsy procedures suggested under "hepatitis, viral" should be followed. note: for special autopsy procedures in postoperative deaths, see chapter . in some instances, procedures described under "death, anesthesia-associated" may be indicated. for a review of investigational procedures and autopsy techniques in operating-room-associated deaths, see ref. ( ) . if the autopsy will involve anatomy or dissection techniques that are unfamiliar, the pathologist should not hesitate to invite the surgeon to the autopsy. in patients who develop a cerebral infarction after open heart surgery, arterial air embolism should be considered as a possible cause. the diagnosis often must be based on excluding other causes because the air has been absorbed prior to death. if a patient dies rapidly, the hospital records may be incomplete or scanty. for example, if a patient bleeds to death despite attempted repair of hepatic lacerations, hospital records may not suffice to reach the correct cause-of-death opinion; personal accounts from the surgeon and anesthesiologist may be needed. autopsy data on patients dying following thoracic surgery may be found in ref ( ) . d death, restaurant (see "obstruction, acute airway.") death, sniffing and spray related terms: glue sniffing; sudden sniffing death syndrome. note: no anatomic abnormalities will be noted at autopsy. sudden death may occur after cardiac dysrhythmia or respiratory arrest. procedures possible or expected findings lungs brain if poison had been inhaled at the time when death occurred, tie main bronchi. submit lungs in glass container for gas analysis. submit samples of small bronchi for histologic study. for removal and specimen preparation, see chapter . submit samples of fresh or frozen brain for toxicologic study. submit samples in glass containers (not plastic) for toxicologic study. trichloroethane, fluorinated refrigerants, and other volatile hydrocarbons are most often involved in the "sudden sniffing death syndrome." spray death may occur in asthma sufferers using pressurized aerosol bronchodilators. freons and related propellants may also be responsible for sudden death. toxic components of glue-such as toluene-accumulate in the brain of glue sniffers. also present in various glues are acetone, aliphatic acetates, cyclohexane, hexane, isopropanol, methylethyl ketone, and methylisobutyl ketone. aerosols may occlude the airway by freezing the larynx. carbon tetrachloride sniffing may cause hepatorenal syndrome (see also under "poisoning, carbon tetrachloride"). death, sudden unexpected, of adult note: medicolegal autopsies are usually indicated, and appropriate procedures should be followed. ifanaphylactic death is suspected, see also under that heading. for all unexpected deaths, the pathologist should learn the circumstances of the death, in order to determine whether the mechanism of death was rapid or slow, and to guide the selection of ancillary tests. whenever paramedics attended a person, the run sheet should be obtained to look for a history of recent drinking or ofchronic alcoholism may be an important clue. the combination of a history ofalcoholism, a negative test for ethanol, and absence ofcardiovascular disease, should suggest alcohol withdrawal as the cause ofa sudden death. the list of"possible or expected findings" below is not complete. for general toxicologic sampling, see chapter . possible associated conditions: atrial septal defect;*bicuspid aortic valve;* coarctation,* hypoplasia, or interruption (type a) of aortic arch; coronary artery from main pulmonary artery; right atrial arch; patent ductal artery;* right pulmonary artery from ascending aorta; subaortic stenosis;* tetralogy of fallot;* ventricular septal defect. * (in approx % of the cases, one or more of these associated conditions are found.) defect, atrial septal note: the basic anomaly is a defect of the atrial septum, usually at the oval fossa (in %). possible complications in unoperated cases include atrial arrhythmias, congestive heart failure; paradoxic embolism; plexogenic pulmonary hypertension « %), and pulmonary artery aneurysm. possible surgical interventions include surgical and transcatheter closure of defect. for deficiency, vitamin c synonyms: hypovitaminosis c; scurvy. external examination and skin other organs bones, joints, and soft tissues record extent and character of skin lesions; prepare sections of skin. describe appearance of gums, and prepare sections. record evidence of bleeding. for removal, prosthetic repair, and specimen preparation of bones and joints, see chapter . hyperkeratotic hair follicles with perifollicular hemorrhages (posterior thighs, anterior forearms, abdomen); petechiae and ecchymoses (inner and posterior thighs); subcutaneous hemorrhages. gingivitis. in rare instances, gastrointestinal or genitourinary hemorrhages. hemorrhages into muscles and joints. subperiosteal hemorrhages occur primarily in distal femora, proximal humeri, tibiae, and costochondral junctions (scorbutic rosary). deficiency, vitamin d synonyms: hypovitaminosis d; rickets. note: features or rickets may be found in familial hypophosphatemia (vitamin d-resistent rickets; fanconi syndrome). vitreous or blood (serum) other organs prepare skeletal roentgenograms. in infants with suspected rickets, record size of anterior fontanelle and shape of head; state of dentition; and shape of costochondral junctions, wrists, long bones, and spine. submit samples for calcium, magnesium, and phosphate determination. procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. weigh parathyroid glands and submit samples for histologic study. submit samples of intestine for histologic study. for removal, prosthetic repair, and specimen preparation, see chapter . in infantile rickets, diagnostic sites for histologic sampling are costochondral junctions, distal ends of radius and ulna, and proximal ends of tibia and humerus. for adults, see under "osteomalacia." in infants, rachitic changes at costochondral junctions; in adults, osteoporosis* and osteomalacia*-with or without pseudofractures (milkman's syndrome ( ) . note: the term spinocerebellar degeneration encompasses a variety of lesions whose classification is controversial. a new approach has come from linkage analysis and molecular biology. for instance, friedreich's ataxia, the classic form of hereditary ataxia, is due to an intronic expansion of a gaa tri-nucleotide repeat. other forms are also identified by their specific gene loci. neuropathologic examination still is important and ample sampling is suggested, which should include cerebral cortex, basal ganglia (caudate nucleus, putamen, and globus pallidus), thalamus, subthalamic nucleus, midbrain (red nucleus and substantia nigra), pons (pontine nuclei), spinal cord (at cer-vical, thoracic, and lumbar levels), optic tract, optic nerves with lateral geniculate nucleus, and sensory and motor peripheral nerves. for removal and specimen preparation, see chapter . enlargement of head. poor demarcation between cortex and gelatinous white matter. extensive demyelination and vacuolation of white matter, particularly subcortically. optic atrophy. degeneration, striatonigral (see "atrophy, multiple system.") related term: thirst. note: possible underlying conditions not related to inaccessibility of water include bums, exposure to heat, gastrointestinal diseases, recent paracentesis, renal diseases, and use of diuretic drugs. see also under "disorder, electrolyte(s)." external examination vitreous urine prepare histologic sections of blisters, ulcers, or skin abrasions. submit sample for sodium, chloride, and urea nitrogen determination. skin turgor may be decreased and eyes may be sunken. microscopic changes help to decide whether skin lesions are antemortem or postmortem. sodium concentrations more than meqll, chloride concentrations more than meq/ and urea nitrogen concentrations between and meq/dl indicate dehydration. absence or minimal amount of urine. dementia (see "disease, alzheimer's.") drug abuse, amphetamine(s) note: methamphetamine abuse may be suggested by poor condition of the dentition. methylenedioxymethamphetamine ("ecstasy") abuse is often suggested by friends with whom the decedent was abusing drugs. follow procedures described under "dependence, drug(s)." drug abuse, cocaine note: cocaine is spontaneously hydrolyzed by blood esterases, even after death. however, one of its major metabolite, benzoylecgonine, is routinely identifiable by immunoassay screening tests. when cocaine is abused concurrently with heroin or other depressant drugs, it may be difficult to ascribe deth to a single agent, unless circumstances clearly point to a rapid cardiac mechanism or a slow brainstem depression mechanism. note: if narcotic paraphernalia and samples of the drug itself are found at the scene of the death, they should be submitted for analysis. helpful information about the nature of a drug may be obtained from witnesses. state crime laboratories may provide much assistance. if name of drug is known, see also under "poisoning,..." the slang name of a drug may be insufficient for identification because these names often are used for different compounds at different times of places. opoid narcotics can be injected intravenously, or subcutaneously, or snorted. death may occur with such speed that the bodies may be found with needles and syringes in the veins or clenched in the hands. drug abuse may be associated with a multitude of local (see below) or systemic complications, including malaria* and tetanus. * as stated in chapter , for a growing number of analytes, most notably tricyclic antidepressants, peripheral blood is preferred over central blood. peripheral blood is aspirated by percutaneous puncture before autopsy, from the femoral vein or the subclavian vein. the authors prefer the femoral approach in order to avoid any question of artifact in the diagnosis of venous air embolism. it may be pru-dent to add naf to some of the samples. related term: childhood dermatomyositis (or polymyositis) associated with vasculitis; dermatomyositis (or polymyositis) associated with neoplasia or collagen vascular disease; primary idiopathic dermatomyositis; primary idiopathic polymyositis. possible associated conditions: carcinoma (lung, stomach, intestine, and prostate in males; breast, ovary, and uterus in females; miscellaneous sites in both sexes); lymphoma* (rare) and other malignancies ( ); lupus erythematosus;* mixed connective tissue disease; progressive systemic sclerosis;* rheumatoid arthritis;* sjogren's syndrome;* and others. vasculitis of childhood polymyositis (dermatomyositis). external examination and skin heart lungs esophagus and gastrointestinal tract photograph grossly involved skin. prepare sections of involved (anterior chest, knuckles, knees) and grossly uninvolved skin and subcutaneous tissue. prepare roentgenograms. submit samples from myocardium for histologic study. perfuse one lung with formalin. submit samples from all segments for histologic study. arteritis* and phlebitis* with thrombosis, fibrosis, and infarctions. steatohepatitis and manifestations of diabetes mellitus* may be found ( ) . myositis with muscular atrophy and fibrosis; vasculitis in childhood cases. polyneuropathy (rare) ( ). arthritis. diabetes mellitus synonyms: type i (insulin-dependent or juvenile-onset) diabetes mellitus; type ii (insulin-independent or adult onset) diabetes mellitus; secondary diabetes mellitus (e.g., due to drugs or pancreatic disease). note: in infants of diabetic mothers, macrosomia and congenital malformations must be expected. record size and weight of placenta and total weight and length, crown to rump length, and crown to heel length of infant. compare with expected measurements (see part iii). expected histologic finding in-clude hyperpla-sia with relative increase ofb cells of the islands of langerhans with interstitial and peri-insular eosinophilic infiltrates, decid-ual changes of the endometrium, enhanced follicle growth in the ovaries, and leydig cell hyperplasia. possible associated conditions: acanthosis nigricans; acro-megaly;* amyotrophic lateral sclerosis; * ataxia telangiectasia;* fanconi's anemia;* friedreich's ataxia;* gout;* hemochro-matosis; *hyperlipoproteinemia; * hyperthroidism;* obesity;* turner's syndrome;* and many others, too numerous to mention. note: the term "caroli's syndrome" often is used for cases that also show histologic features of congenital he-patic fibrosis or other manifestations of fibropolycystic liver disease,* whereas the name "caroli's disease" refers to idiopathic dilatation of intrahepatic bile ducts, without associated abnormalities. possible associated conditions: choledochal cyst* and related extrahepatic biliary abnormalities ( ); congenital hepatic fibrosis; * cysts of kidneys (renal tubular ectasia or medullary sponge kidney; autosomal-recessive polycystic kidney disease, and rarely, autosomal-dominant polycystic kidney disease [ ] )* and of pancreas. record volume of effusions. prepare smears of fresh blood or of buffy coat, or make thick-drop preparation. submit sample for xenodiagnosis or animal inoculation and for serologic study. record weight. in chronic chagas' disease, perfuse intact heart with formalin (chapter ) and slice fixed heart in a frontal plane so as to create anterior and posterior halves. prepare photographs. histologic samples should include conduction system. include several sections of atrial (auricular) walls for histologic study of autonomous ganglia. perfuse at least one lung with formalin. leave affected hollow viscera intact and fill with formalin. cut fixed organs in half, photograph, and cut histologic sections on edge. record liver weight and submit samples for histologic study. record weight. prepare photographs of abnormalities. weigh and examine. prepare histologic sections. for removal and specimen preparation, see chapter . autopsy is desirable in suspected cases because the diagnosis can only be firmly established after neuropathologic examination. serologic studies are not available. unfortunately, all tissues (not just the brain and spinal cord) may remain infectious even after prolonged fixation and histologic processing. thus, the autopsy recommendations for most other infectious diseases do not apply here. this is a reportable disease in some states. special precautions are indicated and therefore, the procedures described here should be followed strictly ( ) ( ) ( ) ( ) : all persons in the autopsy room must wear disposable long-sleeved gowns, gloves, and masks. contamination of the autopsy table should be prevented by covering it with a disposable, non-permeable plastic sheet. autopsy generally should be restricted to the brain. if organs in the chest or abdomen need to be examined, this is best done in situ. to prevent aerosolization of potentially infectious bone dust, a hood or other protective device should be used while opening the skull with a stryker saw. after completing the autopsy, instruments and other potentially contaminated objects should be autoclaved in a steam autoclave ( h at °c). porous load is considered more effective than gravity displacement autoclaves. immerse autopsy instruments in distilled water before and during autoclaving, in order to protect them from corrosion. ifno autoclave is available, chemical disinfection (see below) is a satisfactory alternative. disposable items should be put in a container for infectious hospital waste and ultimately incinerated. contaminated objects not suitable for autoclaving (such as the stryker saw) should be soaked with a nnaoh solution for h (alternatively, nnaoh may be used for h). contaminated surfaces should be thoroughly washed with the same solution. aluminum should be treated for h with a fresh % naoci (sodium hypochlorite) solution with at least , ppm free chloride. wash waters should be collected; if no autoclave is available, n naoh or > volumes of % sodium hypochlorite bleach should be added to the water and left for a minimum of h before being discarded. before removing the body from the autopsy room, it should be sponged with % sodium hypochlorite. to deactivate cjd infectivity, tissue blocks, mm or less in thickness, should be fixed in formalin in a formalin-totissue ratio of at least : for at least h and then soaked in concentrated formic acid ( - %) for i h, followed by another h of formalin fixation. the fixation fluid should be collected and decontaminated, as described earlier for wash water. glassware and tissue carriers should also be decontaminated as previously described. after this deactivation, the tissue blocks can be processed in a routine fashion. at any stage of these procedures, special care must be taken to avoid cuts with potentially contaminated glassware, blades, or other objects. parenteral exposure to potentially contaminated material also should be avoided. remains of patients who have died of the disease should not be accepted for anatomy teaching for students. if specimens are prepared for pathology collections, they should be handled with great caution. morticians and mortuary workers should be warned of possible hazards posed by tissues of patients with transmissible spongiforme encephalopathies; they should be advised about proper use of disinfectants. clinical laboratories that receive autopsy tissues or fluids must be warned about the infectious nature of the material. if possible, decontamination should be done at the site where the autopsy was done. for the shipping of potentially infected material, see chapter . increased concentrations of nse ( ). spongiforme changes, astrocytosis, neuronal loss, amyloid plaque formation, prp deposition, and proliferation of activated microglia ( ). cerebrospinal fluid brain submit sample for neuron-specific enolase (nse). for removal and specimen preparation, see chapter and above under "note." submit fresh-frozen material for confirmation of diagnosis by histoblot technique on protease k-digested frozen tissue or western blot preparations on brain homogenates. immunohistochemical localization ofprp and hla-dr protein on paraffin-embedded tissue is possible. disease, demyelinating (see "degeneration, spongy, of white matter," "encephalomyelitis, all types or type unspecified," "leukodystrophy, globoid cell," "leukodystrophy, sudanophilic," "sclerosis, multiple;' and "sclerosis, schilder's cerebral.") disease, diffuse alveolar synonym: diffuse pulmonary disease. note: autopsy procedures are listed under the more specific diagnoses, such as "hemosiderosis, idiopathic pulmonary," "lipoproteinosis, pulmonary alveolar," "microlithiasis, pulmonary alveolar," "pneumonia, lipoid," and "syndrome, goodpasture's." glycosphingolipid storage in cornea; lens opacities; dilated vessels in conjunctiva and lens; thrombi in blood vessels ( ). disease, fibropolycystic, of the liver and biliary tract note: "fibropolycystic disease of the liver and biliary tract" comprises a group of well defined conditions, which may occur together and hence need a collective designation. the conditions include autosomal-recessive (infantile) and auto-somal dominant (adult) polycystic disease of the liver; caroli's disease or syndrome;* choledochal cyst,* congenital hepatic fibro-sis,* multiple biliary microhamartomas, and related disorders. for autopsy procedures, see also under more specific designations. disease, glycogen storage synonyms: andersen's disease or brancher deficiency (glycogenosis, type iv); cori's or forbes' disease (glycogenosis, type ill); cyclic amp dependent kinase (type x); glycogen synthetase deficiency (type ); hers' disease (glycogenosis, type vi); mcardle's disease (glycogenosis type v); phosphorylase b kinase deficiency (types ixa, b, and c); pompe's disease (glycogenosis, type it); tarui disease (glycogenosis type vii); von gierke's disease (glycogenosis, type ia); x-linked glycogenosis (type vill). note: if the diagnosis had not been confirmed prior to death, samples of liver, skeletal muscle, blood, and fascia (for fibroblast culture, see below) should be snap-frozen for enzyme assay, which will determine the specific deficiency. types ia and b, iii, vi, and hepatic phosphorylase b kinase deficiency (types ixa, b and c) are hepatic-hypoglycemic disorders, whereas types v and vii affect muscle energy processes. type ii also affects the musculature, whereas type iv may cause cirrhosis and death in infancy from extreme hypotonia. determination of type of glycogenosis usually can be based on (i) pattern of glycogen storage in liver, ( ) presence or absence of nuclear hyperglycogenation in liver, ( ) cytoplasmic lipid in liver, ( ) presence or absence of liver cirrhosis, and ( ) presence or absence of glycogen and basophilic deposits in skeletal muscles. possible associated conditions: fanconi syndrome* or gout* with type ia glycogenosis; neutropenia, recurrent infections, and crohn's disease with types ib or ie. glycogen primarily in retinal ganglion cells and ciliary muscle. glycogen in sympathetic nerve ganglia and neurons of cranial nerves in type vii. gouty arthritis. disease, graft-versus-host note: this disease occurs most commonly after bone marrow transplantation. the disease has also occurred after transfusion of viable lymphocytes, for example, to patients with cancer or leukemia. * in patients with graft-versus-host disease (gvhd), autopsy also may reveal recurrence of the underlying disease such as leukemia. possible associated conditions: alphal-antitrypsin deficiency;* amyloidosis;* ankylosing spondylitis;* primary sclerosing cholangitis;* sjogren's syndrome. * see also below under "possible or expected findings." note: in many instances, either chronic ulcerative colitis or crohn's disease* had been diagnosed clinically, but sometimes, the distinction is difficult to make, even at autopsy. many features described below occur in chronic ulcerative colitis but some manifestations of crohn's disease or conditions that may occur in all types of inflammatory bowel disease also are listed so that both positive and negative findings can be recorded properly. osteoporosis;* ankylosing spondylitis;* arthritis of peripheral joints; periarthritis; hypertrophic osteoarthropathy;* tendinitis (particularly of ankle and achilles tendons). disease, iron storage (see "hemochromatosis.") related terms: atherosclerotic heart disease. note: the most common anatomic finding at autopsy in subjects older than yr is coronary atherosclerosis. unusual under-lying or associated conditions include chronic aortic stenosis or regurgitation; coronary artery anomalies; coronary artery dissection; coronary embolism; coronary ostial stenosis (due to calcification of aortic sinotubular junction or, rarely, to syphilitic aortitis); coronary vasculitis (for instance, in polyarteritis nodosa* or acute hypersensitivity arteritis); hyperthyroidism,* gastrointestinal hemorrhage; * hypothyroidism, * idiopathic arterial calcification of infancy; intramural coronary amyloidosis; pheochromocytoma, polycythemia vera; * pseudoxanthoma elasticum,* radiationinduced coronary stenosis; severe pulmonary hypertension (with right ventricular ischemia); sickle cell disease;* and others. if bypass surgery had been performed, see "surgery, coronary bypass." macular rash ( ). multifocal fibrinopurulent pneumonia with sparing of the bronchi and bronchioles. exudate is rich in phagocytes, fibrin, and karyorrhectic debris. synonym: lyme arthritis note: this infection is caused by the spirochete, borrelia burgdoiferi, which is transmitted from rodents to human by the hard deer ticks, ixodes dammini, . ricinus, and others. brain and spinal cord for removal and specimen preparation, see chapter . request luxol fast blue stain for myelin. symmetric and zonal demyelination in corpus callosum, anterior commissure, optic chiasm, optic tracts, and white matter of frontal lobes. external examination and skin; oral cavity lungs aorta record distribution of skin lesions and submit tissue samples for histologic study. for preparation of angiograms of the pulmonary arterial and venous vasculature, see chapter . if aneurysm or dissection is present, follow procedures described under those headings. telangiectatic (often papular) lesions most commonly found in cheeks, scalp, nasal orifices, oral cavity, ears, neck, shoulders, fingers, toes, and nail beds. cyanosis and clubbing may be prominent. arteriovenous malformations/fistulas. aneurysm; * aortic dissection. * if cirrhosis is present, prepare angiograms of hepatic arteries and veins (chapter ). photograph and prepare sections of angiomatous lesions. note: parkinson's syndrome is caused by conditions that may simulate parkinson's disease; these include carbon monoxide* and manganese poisoning, corticobasal degeneration, druginduced parkinsonism, huntington's disease, multiple system atrophy,* progressive supranuclear palsy* (steele-richardson-olszewski syndrome), space-occupying lesions (rare), trauma (dementia pugilistica), and causes related to tumors and vascular diseases. brain for removal and specimen preparation, see chapter . histologic sections should include midbrain (substantia nigra), upper pons (locus ceruleus), medulla, nucleus basalis (substantia innominata), and basal ganglia. if parkinsonian syndrome was diagnosed, follow procedures described under the name of the suspected underlying condition (see above under "note"). depigmentation of substantia nigra and locus coeruleus; neuronal loss and reactive gliosis; eosinophilic intracytoplasmic inclusion bodies (lewy bodies) in some of the surviving neurons; no significant changes in basal ganglia. disease, pelizaeus-merzbacher synonyms: sudanophilic (orthochromatic) leukodystrophy. brain and spinal cord for removal and specimen preparation, see chapter . request luxol fast blueipas stain for myelin and bielschowsky's stain for axons. prepare frozen sections for sudan stain. brain generally atrophic. myelin loss in centrum ovale, cerebellum, and part of brain stem, with a tigroid pattern of residual myelin near vessels. axons are preserved. diffuse gliosis with relatively few lipoid-containing macrophages, compared to the myelin loss. lipoid material stains with sudan. brain and spinal cord for removal and specimen preparation, see chapter . request silver stains (bielchowsky or bodian stain). histochemical stains in pick's cells and bodies reveal phosphorylated neurofilaments, ubiquitin, and tubulin. some tissue should be kept frozen for biochemical studies. severe cerebral atrophy, involving primarily frontal and anterior temporal lobes (knifeblade atrophy; walnut brain). microscopically, severe neuronal loss accompanied by astrocytosis. characteristic argyrophilic, intracytoplasmic inclusions (pick's bodies), particularly in hippocampus and swollen, distended "ballooned" neurons (pick's cells). these changes are not always present. external examination, skin, and adipose tissue blood cerebrospinal fluid heart liver and kidneys brain, spinal cord, and peripheral nerves eyes submit sample for determinaion of phytanic acid concentration and for molecular studies. for obtaining a sample, see chapter . sample for histologic study. for removal and specimen preparation, see chapter . for removal and specimen preparation, see chapter . ichthyosis. phytanic acid accumulation in adipose tissues. phytanic acidemia, mutation of phyh or pex ( ). increased protein concentrations. cardiomyopathy.* phytanic acid accumulation. axonal neuropathy. retinitis pigmentosa. hypoalphalipoproteinemia. lymphadenopathy with diffuse deposition of cholesterol esters. premature atherosclerotic cardiovascular disease ( ). hepatosplenomegaly with foam cells. enlarged tonsils with characteristic orange discoloration. polyneuropathy ( ) . in adults, corneal infiltrates. foam cells. request pas stain. in granulomas, bacilli are not always pas positive ( ) . section all grossly involved tissues for histologic examination. submit section for electron microscopy. emaciation. hyperpigmentation, particularly of exposed skin and in scars. hyperkeratosis. arthritis involving ankles, knees, shoulders, and wrists. ascites; fibrinous peritonitis. * nodules in peritoneum containing sickle-form particlecontaining cells (spc cells submit sample for determination of sodium, potassium, chloride, glucose, urea nitrogen, and creatinine concentrations. calcium and phosphate concentrations can also be tested. if sample is small, indicate priority for testing. if indicated, submit sample for chemical study. submit tissue samples for histologic study. considerably increased or decreased values for sodium (more than meqll or less than meqll) and chloride (more than meqll or less than meqll) indicate that changes were present before death. for further interpretation, see chapter . postmortem electrolyte concentrations are quite unreliable. may be useful for calcium determination. vacuolar nephropathy (vacuolar changes in proximal convoluted tubules) in potassium deficiency (may also occur after infusion of hypertonic solutions). disorder, hemorrhagic (see "coagulation, disseminated intravascular," ''disease, christmas:' ''disease, von willebrand's," "hemophilia," and "purpura,.••") disorder, inherited, of phagocyte function note: several conditions represent phagocyte function disorders. autopsy procedures for one of these disorders can be found under "disease, chronic granulomatous." consult this entry for other phagocyte function disorders. synonyms and related terms: fabry's disease* (angiokeratoma corporis diffusum); gangliosidosis;* gaucher's disease;* glycogenosis,* type ii; leukodystrophies (krabbe's or globoidcell,* metachromatic leukoencephalopathy*); mucopolysaccharidoses* (hunter, hurler, morquio, and sanfilippo disease); mucolipidosis; niemann pick disease* (type a, b, c, or sphingomyelinase deficiency); neuraminidase deficiency; neuronal ceroid lipofuscinosis (batten's disease or kufs' disease). hypopharyngeal pulsion diverticulum (zenker's diverticulum) at lower margin of inferior constrictor muscle of pharynx. traction diverticulum at midesophagus after an inflammatory process-for instance, tuberculous lymphadenitis. epiphrenic diverticulum may also occur. luxtacardiac or juxtapyloric diverticulum. heterotopic tissue in meckel's diverticulum, with or without peptic ulceration. colonic muscular hypertrophy and stenosis, usually in sigmoid colon. diverticulitis with perforation, fistulas, or peritonitis. * diving (see "accident, diving (skin or scuba).") related terms: dry drowning; fresh-water drowning; near-drowning; salt (sea)-water drowning (see the following table). primary drowning ("immediate drowning") deaths occurring within minutes after immersion, before or without resuscitative measures deaths from hypoxia and acidosis caused by glottal spasm on breath holding. there may be no evidence of water entering stomach or lungs and no appreciable morphologic changes at autopsy. note: the diagnosis is one of exclusion. the pathologist should help the police to determine: i) how did the person (or dead body) get in the water, and ) why could that person not get out of the water? it is not enough to ask if a person could swim but investigators should find out how well (what strokes did the victim know?) and how far he or she could swim. the inquiry must include the depth of the water and must address hazards such as undertow or underwater debris, and the behavior deaths occurring from within min to several weeks after resuscitation, because of metabolic acidosis, pulmonary edema, or infective or chemical pneumonitis deaths from hypoxia and acidosis caused by obstruction of airway by water related to: hypervolemia hemolysis hyponatremia hypochloremia hyperkalemia of the victim immediately before submerging. deaths of adults in bathtubs and swimming pools are usually from natural, cardiac causes, or they are suicides, unless the victim was drunk. diatom tests ( ) have not proven useful in the united states but there is enthusiasm for such tests among european pathologists. the distinction between hyponatremic deaths in fresh water and hypernatremic deaths in salt water derives from experimental studies; in practice, one cannot reliably predict the salinity of the immersion medium from autopsy studies. because many bodies of drowning victims are recovered only after the body floats to the surface, decomposition will often obscure even the nondiagnostic findings such as pleural effusions, which are often associated with drowning. external examination and skin (wounds) organ samples for diatom search serosal surfaces and cavities if identity of drowning victim is not known, record identifying features as described in chapter . prepare dental and whole-body roentgenograms. submit tissue samples for histologic study of wounds. inspect inside of hands. collect fingernail scrapings. record appearance and contents of body orifices. record features indicative of drowning. photograph face from front and in profile. take pictures of all injuries, with and without scale and autopsy number. remove vitreous for analysis. if diatom search is intended, clean body thoroughly before dissection to avoid contamination of organs and body fluids with algae and diatoms (see below). submit sample for toxicologic study. sample early during autopsy, before carrying out other dissections. use fresh instruments for removal of specimens to avoid contamination. submit subpleural portion of lung: subcapsular portions of liver, spleen, and kidneys; bone marrow; and brain. store samples in clean glass jars. for technique of diatom detection, see below. record volume of fluid in pleural spaces. photograph petechial hemorrhages. photograph layerwise neck dissection if strangulation* is suspected. open airways posteriorly, and photograph, remove and save mud, algae, and any other material in tracheobronchial tree. record size and weight of lungs. there may be wounds that were inflicted before drowning occurred-for instance, in shipwrecks or vehicular and diving accidents. other wounds may be inflicted after deathfor instance, from ship propellers or marine animals. sometimes, premortem and postmortem wounds can be distinguished histologically. object (hair?) held by hands in cadaveric spasm. cutis anserina and "washerwoman" changes of hands and feet are of no diagnostic help. foreign bodies; semen (see also under "rape"). foam cap over mouth and nose. in the autopsy room, water running from nose and mouth is usually pulmonary edema or water from the stomach. high concentrations of alcohol indicate intoxication (see under "alcoholism and alcohol intoxication"). evidence of alcohol intoxication may be found. diatoms may occur in the liver and in other organs of persons who have died from causes other than drowning. comparison with diatoms in water sample from area of drowning may be helpful. penny-sized or smaller hemorrhages may indicate violent respiratory efforts or merely intense lividity. presence of pleural fluid suggests drowning. for diatom detection (l) , boil - g oftissue for -- min in rnl of concentrated nitric acid and . rnl of concentrated sulfuric acid. then, add sodium nitrate in small quantities until the black color of the charred organic matter has been dispelled. it may be necessary to warm the acid-digested material with weak sodium hydroxide, but the material must soon be washed free from alkali to avoid dissolving the diatoms. the diatoms should be washed, concentrated, and stored in distilled water. for examination, allow a drop of the concentrate to evaporate on a slide, and then mount it in a resin of high refractive index. all equipment must be well-cleaned, and distilled water must be used for all solutions. there are several variations and adaptations of this method. drug abuse, amphetamine(s) note: methamphetamine abuse may be suggested by poor condition of the dentition. methylenedioxymethamphetamine ("ecstasy") abuse is often suggested by friends with whom the decedent was abusing drugs. follow procedures described under "dependence, drug(s)." ductus arteriosus, patent (see "artery, patent ductal.") synonyms and related terms. achondroplastic dwarf; asexual dwarf; ateliotic dwarf; micromelic dwarf; normal dwarf; pituitary dwarf; true dwarf; and many other terms, too numerous to mention. external examination bones and joints record height and weight. prepare skeletal roentgenograms. for removal, prosthetic repair, and specimen preparation, see chapter . growth retardation. abnormal growth of epiphyseal cartilage with enlargement of metaphysis. long bones and pelvis most commonly affected. cavernous hemangiomas (maffucci's syndrome). see above under "external examination." chondrosarcoma. dyscrasia, plasma cell note: these conditions are characterized by abnormally proliferated b-immunocytes that produce a monoclonal immunoglobulin. multiple myeloma, * plasma cell leukemia, plasma-cytoma, and waldenstrom's macroglobulinemia* as well as heavy-chain diseases and monoclonal gammopathies of unknown type belong to this disease family. amyloidosis* is closely related to these conditions. for autopsy procedures, see under "amyloidosis," "macroglobulinemia," or "multiple myeloma" and under name of condition that may have caused the plasma cell dyscrasia. such conditions include carcinoma (colon, breast, or biliary tract), gaucher's disease,* hyperlipoproteinemia, * infectious or noninfectious chronic inflammatory diseases, and previous cardiac surgery. synonym: shigella dysentery. note: (i) collect all tissues that appear to be infected. blood bowel eyes joints submit sample for culture and for serologic study. submit sample of feces or preferably bloodtinged mucus for culture. if bacteriologic diagnosis has already been confirmed, pin colon on corkboard, photograph, and fix in formalin for histologic study. submit sample of vitreous for study of sodium, potassium, chloride, and urea nitrogen concentrations. for removal and specimen preparation of eyes, see chapter . for removal, prosthetic repair, and specimen preparation, see chapter . escherichia coli septicemia. colitis with microabscesses; transverse shallow ulcers and hemorrhages, most often in terminal ileum and colon. dehydration* pattern of electrolytes and urea nitrogen. serous arthritis* of knee joints is a late complication. external examination record extent of pigmentation, facial features, and primary and secondary sex characteristics. prepare skeletal roentgenograms. for removal, prosthetic repair, and specimen preparation, see chapter . record size of apertures of cranial nerves in base of skull. unilateral skin pigmentation and precocious puberty in females (albright's syndrome), less commonly in males. synonyms and related terms: becker's muscular dystrophy; congenital muscular dystrophy; duchenne's progressive muscular dystrophy; dystrophinopathy; em-ery-dreifuss mucular dystrophy; facioscapulohumeral dystrophy; limb girdle dystrophy; myotonic muscular dystrophy. external examination record pattern of scalp hair. record status of skeletal musculature. obtain sections for histologic examination. dystrophin staining of the sarcolemma is absent in duchenne's muscular dystrophy and patchy in becker's dystrophy. frontal baldness (in myotonic muscular dystrophy). atrophy and wasting of muscles (generalized or local: predominantly distal in myotonic muscular dystrophy). pseudohypertrophy of calf muscles in duchenne's muscular dystrophy. dystrophic changes include variations in fiber size, fiber degeneration and regeneration, peri-and endomysial fibrosis, and fatty replacement of muscle. the liver, especially the right lobe, is the most common site of involvement. secondary infection or calcification may be present. the lung is the second most common site of involvement. fluid and air may be visible on the roentgenogram. cysts may be present in the abdominal cavity, muscles, kidneys, spleen, bones, heart, and brain. eosinophilia. edema, angioneurotic synonym: angioedema. note: possible causes and suggested autopsy procedures are described under "death, anaphylactic." related term: silo-filler's disease. n<yfe: this condition is causedby inhalationoftoxic gases, such as oxides of nitrogen (silo-filler's disease) and phosgene (coci ). see also "bronchitis, acute chemical" and "poisoning, gas." ( ) gunshot and shotgun wounds of the head involving dural sinuses; ( ) injury to large veins, particularly cranial sinuses (during neurosurgical procedures) and veins ofthe neck (knife wounds, surgery) or uterus (criminal abortion); ( ) insufflation offallopian tubes (particularly in pregnancy or during menstrual period); ( ) infusion of blood components or crystalloid; ( ) malfunctioning of dialysis machines; ( ) subclavian vein catheterization in the semi-fowler position; and ( ) fracture in the hub of a central venous catheter used for parenteral nutrition. possible causes of arterial air embolism include: ( ) open heart surgery involving the aorta, left atrium, or left ventricle; ( ) positive-pressure ventilation in newborn infants; and ( ) underwater ascent with closed glottis (see accident, diving) ifair embolism is suspected, the autopsy should be performed as soon after death as possible. decomposition gases may be produced within a few hours. roentgenography of the whole body may detect large quantities of air, and the roentgenograms may serve as a guide to the most advantageous way of dissection. the postmortem diagnosis of arterial air embolism is made largely on the basis of medical history and circumstances. the autopsy serves to rule out competing conditions. the diagnosis of venous air embolism is made by chest roentgenography before the autopsy ( ). the air in the right chambers of the heart can be confirmed at autopsy by aspiration into a syringe. the formerly recommended procedure of clamping the internal mammary vessels below the sternoclavicular joints, and then cutting across the sternum distal to these clamped vessels so that the sternoclavicular joint area remains intact, is designed to prevent the production of a vacuum that pulls air into the veins. the procedure is not necessary if the air is welldocumented by roentgenography before autopsy. at autopsy, a large fatal pulmonary air embolism is readily apparent. the right atrium and ventricle are distended with fine, frothy, brightred blood, which also may distend the pulmonary arteries and superior vena cava. microbiologic examination ofblood and pericardial sac contents may help to rule out the presence of gas-forming bacteria that may simulate air embolism (fig. ii-i). however, the presence of putrefactive emphysema in any part of the body points toward a bacterial origin of the gas. differentiation of air and decomposition gases at the autopsy table with the pyrogallol test is described below. a % pyrogallol solution is prepared (it should be waterclear). two lo-ml syringes (syringe a and syringe b) are loaded with ml of the pyrogallol solution in each, without permitting any air to enter the system. immediately before the solution is used, drops of . n naoh is aspirated through the needle of syringe a to adjust the ph to about ( drop per ml of solution); the mixture will turn faint yellow. six ml of gas is then aspirated from the heart or blood vessels. the needle is immediately sealed with a cork or replaced by a cap, and the syringe is vigorously shaken for about min. in the presence of air, the pyrogallol solution will turn brown. if the solution remains clear, decomposition gases were present. in the latter instance, drops of . n naoh and ml of room air should be aspirated into syringe b, which is then also sealed and shaken for min. the mixture should turn brown, thus serving as a fig. · . gas-forming bacteria simulating air embolism. the pericardial sac is opened and filled with water. the heart is kept submerged with a pair of scissors. the coronary arteries have been incised with a scalpel. note gas bubbles and foam on the water surface. no discoloration of % pyrogallol was noted. blood cultures were positive for enterococcus organisms. microscopically, gram-negative rods were found in most tissues. control that the pyrogallol solution had been properly prepared. syringe b may also serve as a reserve. if only one syringe is used, the decomposition gas can be expel ed and room air can be aspirated for the control test. if only small amounts of gas can be aspirated, the volume of the pyrogallol solution should be decreased so that the gas-fluid volume ratio is at least : . if no bacterial gas formation is present, the edges of the pericardial incision are elevated and the pericardial sac is fil ed with water. clamping of the ascending aorta and venae cavae prevents the escape of gas into these vessels. the heart is held under water while the coronary arteries are incised, and the escape of bubbles is recorded. when the right coronary artery is opened, care must be taken that the right atrium is not incised. air in the coronary arteries indicates systemic embolism. the heart chambers are then incised. when there is gas in any of the arteries or heart chambers, gas bubbles rise to the surface of the water in the pericardial sac ("bubble test"). sometimes the vessels have to be somewhat compressed in order to cause the gas to escape. because large amounts of air or other gases cause the heart to float, it must be kept submerged before the vessels and chambers are incised. basically the same procedure is used for demonstrating the presence of gas in the superior or inferior vena cava and the pelvic veins (for example, in cases of criminal abortion). in this situation, the abdominal cavity is filled with water and the inferior vena cava and its tributaries are incised. in support of the diagnosis of systemic arterial air embolism, the skull vault may be removed without puncturing the meninges, so that the cerebral arteries can be inspected for gas bubbles. the demonstration of gas bubbles in the meningeal vessels and in the circle of willis is meaningful only when the neck vessels are still intact and the internal carotid artery and basilar artery have been clamped before the brain is removed. in acute cases, gas bubbles will be visible within the cerebral vessels. they are released under water when the clamps are removed and the vessels are slightly compressed. for the collection of gas from blood vessels or cavities, a system oflittle quantitative reliability is an air-tight, water-filled glass syringe with a needle. the needle is inserted into the vessel or cavity in question and gas is carefully aspirated. a combined qualitative and quantitative method has been described by kulka ( , ) (see fig. - ). he devised an apparatus for gas collection and described it as shown in fig. - and caption. the entire system is filled with mineral oil so that, when the funnel is level with the upright bottle, the oil fills only about half of the funnel. in operation, the funnel is first raised to a position - cm above the level of the upright bottle. all the cocks are opened and the position is held until every trace of gas has been driven from the system through the needle, which is thereby coated on the inside by a film of oil. after all air has been expelled, the cocks are closed and the funnel is lowered to its original position. as a precautionary measure and control, the air-tightness of the whole system should be tested before operation. this is done by inserting the needle into musculature or skin and attempting aspiration in the manner described in the next paragraph. to make the test, the bottle is inverted and the needle is inserted into the cavity in question. when the needle is in position, all cocks are opened. the funnel is lowered about - cm, or until adequate suction is created. this aspirates the contents of the cavity, which may consist of air or other gases, either pure or mixed with blood or other liquid. any gas or liquid entering this system may be observed through the wall of the short bent glass tubing. in a positive test, gas bubbles will collect in the bottle above the level of the oil. if desired, this gas can be saved for further examination by closing all the cocks and returning the bottle to its upright position ( ). the volume ofintravenous air needed to cause death in adults depends of the cross sectional area of the cardiac cavity or great vessel that is the site of the gas lock, which in tum depends on the position of the decedent at the time of the embolism ( ). ml of gas can pass through an intravenous hub in just a few seconds. small amounts of air entering the systemic circulation may cause death within minutes. delayed air embolism with fatal outcome may also occur. other organs if purpura is present, prepare photographs and record extent. submit sample (from right atrium) for microbiologic study. collect blood from right atrium and right ventricle. after the heart has been removed, allow blood in pulmonary vessels to pool in the pericardial sac. centrifuge this blood and submit sample of flocculent layer above buffy coat for microscopic study ( ) . submit a section of lung for bacteriologic study. dissect pulmonary arteries; prepare histologic sections of all lobes; request mucicarmine stain and the aldan blue and phloxinetartrazine stain of lendrum. also request sudan stain on frozen sections. skin purpura. vernix, lanugo hairs, and meconium can be found in pericardial blood pool. meconium-type material in blood vessels. in histologic sections, squamous epithelium, meconium, and fat from vernix caseosa. complete or incomplete lower uterine tear; chorioamnionitis. manifestations ofdisseminated intravascular coagulation* and fibrinolysis. intrauterine pneumonia. large amounts of debris in the blood vessels of all sections of the lungs may be considered to be lethal if there is no other cause of death. small amounts in one or more blocks of pulmonary tissue are more likely incidental ( ). a small uterine tear is more likely followed by a fatal amniotic fluid embolization than is a large tear, which may result in fatal hemorrhage or fibrinogen depletion. chorioamnionitis, intrauterine pneumonia, and positive lung cultures of the mother indicate infection of the amniotic fluid. record weight and sample for histologic study. for removal and specimen preparation, see chapter . photograph horizontal sections through brain, brain stem, and spinal cord. prepare frozen sections and request sudan stain. prepare frozen sections of one-half of gland and paraffin sections of the other half. petechial hemorrhages of skin (chest, neck, and face). wounds; other traumatic lesions. bone fractures. petechiae of conjunctivas and retinas. fat may accumulate on surface ofblood pool. no useful technique is available for estimating the amount of fat globules in the blood. fat emboli in lumen of small vessels and in pulmonary air spaces. severe fatty changes may be the cause offat embolism. fractures are the most common cause of fat embolism. petechial hemorrhages, fat emboli ( ). abdominal cavity submit sample of subphrenic exudate for gram stain and cultures. record location and volume of subphrenic exudate. possible causes of subphrenic empyema include appendicitis, cholecystitis, * diverticulitis, intrahepatic abscess, pancreatitis,* ruptured viscus; penetrating abdominal wound(s), perforated ulcer of stomach or duodenum,* and other conditions. pleural cavities and lungs e procedures record volume of effusion or exudate in pleural space. basal pleuritis and pneumonia, adjacent to empyema. encephalitis, au types or type unspecified synonyms and related terms: acute disseminated encephalomyelitis;* acute hemorrhagic encephalitis; acute infective encephalitis or encephalomyelitis; acute poliovirus encephalitis or encephalomyelitis; amoebic encephalitis; arbovirus encephalitis (japanese encephalitis; eastern encephalitis, western encephalitis, venezuelan equine encephalitis, st. louis encep-halitis); bulbar encephalitis;* brain stem encephalitis;* herpes encephalitis (cytomegalovirus encephalits, epstein-barr virus encephalitis, varicella-zoster encephalitis); herpes simplex ence-phalitis; hiv encephalitis; measles encephalitis; measles inclusionbody encephalitis; postinfectiousencephalitis; postvac-cinal encephalitis; progressive multifocal leukoencephalitis or leukoencephalopathy; rabies* encephalitis; subacute encephalitis; subacute sclerosing panencephalitis; viral encephalitis, west nile encephalitis and other terms ( ), too numerous to mention. see also under "note" and under "possible or expected findings." note: if the condition that caused the encephalitis is known, see also under that heading. if the cause of the encephalitis is unknown, submit samples of tissue for microbiologic and toxicologic study, particularly if there is a suspicion of lead poisoning.* see also under "encephalitis, brain stem," "encephalomyelitis,...," "encephalopathy" and "myelopathy, myelitis." encephalitis, brain stem synonyms and related terms: brain stem abscess; infectious brain stem encephalitis; listeria monocytogenes brain stem encephalitis; viral brain stem encephalitis. note: see also under "encephalitis, limbic." brain for removal and specimen preparation, see chapter . necrotizing encephalitis, with or without abscess formation ( enterocolitis, other types or type undetermined note: a multitude of infectious and noninfectious agents may cause inflammation of the small bowel, large bowel, or both. if the condition is not listed under "colitis," "enteritis," or "enterocolitis" or under another specific heading such as "dysentery, bacillary," obtain sufficient material for microbio-logic and histologic study to identify organisms such as clos-tridium, chlamydia, shigella, salmonella, yersinia, helicobacter, verotoxic e. coli, and others. if lymphogranuloma venereum,* or tuberculosis* are suspected, see also under these headings. see also under "disease, inflammatory bowel" and "disease, crohn's." synonyms and related terms: c. difficile colitis; darmbrand; hemorrhagic necrosis (gangrene; infarction) of intestine; ischemic enteritis or enterocolitis;* neutropenic enterocolitis;* pseudomembranous colitis. note: the name "pseudomembranous enterocolitis" is descriptive; the condition may be infectious, ischemic, or both. if the intestinal changes are clearly ischemic, see above under "enterocolitis, ischemic." if the cause is in doubt and if pseudomembranes can be identified, follow the procedures described here. (l) collect all tissues that appear to be infected. for other infectious intestinal diseases, see under specific names, such as "enterocolitis, neutropenic" or "enterocolitis, staphylococcal." intestinal tract and mesentery other organs collect material from pseudomembranes for culture and for c. difficile toxin assay. sample intestinal wall with pseudomembranes for histologic study. if the condition is suspected to be caused by ischemia, follow procedures described above under "enterocolitis, ischemic." note: myoclonic seizures also have been described in a number of progressive encephalopathies with complex neurological symptoms, such as gmi and gm gangliosidosis,* and niemann-pick* and krabbe's disease but also acquired disorders, including alzheimer's disease,* creutzfeldt-jakob epilepsy, myoclonus (continued) disease,* posthypoxic encephalopathy, and subacute sclerosing panencephalitis. mitochondrial encephalomyopathy also can present with myoclonus epilepsy and a mitochondrial myopathy with ragged red fibers (merrf syndrome) in skeletal muscles. lafora-body-type material in the heart, liver, retinas, peripheral nerves, skeletal muscles, and sweat gland ducts (especially axillary). ragged red fibers in mitochondrial myopathies. epilepsy, symptomatic note: possible causes or underlying conditions include cerebrovascular diseases, congenital malformations ofthe brain, degenerative and demyelinating diseases of the brain, head injury,* intracranial and cerebral infections, toxic or metabolic disorders (alcoholism,* barbiturate,* carbon monoxide,* and lead poisoning,* hemodilution, hypocalcemia, or hypoglycemia),* and tumors of the brain. * f failure, congestive heart note: coronary atherosclerosis and manifestations of ischemic heart disease, * valvular heart disease, congenital cardio-vascular diseases, and manifestations of systemic or pulmonary hypertension are the most frequent findings in patients dying of or with congestive cardiac failure. other causes include cardiomyopathies* and secondary myocardial disease (such as amyloid or pericardial constriction). if the cause of the congestive cardiac failure is unknown or not immediately evident after dissection of the heart and of the great vessels, myocardium and other appropriate tissues may be submitted for microbiologic study-including viral cultures-and for electron microscopy. specimens can also be snap-frozen for possible immunofluorescent, biochemical, or histochemical studies, particularly of the myocardium. possible causes of congestive cardiac failure are too numerous to mention. dilatation of heart, with or without mural thrombosis. myocardium may be soft, normal, or firm. pulmonary congestion, with or without hemosiderosis; congestion of viscera with organomegaly. other organ manifestations include bowel edema or hemorrhagic enteropathy (without mechanical vascular occlusion) and zonal hepatic steatosis, fibrosis, or necrosis, with or withoutevidence of liver failure. acute renal tubular necrosis may be present also. synonyms and related terms: acute kidney failure; chronic kidney failure; renal failure; uremia. note: if acute kidney failure had been diagnosed, the autopsy procedures will depend on the expected causes, such as poisoning with ethylene glycol,* lead,* mercury,* or methyl alcohol;* disseminated intravascular coagulation* and its various underlying conditions; glomerulonephritis* and its various underlying conditions; diabetes mellitus;* or multiple myeloma. * the procedures described below deal primarily with chronic renal failure. if the patient had had dialysis, see also under "dialysis (for chronic renal failure )." if transplantation had been carried out, see also under "transplantation, kidney." lassa fever is a highly communicable disease and autopsy studies are not recommended in the usually available surround-ings. if lassa fever is suspected, contact the state health department and the centers for disease control and prevention, atlanta, ga, for disposition and further studies ( ).ifan autopsy is done, disinfection can be accomplished by washing instruments with . % phenol in detergent (i.e., lysol), . % hypochlorite solution, formalin, or paracetic acid. for shipping procedures, see chapter . this is not a reportable disease. synonyms: borreliosis; louseborne (epidemic) relapsing fever; tickborne (endemic) relapsing fever. note: (i) collect all tissues that appear to be infected. ( ) rat/mouse inoculation with infected blood is the most sensitive method for the detection of the organism. consult the state health department. ( ) before the autopsy, consultation with the microbiology laboratory is advised. ( ) request direct dark-field examination and giemsa or wright stain. ( ) no special precautions are indicated. ( ) serologic studies are of questionable value due to the antigenic variability of the organism. ( ) fever, scarlet note: usually, this disease is a nonfatal group a streptococcal tonsillitis and pharyngitis with skin rash. potentially fatal complications include suppurative otitis media,* mastoiditis, and pharyngeal abscess ( ), with or without septicemia. ( ) collect all tissues that appear to be infected. ( ) atresia, but may also be seen with viral myocarditis, type ii glycogen storage disease (pompe disease) and carnitine deficiency. thus, a metabolic disorder must be considered. it is ideal to perform the autopsy as soon after death as possible ( ) . urine plasma for removal, prosthetic repair, and specimen preparation, see chapter . malnutrition ( ) muscle necrosis (clostridial myonecrosis) and accumulation of gas; little leukocytic infiltration. other organs g procedures depend on expected findings or grossly identified abnonnalities as listed in right-hand column. see also above under "note" and under "skeletal muscles." synonyms and related terms: acute postinfectious glomerulonephritis (nonstreptococcal postinfectious glomerulonephritis;*minimalchangedisease;mesangialproliferativeglomerulonephritis;* focal and segmental glomerulosclerosis with hyalinosis (focal sclerosis); poststreptococcal glomerulonephritis; idiopathic nephrotic syndrome; iga nephropathy (berger's disease); membranous glomerulonephritis; membranoproliferative glomerulonephritis; mesangial proliferative glomerulonephritis; rapidly progressive glomerulonephritis (associated with systemic infectious or immunologic multisystem diseases; drug idiosyncrasy; or as primary crescentic glomerulonephritis or superimposed on another primary glomerular disease). possible associated conditions: acquired immunodeficiency syndrome (aids);* alport's syndrome;* amyloidosis; anaphylactoid purpura; bee stings; chronic allograft rejection; dennatomyositis;* dennatitis herpetiformis; diabetes mellitus;* drug dependence;*fabry's disease;* goodpasture's syndrome;* guillain-barre syndrome;* henoch-schonlein purpura;* hemolytic uremic syndrome;* infective endocarditis;* leprosy;* malig-nancies;mixedconnectivetissuedisease;myxedema;polyarteritis nodosa;* rheumatoid arthritis;* preeclamptic toxemia; renovascular hypertension; sarcoidosis;* serum sickness;* sjogren's syndrome;*syphilis;*systemiclupuserythematosus;*systemic sclerosis;* thrombotic thrombocytopenic purpura;* thyroiditis;* vasculitis; viral hepatitis;* wegener's granulomatosis;* and many other conditions. urate deposits in scleras and corneas. granulocytopenia note: midline granulomas may belong to the angiocentric immunoproliferative lesions, which are related to lymphomatoid granulomatosis* and malignant lymphoma. the name "idiopathic midline granuloma" should be reserved for the few cases without evidence of malignant lymphoma or wegener's granulomatosis* ( ). the diagnosis of idiopathic midline granuloma also can be ruled out if studies reveal fungal organisms or features of leishmaniasis;* leprosy,* rhinoscleroma, pseudotumor of the orbit or tuberculosis. * complications of nasal cocaine abuse also may mimic midline granuloma ( ). eosinophilic pneumonitis (degenerating eosinophils with charcot-leyden crystals) and granulomas. angiitis (mostly arteritis), typically with giant cells in tunica media ( ) . findings resemble those in polyarteritis nodosa. * heart ( ), grastrointestinal tract ( ), skin, muscles, and joints are commonly involved. however, renal disease is often (but not always) mild or absent. necrotizing vasculitis of small arteries and veins is present, with extravascular granulomas and eosinophilic infiltration of vessels and perivascular tissues. optic neuritis may be found ( ). may be affected by the vasculitis ( ). pcr studies on paraffin sections via rna in situ hybridization may confirm presence of epstein-barr virus-positive b-cell proliferations combined with dense t-cell accumulations ( ) ( ) ( ) . the condition closely resembles angiocentric t-/nk cell lymphoma ( ). infiltration of lymphocytoid cells, plasma cells, and macrophages with necroses and granulomatous features, which are found primarily in the vicinity of blood vessels. special stains may reveal evidence of infection. lymphoreticular and granulomatous infiltrates (see "lung"). lymphoreticular and granulomatous infiltrates (see "lung"). characteristic infiltrates may be present in all organs and tissues. involvement of spleen, lymph nodes, and bone marrow is uncommon. in rare instances, the disease is confined to the abdomen. in most instances, characteristic infiltrates are present ( ). septicemia; circulating immunoglobulin complexes, and antiendothelial antibodies ( ). angiocentric granulomatosis ( ) hemorrhage, intracranial (see under "hematoma, subdural" and under name of suspected underlying condition, such as "aneurysm, cerebral artery," "infarction, cerebral," "injury, head," and "tumor of the brain." hemorrhage, subarachnoid (see under name of suspected underlying condition, such as "aneurysm, cere-bral artery," "infarction, cerebral," "injury, head," and "tumor of the brain." hemosiderosis, idiopathic pulmonary note: this diagnosis is made by exclusion; involvement of organs other than the lungs (see below under "kidneys") suggests another disease. hepatitis, chronic synonyms and related terms: autoimmune hepatitis; chronic viral hepatitis b (with or without d) and c. note: the term "chronic hepatitis" is not a complete etiologic diagnosis and related names such as chronic active (aggressive) hepatitis, chronic active liver disease, and chronic per-sistent hepatitis are obsolete. most cases in these categories represent autoimmune hepatitis or chronic viral hepatitis b or c. many other liver diseases, including drug-induced hepatitis, inborn errors ofmetabolism (e.g., alphal-antitrypsin deficiency* or wilson's disease*), developmental disorders, and chronic biliary diseases such as primary biliary cirrhosis or primary sclerosing cholangitis also may present as chronic hepatitis. ifchronic hepatitis was the cause ofdeath, submassive hepatic necrosis or cirrhosis* is usually present, often with manifestations ofportal hypertension,*hepatic encephalopathy, hepatorenal syndrome,* and with ascites or spontaneous bacterial peritonitis. possible associated conditions: see also below under "possible or expected findings." conditions that may be associated with hepatitis c include ( ): autoimmune hepatitis; behcet's disease; diabetes mellitus (type );* glomerulonephritis;* guillain-barre syndrome;* idiopathic pulmonary fibrosis; idiopathic thrombocytopenic purpura; iga deficiency; lichen planus; mixed essential cryoglobulinemia; mooren's corneal ulcers; polyarthritis; porphyria cutanea tarda;* thyroiditis. * note: coinfection with other hepatitis viruses (e.g., c and g) and/or systemic viruses such as the immunodeficiency virus are common, particularly in drug addicts ( , ) . in many cases of fulminant hepatitis, tests for known hepatitis viruses are negative ( , ) . homicide note: not all of the following procedures can be carried out or will be required in all cases, nor will this checklist be sufficient in all instances. consult also the entries indicating the cause of death, such as "injury, firearm" or "injury, stabbing." if the victim is an infant, see all under "infanticide." before the body arrives or before autopsy is begun: . investigate the scene where the body was found and where the crime may have been committed (these may be two separate locations). if this is not possible, study the report and photographs submitted by the investigator or the police. study all available information. request previous medical records and roentgenograms of the victim. . emphasize to first responders and autopsy personnel that the body of the victim should not be undressed, washed, or otherwise disturbed until it has been inspected by the pathologist. embalming is not permitted before completion of the autopsy. advise first responders or transporters to put paper bags over the hands of the victim. this will protect possible evidence, such as hair from the assailant. . prepare record sheets to document the time of arrival and release of the body; chain of custody for the body and specimens; the name, age, sex, and other information about the victim; the names ofthe technician, pathologist and guests; and the specimens taken. . prepare roentgenographic and photographic equipment. after body arrives but before autopsy is begun: . if the body is left unguarded-for instance, overnight-a lock should be placed on the refrigerator or cool room where the deceased is kept, and the key should be retained by the technician. . if the pathologist is not accustomed to a busy autopsy room, he or she should feel free to ask all persons other than the pathologist(s), technicians, and law enforcement personnel who are not immediately involved in the actual performance of the autopsy to leave the morgue. . the body temperature of the victim can be recorded at the autopsy facility, but this is rarely useful, particularly if the victim seems to have been deadfor longer than a day ortwo or ifthe body had previously been stored in arefrigeratoror cool room. insert thermometer deep into the anus (about - cm). record temperature and manner and time of procedure. . aspirate vitreous from one or both eyes and store the specimen in a refrigerator. . prepare roentgenograms. in each case, a decision must be made whether roentgenograms should be made of the entire body or only of parts of it-or not at all-and whether they should be made before the victim is undressed, after the victim is undressed, or at both times. . take overall survey photographs that depict the anterior surfaces of the body as it is on arrival to the facility, before any undressing, manipulation for roentgenographs, or cleaning. . take close-up photographs of any trace evidence such as fibers or flakes of gunpowder before undressing or cleaning. . photograph the face of the victim for identification purposes after it has been cleaned of any blood and foreign matter. to avoid perspective distortion from wide angle lenses, the lens should be about ft from the face. . photograph all injuries and other forensically significant findings after blood and foreign matter have been cleaned off the body. a moist towel is useful; brushes are too abrasive. two photographs should be taken of each finding. one should include the autopsy number and a scale. a second photograph should be taken without any extraneous objects. collection and documentation of evidence: . fingerprinting can be done before or after the autopsy but should be done in all homicide cases. in cases involving close contact between assailant and victim, fingernail scrapings or clippings (use a new clipper) and hair with roots should be collected, and its source should be identified (hair pulled from scalp, axillae, and pubis is identified as that of the victim; hair in hands or under fingernails or on clothing of victim may be from the assailant). hair exemplars can be collected in cases of homicide by firearm but are rarely of use. if the body is decomposed or mutilated or for any other reason has not been identified, prepare roentgenograms ofthe head, neck, and torso before the autopsy (the radiographic appearance is altered by the autopsy). these can be used for comparison purposes if antemortem films are located. if films of the extremities are needed they can be prepared after the autopsy. dental roentgeno-.grams are taken, usually after the autopsy, when investigators have located antemortem dental records. detailed descriptions and sizes of clothing, with photographs of clothing, can be useful then there is no putative identity. . in cases in which sexual assault may have been involved, collect pieces of clothing that may contain seminal fluid stains. follow procedures described under "rape." . preserve clothing or part of clothing as evidence. wet clothing should be dried before it is stored in labeled and sealed bags. the autopsy: . the measured length and weight, extent of rigor, and color and distribution of livor should be recorded, along with the state of preservation, nutrition, and hydration. do not omit examination of the hands, particularly of the volar surfaces. . if air embolism is suspected, see procedure described under part ii "embolism, air". if pneumothorax is suspected, see procedure described under part ii "pneumothorax". if there is evidence of strangulation or other neck injury, perform a layerwise dissection of the neck that includes the hyoid bone and tongue. . prepare diagrams of wounds and identify their location by anatomic region. for wounds of the torso, state the distance from the soles of a foot and the distance laterally from to the right or left of the midline. . submit samples of wounds for histologic study when there is any question of the age of the wounds. . the records should show when body fluids and tissues were collected for laboratory analysis, the volume and appearance of such specimens, and what was done with them. if most of the toxicology specimens are collected immediately after the internal examination has commenced, the time of the internal examination serves as the collection time. in all instances, the following items should be collected: blood ofvictim for dna comparison and toxicologic study (determination of alcohol, carbon monoxide, and drug concentrations will be requested most frequently); vitreous; urine; gastric contents; at least one solid organ specimen for toxicologic study (liver and brain have the most comparison data if an extracerebral congenital malformation is suspected, follow procedures described under "malformation, arnold-chiari." if cerebrospinal fluid is aspirated with a syringe, record volume. record weight of brain; record size of brain in relation to inner dimensions of skull. describe size of ventricles. other procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. if a surgical shunt is present, its location should be recorded and both ends of the implanted specimen that was used for shunting may be submitted for microbiologic study. if the shunt is not patent, record site and nature of obstruction. related terms: antibody-mediated hydrops fetalis; erythroblastosis fetalis;* nonimmune hydrops fetalis. note: the classic example of antibody mediated (rh incompatibility) hydrops fetalis is hemolytic disease of the newborn (erythroblastosis fetalis*). however, nonimmune hydrops fetalis also may be caused by hematologic disorders, e.g., alpha-thalassemia* ( ) or it may have no known cause. infec-tions, e.g., with human parvovirus bl ( ), cytomegalovirus, or syphilis;* heart and vascular diseases ( ) (cardiac tumors, cardi-omyopathy,* myocarditis,* arterial calcification, and others); storage disease ( ); tumors (including neonatal leukemia*); and many other fetal (e.g., congenital chylothorax* or lymphatic dysplasia; pulmonary sequestration and cystic adenomatoid malformation) or maternal conditions, e.g., maternal thyrotoxicosis, also may cause nonimmune hydrops fetalis. if coronary insufficiency or myocardial infarction is suspected, follow procedures described under "disease, ischemic heart." for coronary arteriography, see chapter to. record distribution of atherosclerotic lesions in aorta. samples for histologic study should include aorta, coronary arteries, and peripheral arteries. see also above under "heart." submit samples of head, corpus, and tail for histologic study. if applicable, see also under "diabetes mellitus." for special procedures and stains, see above under "heart." other procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. for removal and specimen preparation, see chapter . obesity* is a common finding. eruptive xanthomas (palms, elbows, knees) in some but not all types of hyperlipoproteinemia. most prominent in apoprotein cii deficiency. xanthomas of tendons (knees, elbows, dorsum of hands), xanthelasmas, and arcus comeae in familial hypercholesterolemia. gangrene of lower extremities (see below under "arteries"). severe coronary atherosclerosis and myocardial infarcts in type hyperlipoproteinemia and multiple lipoprotein-type hyperlipidemia. atherosclerosis of abdominal aorta and its branches and of carotid arteries. coronary atherosclerosis (see above). pancreatitis* in familial apoprotein cii deficiency. foam cells with triglycerides in liver, spleen, and bone marrow in familial lipoprotein lipase deficiency. manifestations of diabetes mellitus* and hypothyroidism* in some cases of type hyperlipoproteinemia. cerebral infarct (stroke*) in type hyperlipoproteinemia. hypernatremia (see "disorder, electrolyte(s).") hyperoxaluria synonyms and related terms: oxalosis; primary hyperoxaluria type i (a anineglyoxylate aminotransferase deficiency); primary hyperoxaluria type ii (d-glyceric acid dehydrogenase deficiency); secondary hyperoxaluria (see under "note"). note: in ethylene glycol poisoning,*oxalatecrystals in media ofsmall arteries, with associated ischemic lesions. similardeposits may occur after long-term hemodialysis ( ). these conditions must be distinguished from the genetic disease. also, oxa-iate nephropathy may be a complication of short bowel syndrome. if patient with congenital hyperoxaluria underwent liver or combined liverlkidney transplantation ( ) , see also under these headings. remove vitreous for biochemical evalvation. submit tissue (i.e., fascia lata) for karyotype analysis. polycystic ovaries, testicular adrenal rests ( note: there are no diagnostic autopsy findings for hypoxia. possible causes of acute hypoxia include anesthesia-associated death,' compression of the torso by a vehicle, diving accident,' exposure to toxic gases-forinstance, carbon monoxide poisoning;' mechanical failure ofan oxygen supply system, as in an airplane; and sudden mechanical airway obstruction,' as in aspiration ofa foreign body orstrangulation. causes ofchronic asphyxia include prolonged exposure to high altitude and chronic pulmonary disease. profound medial hypertrophy of small pulmonary arteries and of pulmonary veins from chronic exposure to hypoxia. acute pulmonary edema may also occur in chronic hypoxia. procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. malnutrition. empty and collapsed colon; small amounts of gray and dry meconium in terminal ileum; meconium masses in dilated and hypertrophied mid-ileum; liquid contents in proximal intestine ( ); appendicitis; bowel perforation. glandular inspissation and atrophy of intestinal mucosa. volvulus, infarction, necrosis, perforation, peritonitis, and acquired intestinal atresia may be present. absence of neural plexuses in congential megacolon* (hirschsprung's disease). manifestations of cystic fibrosis, with or without cirrhosis;* biliary atresia* ( ). immunodeficiency (see "syndrome, acquired immunodeficiency" and "syndrome, primary immunodeficiency.") impression, basilar note: basilar impression constitutes an upward bulging of the margins of the foramen magnum. when occipital condyles are displaced above the plane of the foramen magnum, basilar invagination is present. possible associated conditions: klippel-feil syndrome;* osteogenesis imperfecta;* osteomalacia;* paget's disease of bone;* rheumatoid arthritis; rickets; syringobulbia; syringomylia. * compression and secondary ischemic injury to lower brain stem and spinal cord. see also above under "skull and spine" and under "possible associated conditions." incompetence,••• (see "insufficiency,..•") related terms: battered-child syndrome; child-abuse or child-neglect death. note: vitreous should not be aspirated until the skull has been opened and trauma ruled out, because there is a risk of artifactual damage to the retina. in the presence of craniocerebral trauma the pathologist can opt to examine the eyes by removal, fixation, and histologic study; or retinal photography. follow general procedures described under "homicide" and, if necessary,. the procedures described under "rape." external examination see above under "note." prepare roentgenograms of entire body. see above under "note." in other situations, or after study of the fundus, submit sample of vitreous for chemical and possible toxicologic study. umbilical cord attachment prepare histologic sections of umbilicus. and end of umbilical cord blood submit sample for toxicologic study. consider retaining dried blood on filter paper for possible dna testing for paternity investigation. if there is a possibility that the cadaver represents a stillbirth, see part ii, "stillbirth". if infant is thought to have died shortly after birth, determine the location of air in the intestinal tract; roentgenograms may be helpful. save stomach contents and record amount. other organs see also under "homicide." in the abused child, bruises, hematomas, burn marks, or other patterned injuries. in any child, diaper rash, mongolian spots, self-inflicted fingernail scratches, skin infections, and emaciation. in the previously battered child, fractures in various states of healing. in hypertonic dehydration,' sodium concentrations more than meq/l. this may be caused by organic disease, improper medical treatment, or physical neglect. if infant was born alive, inflammatory changes may be found, depending on survival interval. for the hydrostatic lung test, see "stillbirth." the test is unreliable. extraneous material in air passages indicates that child was alive. air reaches the stomach after min, the small intestine after - h, the colon after - h, and the rectum after l h. there is no difference in the speed of gas propulsion between full-term and premature infants. bacterial gas production and previous resuscitation attempts are potential sources of errors. in questionable stillbirth, presence of milk proves that infant was alive and had been nursed. traumatic lesions and signs of neglect may be present. unilateral ulcers in visceral zoster. in rare instances, diffuse meningoencephalitis may occur. limited necrotic and inflammatory lesions in the cord or brain stem at the level of the affected ganglion are common. posterior hom myelitis ( ). ganglion cell necrosis; lymphocytic infiltration, hemorrhage, and, later, fibrosis. as a rule, only one ganglion is severely involved, but less severe lesions may occur in the ganglia that are immediately adjacent. diffuse lymphocytic infiltration; demyelination; axonal destruction; fibrosis. conjunctivitis; keratitis; iridocyclitis; retrobulbar neuritis; neuroretinitis; occlusion of retinal vessels. evidence of hematologic malignancies ( ) or other conditions, as listed above under "note." infection, middle ear (see "otitis media.") infection, pneumocystis carinii synonym: pneumocystosis. note: ( ) collect all tissues that appear to be infected. ( ) this organism cannot be cultured at present but is frequently associated with viral, bacterial, or fungal infections that can be diagnosed by culture. ( ) for rapid staining of aspirates, use gram-weigert stain, which will stain cysts of pneumocystis carinii and also fungi and bacteria. pneumocystis carinii is best demonstrated with grocott's methenamine silver stain. ( ) no special precautions are indicated. ( ) usually, no serologic studies are available. ( ) this is not a reportable disease. injury, fire (see "burns.") injury, firearm note: protect all drains of autopsy table, which will prevent accidental loss of bullets or bullet fragments. recovery of bullet fragments and pellets can also be improved by passing tissue fragments, blood, and other appropriate materials through a fine nonmetallic sieve. caution must be exercised because sharp edges andjagged projections ofbullets and bullet fragments may cause injury ( ). this applies particularly to the black talon bullet. bullets and bullet fragments should not be touched with forceps or other metal instruments that may produce artifactual markings; they must be placed in properly labeled evidence containers, and the chain of custody must be preserved. from a birdshot wound, at least pellets should be recovered. the firearms examiner will divide the total pellet weight by and derive median pellet weight. from a buckshot wound, all pellets should be recovered. in many of these cases, procedures described under "homicide" must also be followed. do not excise wounds before completion of autopsy (see below). as an academic exercise, excised firearm wounds may be used later for analysis of metal traces by neutron activation or for tests for carboxyhemoglobin. in practice, however, a high quality photograph is adequate to establish the presence or absence of gunpowder stippling or soot deposition toward the end of establishing the range of fire. the dimensions of the stippled areas and soot deposits should be recorded. after completion of external examination, dry the wet clothing and keep as evidence. clothing may also be used for possible test firing or for stain, powder, or particle analysis. external examination if identity of victim is unknown, follow procedures described in chapter . prepare initial roentgenograms of the body regions that have been shot, before disrobing of body, and then lateral films of body regions with bullets. follow up with films of other body regions as needed in the course of autopsy. if the body is decomposed to the extent that the external examination cannot be relied upon to determine cutaneous gunshot perforations, prepare roentgenograms of the entire body before autopsy. record location and number of bullet holes in all layers of garments, indicating whether the involved area is bloodstained or shows gunpowder or soot. if there are several cutaneous perforations, number or letter each consecutively and refer to these numbers in all records. location of bullet holes should be described by recording distance from soles of feet or top of head, and from midline of body. prepare diagrams and photograph holes, with and without labels. photographs should show surrounding garments and extent of blood stains. in hairy areas, shave around bullet wounds for better photographic documentation. for evaluation of distance from where a shot had been fired, see fig. additional roentgenograms during the autopsy may be needed to find bullets embolized to the femoral veins or down the spinal canal. systemic roentgenograms may reveal bullets from obscure entrance wounds, particularly in decomposed bodies, old bullets, bullets in the skin flaps reflected at autopsy, or bullets external to the body in clothing. bullet(s) may have been arrested in hollow viscus or blood vessel and may have been transported to distant sites by peristalsis or bloodstream ("bullet embolus"). bullets may be deflected in unexpected directions from bony surfaces. soot ("smudging") and gunpowder stippling may occur around entrance wound, depending on the muzzle-to-skin distance. there may also be an impression from a recoiling handgun or from a power piston. wounds may be stellate, round, jagged, or slit-like, with or without wide margins of abrasion. primer residues on hand of suicide victim after use of a handgun. fatal secondary injuries, particularly hemorrhages. alcohol intoxication is a frequent finding. fig. · . bullet wounds. differences in the appearance of cutaneous wounds of entrance and of exit and differences in wounds of entrance according to the distance between muzzle and skin at the moment of fire. entrance wounds often differ from exit wounds in that the former are usually surrounded by a narrow zone of abrasion. if the muzzle of a gun is in close contact with the skin at the moment of fire, the combustion products will be blown into the wound and will not be visible on the surface. for close-range wounds, the stippling of the skin around the wound, produced by particles of burned and unburned powder, becomes progressively more dispersed as the range of fire increases and is ordinarily not perceptible if the range has been greater than inches ( synonyms and related terms: acute radiation syndrome; chronic (delayed) radiation injury; radiation enterocolitis; radiation nephritis; radiation pneumonitis. note: procedures and expected findings depend on type of radiation damage, whether acute or chronic, localized or whole-body irradiation. if suspected radiation injury was associated with the administration of radionuclides such as p, , or au, follow procedures suggested in chapter . in fatal whole-body irradiation, findings are most likely related to bone marrow injury, and the suggested procedures and expected findings are those described under "pancytopenia." record extent of oropharyngeal and intestinal ulcerations and hemorrhages. late complications include malignant tumors (carcinoma, leukemia,* lymphoma*), manifestations of hypothyroidism,* and cataracts. organ changes in localized radiation injury depend on site of irradiation (lung, brain, kidney, intestine). the skin is involved in both acute (erythema) and chronic (atrophy, epilation) radiation injury. related terms: cutting injury; knife injury. note: in many of these cases, procedures described under "homicide" must also be followed. insuffiency, adrenal synonyms and related terms: adrenocortical insufficiency; polyglandular autoimmune syndrome (type i, usually in childhood, with parathyroid insufficiency and chronic mucocutaneous moniliasis; type ii, usually in adults, with two or more autoimmune endocrine disorders such as thyroiditis and diabetes mellitus*); primary adrenal insufficiency (addison's disease); secondary adrenal insufficiency (in hypothalamic-pituitary disease or steroid induced); x-linked adrenal hypoplasia. is caused by anatomic changes (see below under "adrenal glands"), drugs (enzyme inhibitors or cytotoxic drugs), or acth-blocking antibodies. possible associated conditions: aids with systemic infections ( ); antiphospholipidantibody syndrome ( ); autoimmune hepatitis; chronic lymphocytic thyroiditis; type i diabetes mellitus;* hypoparathyroidism;* hypothyroidism;* megaloblastic anemia;* surgical procedures, such as heart surgery or orthopedic procedures. large cell lymphoma ( ). should be removed as soon as possible (before embalming), either from cervical midline incision or from chest (neck of cadaver must be well-extended). photograph obstruction before larynx is opened, and inside of larynx and epiglottis after larynx is is opened in posterior midline. make gram-stained touch preparations. make histologic sections of larynx and epiglottis. hemophilus injluenzae (cannot always be isolated from larynx synonyms and related terms: acute or chronic lymphocytic leukemia, acute or chronic myelogenous leukemia, and hairy cell leukemia. multiple subtypes have been identified; they are characterized by cell surface markers, chromosomal abnormalities, staining reactions, and morphologic features. note: at the time of autopsy, most leukemias have been properly classified and often treated. in these cases, the goal of the autopsy is to document the extent of the disease and the presence of complications. if the leukemia had not been classified or if the features might have changed from the time of the last work-up (e.g., in suspected cases of superimposed diffuse large cell lymphoma [richter's syndrome]), material should be snap-frozen and studied in more detail. if the patient was treated by bone marrow transplantation,* see also under that heading. possible associated conditions: agnogenic myeloid metaplasia with myelofibrosis; bloom's syndrome;* cutaneous mastocytosis; down's syndrome;* immunodeficiency syndromes* (bruton's disease; louis-bar syndrome; wiskott-aldrich syndrome); infantile genetic agranulocytosis; klinefelter's syndrome;* lymphoma;* multiple myeloma;* polycythemia;* and many others. for sampling and specimen preparation, see chapter . increased protein concentration. material in sediment stains red with toluidine blue. metachromatic material. arylsulfatase-a deficiency. excessive loss of myelin with large amounts of metachromatic material (see above under "urine") in white matter and also in some neurons. demyelination with metachromatic material (see above under "urine"). leukoencephalopathy, progressive multifocal possible associated conditions: aids* and other immunosuppressed states; carcinoma; malignant myeloproliferative or lymphoproliferative disorder; sarcoidosis;* tuberculosis. * procedures submit portion of fresh brain for viral culture. fix remainder of brain and spinal cord in formalin and submit samples for histologic study. in situ hybridization for ic virus is available on paraffin-embedded tissue. small patches of demyelination with tendency to form confluent areas in the cerebral white matter. white matter necrosis may be present. eosinophilic intranuclear inclusions occur in affected oligodendroglia cells. subsequently, large, bizarre astrocytes develop. no inflammatory (lymphocyte) cell reaction is present. lightning (see "injury, lightning.") lipoproteinemia (see "hyperlipoproteinemia.") lipoproteinosis, pulmonary alveolar synonym: idiopathic alveolar lipoproteinosis. note: the condition has been described in allografts ( ); in such cases, see also under "transplantation, lung." elephantiasis of penis, scrotum, or vulva (in chronic cases); skin rash (l) and conjunctivitis (in acute cases); genital ulcers. suppurative or fibrosing inguinal, iliac, or pelvic lymphadenitis, with or without sinus tracts. rectal strictures and fistulas in chronic cases ( ) . systemic involvement in the acute stage with pericarditis,* and arthritis,* and meningitis,* proctitis ( ) lymphoma synonyms and related terms: aids-related lymphoma; adult t-cellieukemiallymphoma; angioimmunoblastic lymphadenopathy; b-celllymphoma; burkitt's lymphoma; cutaneous t-cell lymphoma (includes mycosis fungoides and sezary syndrome); hodgkin's disease; non-hodgkin's lymphoma (low grade, intermediate grade, high grade, all with multiple subtypes too numerous to list, which vary in natural history); natural killer cell neoplasms; post-transplant lymphoproliferative disorders (ptld); t-cell lymphoma. note: at the time of autopsy, most lymphomas have been properly classified and often treated. in these cases, the goal of the autopsy is to document the extent ofthe disease and the presence of complications. if the lymphoma had not been classified or if the features might have changed from the time of the last work-up, material should be snap-frozen and studied in more detail. if the patient was treated by bone marrow transplantation, * see also under that heading. for current terminologies of hodgkin's disease and non-hodgkin lymphomas as well as for staging criteria, appropriate hematologic textbooks should be consulted. possible associated conditions: acquired immunodeficiency diseases (e.g., after organ transplantation; human immunodeficiency virus-l infection); autoimmune disease (e.g., celiac sprue,*rheumatoid arthritis,* systemic lupus erythematosus,* or sjogren's syndrome*); chemotherapy with or without radiation treatment; epstein-barr virus infection; human t-cell leukemia virus infection; inherited diseases with immunodeficiency (e.g., klinefelter syndrome; * common variable immunodeficiency disease, wiskott-aldrich syndrome); radiation. the lesions are found most commonly in the urinary bladder and other parts of the urinary tract, but may also occur at many other sites, including skin ( ) and upper respiratory tract ( ). note: (i) collect all tissues that appear to be infected. ( ) usually, cultures are not indicated. ( ) request giernsa or wright stain. tissue blocks should be rinsed of blood and be as thin as possible before fixation in refrigerated buffered and neutral % formalin solution. this is to avoid precipitationofformalin pigment that may be confused with malaria pigment. the formalin solution should be used in a ratio of parts formalin to one part tissue and should be agitated every few hours. if one is dealing with tissues that contain formalin pigment, this pigment may be removed with a bleaching solution. ( ) usually, no special precautions are indicated. ( ) possible associated conditions: histoplasmosis* and other chronic fungal infections; immune connective tissue diseases such as rheumatoid arthritis* (l) ; malignancies (l); sarcoidosis;* silicosis;* tuberculosis* (l) . note record location and extent of skin changes or skin defects on back. prepare skeletal roentgenograms. if meningitis is suspected, submit sample of cerebrospinal fluid for culture. dse the posterior approach to remove the spinal cord. atrophic skin over meningocele, lacking rete pegs and skin appendages; skin defect in complete rachischisis. bony defects of spine. in meningocele and spina bifida occulta, arachnoid and dura herniate through the vertebral defect. spinal cord and roots are generally not involved. lumbosacral mass in meningomyelocele, with a highly vascular mass (area medullovasculosa) in spina bifida aperta. neural defects in complete rachischisis. diastematomyelia, hydrocephalus ( ) . pyelonephritis;* enlarged urinary bladder ("neurogenic bladder"). external examination heart lungs procedures prepare chest roentgenogram. procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. record weights. prepare photographs and roentgenograms of fresh and fixed lung specimens. perfuse one lung with formalin. for preparation of paper-mounted sections, see chapter . submit one lobe for microbiologic and chemical study. decalcify tissue for histologic study. request van gieson's, hale's colloidal iron, pas, and sudan stains. miliary mottling in lower lung fields. mitral stenosis* may be a cause of microlithiasis (mostly intra-alveolar ossification). * cor pulmonale and heart failure ( ) (l) , see also under that heading. ( ) collect all tissues that appear to be infected. ( ) viral isolation, especially with ebv, is not diagnostically useful, due to long incubation periods. ( ) note: these diseases are characterized by a deficiency of a variety of hydrolases, resulting in accumulation of gly-cosaminoglycans (mucopolysaccharides) and glycolipids within lysosomes of fibroblasts, macrophages, white cells, and parenchymal cells of many organs ( , ) . mucopolysaccharides are also excreted in the urine. the general approach is similar in all types. formalin may dissolve all of the stored material and leave empty vacuoles in the involved cells. therefore, frozen sections should be utilized or tissues should be fixed in absolute alcohol. the accumulated material will show intense metachromasia if stained with toluidine blue. it will also stain with pas, alcian blue, and colloidal iron. oil red a will also stain the material from frozen sections. for specimen preparation for elec-tron microscopy, see chapter is. characteristic external features include dwarfism; thickened long bones; coarse facial features; coarse hair; macrocephaly; prognathism; hypertelorism; malformed teeth, and scaphocephalic skull with hyperostosis of sagittal suture; short neck; chest deformity; umbilical and inguinal hernias; lower thoracic and lumbar gibbus; genu valgum and coxa valga; pes planus and other joint deformities; wide hands (clawhands) and feet. coarse thickened skin, covered with lanugo-like hair. hyperlordosis; ovoid deformities of vertebrae; odontoid hypoplasia; large, shoe-shaped sella turcica; kyphosis; hypoplasia of femoral heads; osteoporosis.* if patient underwent bone marrow transplantation ( ), see also under that heading. chronic upper respiratory infections. large cytoplasmic granules in neutrophils. storage of mucopolysaccharides in osteocytes, chondrocytes, and fibroblasts of periosteum, tendons, fasciae, and other connective tissues; dysostosis multiplex. other tissues histologic sampling cannot be excessive. ( ) collect all tissues that appear to be infected. ( ), see also under that heading. blood; other organs and tissues record extent of skin lesions and prepare photographs; prepare sections of affected and unaffected skin. request gram stain of sections and smears. submit samples of blood and grossly affected organs or tissues for microbiologic study. other procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. extensive epidermal necrosis. shedding of granular and horny layers of epidermis. septicemia; staphylococcal infection of nose, throat, ears, eyes, heart valves, urogenital tract, and other sites. bronchial epithelial detachment and bacterial pneumonia ( ). synonyms and related terms: acute kidney failure; *acute tubular necrosis; lower nephron nephrosis. note: the morphologic diagnosis of this condition may be difficult to discern from autolysis. the features that suggest tubular necrosis are: tubular dilation with epithelial flattening, intertubular edema and necrotic epithelial cells in the collecting ducts. the autopsy should be performed as soon as possible. needle specimens of the kidneys obtained in the immediate postmortem period may yield acceptable material. ifnephrotoxic drugs or chemicals are thought to be responsible for tubular necrosis, submit samples for toxicologic study (see also under synonyms and related terms: multiple endocrine neoplasia (men), type (parathyroid hyperplasia or adenoma; pancreatic islet cell hyperplasia, adenoma, or carcinoma; pituitary hyperplasia or adenoma); or type a (medullary thyroid carcinoma, parathyroid hyperplasia or adenoma; and pheochromocytoma); or type b (medullary thyroid carcinoma, pheochromocytoma, mucosal and gastrointestinal neuromas, and marfanoid features). note: in men, type , foregut carcinoids and subcutaneous and visceral lipomas also may be found. in type a, cutaneous lichen amyloidosis may be observed. mixed syndromes include (i) familial pheochromocytoma and islet cell tumor, ( ) von hippel-lindau syndrome, pheochromocytoma and islet cell tumor, ( ) neurofibromatosis* with features of men or , and myxomas, spotty skin pigmentation, and generalized endocrine overactivity (carney complex). in all instances, the autopsy should be done as soon as possible so that tissues for biochemical study can be frozen without delay. nephritis note: see under specific designation, such as "glomerulonephritis" and "pyelonephritis," or under name of suspected underlying condition, such as "gout" or "lupus erythematosus, systemic." nephroblastoma (see "tumor of the kidney(s).") synonyms and related terms: renal stones; urolithiasis. possible associated conditions: carcinomatosis; cushing's syndrome;* cystinuria;* fanconi syndrome;* hyperoxaluria;* hypervitaminosis d;* gout;* multiple myeloma;* osteoporosis;* polycystic renal disease;* primary hyperparathyroidism;* rheumatoid arthritis* ( ); sarcoidosis* ( ). kidneys ureters, urinary bladder, and urethra other organs remove kidneys, ureters, and urinary bladder en bloc. excise and bivalve kidneys to reveal renal pelves. open ureters. record size, number, and appearance of stones, and save for chemical analysis ( ). record heart weight. dissect and record weights of all parathyroid glands. other procedures depend on suspected underlying conditions, as listed above under "possible associated conditions." nephropathy note: see under name of suspected underlying condition, such as "diabetes mellitus," "disorder, electrolyte(s)" (hypercalcemia, potassium depletion), "gout," "hypertension (arterial), all types or type unspecified," or "poisoning,.. ." (heavy metal). if kidney failure was present, procedures under that heading should also be followed. renal tissue may need decalcification or fixation in water-free solution. ( ) note: in this condition, lesions in the brain and cranial nerves, spinal cord, and spinal roots may be schwannomas (including bilateral vestibular schwannomas); multiple meningiomas; gliomas (generally of spinal cord), mostly ependymomas ( %) and pilocytic astrocytomas; or schwannosis of spinal dorsal root entry zones. intracortical meningioangiomatosis; glial hamartia (intracortical, basal ganglia, thalamus, cerebellum, and dorsal horns of spinal cord) and cerebral calcifications also may be found. cutaneous abscess (j). acute necrotizing nocardial pneumonia with abscesses or sinus formation into surrounding tissues; empyema. pulmonary and mediastinallymph nodes other organs submit samples for culture and histologic study. submit samples from all organs and tissues with suspicious gross lesions for culture and histologic study. poorly encapsulated abscesses of any organ; endocarditis ( ) . related term: total parenteral nutrition. note: air embolism* or blood loss may have occurred if line became detached from the catheter hub. metabolic complications such as fluid overload or disturbances of acid-base and electrolyte balance often cannot be diagnosed reliably at autopsy. the disease(s) that may have necessitated parenteral nutrition therapy are not considered here; they include the acquired immunodeficiency syndrome (aids);* cancer cachexia; inflammatory bowel disease; liver or kidney failure;* severe pancreatitis;* short bowel syndrome, and others. obstruction, acute airway synonyms and related terms: aspiration; bolus death; "cafe coronary"; croup; restaurant death. note: ifpermission has been obtained, remove neck organs through straight incision from chest to chin to avoid dislodging a foreign body. ifdissection has to be accomplished from chest, neck of unembalmed cadaver should remain well extended during procedure. inspect larynx and trachea from above and below, respectively, before opening them carefully along the posterior midline. for removal, prosthetic repair, and specimen preparation, see chapter . submit samples of osteocartilaginous and synovial tissues for histologic study; sagittal or frontal saw section through spine provides for best routine evaluation. heberden's nodes at interphalangeal joints of fingers. degenerative changes, primarily of spine, hip joints, and knee joints. histologic and macroscopic degeneration of cartilage; exposure of subchrondral bone; formation of marginal osteophytes; bone cysts; synovial fibrosis; hypertrophic synovitis. o procedures if artificial joints (e.g., hip or knee) had been implanted, record state of implant site. loose joint prostheses. synonyms: hypertrophic pulmonary osteoarthropathy; pac-hydermoperiostosis (idiopathic hypertrophic osteoarthropathy [ j). note: in all instances, an underlying disease must be identified; they include cardiac disease (cyanotic congenital heart dis-ease with right-to-left shunt; infective endocarditis*); gastroeso-phageal reflux ( ); neoplasms of lungs, esophagus, intestine, and liver; chronic liver disease; inflammatory bowel disease;* pulmonary disease (e.g., abscess;* bronchiectasis;* cystic fibrosis;* emp-yema;* emphysema;* lipoid pneumonia* ( ); pneumocystis pneumonia; sarcoidosis;* tuberculosis*); hyperthyroidism. * external examination elastic arteries other organs prepare roentgenograms of extremities. for removal, prosthetic repair, and specimen preparation, see chapter . record presence of aneurysms (thoracic aorta, subclavian) or arteriovenous fistula of brachial vessels. if abdominal aortic aneurysm had been surgically repaired, search for evidence of graft infection ( ) . procedures depend on expected findings or grossly identified abnormalities as listed above under "note." clubbing of fingers or toes (see below under "elastic arteries"); swelling of extremities. periosteal new bone formation in distal shafts of bones of forearms and legs. all bones of extremities may be involved. see above under "external examination." unilateral clubbing. clubbing of toes but not of fingers. swelling; fistulas. bone defect(s) and focal osteosclerosis. bacterial or fungal infections, most common in metaphyseal region of bones; paravertebral or psoas abscess in osteomyelitis of spine. bacterial or fungal osteomyelitis, with or without cavitation and fistulas. trauma, surgery, insertion of prosthesis, generalized ( ) or contiguous infection, inflammatory bowel disease ( ), diabetes mellitus,* and peripheral vascular diseases, deep vein thrombosis ( ). synonyms and related terms: aseptic necrosis of bone; avascular necrosis of bone; idiopathic osteonecrosis; perthes' dis-ease; postfracture osteonecrosis; renal transplant associated osteonecrosis. note: possible underlying conditions include chronic alcoholism,* decompression sickness,* diseases that have been treated with high doses of corticosteroids ( ), gaucher's disease,* human immunodeficiency virus infection* ( ), tuberculosis* ( ), sickle cell disease, * systemic lupus erythematosus,* tuberculosis* ( ), and bisphosphonate therapy ( ). other organs submit sample for biochemical study. for removal, prosthetic repair, and specimen preparation, see chapter . if osteonecrosis is suspected because one of the possible underlying conditions (see above) is present, prepare saw sections through femoral heads, medial femoral condyles, and heads of humeri. procedures depend on suspected underlying conditions, as listed above under "note." osteopetrosis synonymsand related terms: albers-schonberg disease; autosomal-dominantosteopetrosis; carbonic anhydrase-it deficiency; malignant infantile osteopetrosis ( ); marble bone disease. note: manifestations of renal tubular acidosis may have been a clinical complication. possible associated conditions: bone marrow transplantation* (for infantile osteopetrosis). anemia, recurrent infections, bleeding, and bruises may have resulted from myelophthisis associated with osteopetrosis. upper airway obstruction in malignant infantile osteopetrosis may have necessitated tracheostomy ( ). in heritable osteoporotic disorders of connective tissue (osteogenesis imperfecta,* marfan' s syndrome,* and others) are pre-sented separately. for"osteomalacia," see above. for "osteodystrophy," see under "failure, kidney." possible associated conditions: acromegaly;* chronic alco-holism;* chronic obstructive pulmonary disease; chronic kidney failure* ( ); cushing's syndrome;* debilitating disease (various kinds, often with immobilization); diabetes mellitus* ( ); epilepsy;* hyperthyroidism ( );* hypogonadism; malabsorption syndrome;* malnutrition;* primary biliary cirrhosis;*rheu-matoid arthritis;* scurvy;* steroid therapy or anticonvulsant medication. normal parathyroid glands in uncomplicated cases. hyperparathyroidism* ( ) (without osteitis fibrosa) may be present, however. features of osteitis fibrosa (osteoclastic osteoporosis; see "hyperparathyroidism") or osteomalacia* exclude the diagnosis of uncomplicated osteoporosis. osteoporosis, which may be localized, occurs in various neoplastic (e.g., mastocytosis) and inflammatory diseases. external examination brain and base of skull with middle ears examine external ear canal. for removal and specimen preparation, see chapter . culture any lesion appearing to be infectious. draining, foul-smelling greasy material associated with acquired cholesteatoma. bacterial or viral infection, with or without mastoid osteitis. neck abscess, sinus thrombosis ( ) and brain abscess* and meningitis* may complicate otitis media. otosclerosis ( , ) note: otosclerosis may be a measles-virus-associated disease ( ) and therefore, studies to rule out a past infection may be indicated. for removal and specimen preparation, see chapter . for current methods of temporal bone studies, see ref. ( ) . spongy bone in the capsule of the labyrinth. trabeculae of woven bone show pagetoid changes. if stapedectomy had been done, a prosthesis may be in place. brain is externally normal or mildly atrophic. characteristic is midbrain atrophy with aqueduct dilatation and depigmentation of the substantia nigra. neuronal loss with reactive gliosis, associated with neurofibrillary tangles (globose tangles). primarily affected are globus pallidus, subthalamic nucleus, red nucleus, substantia nigra, tectum, periaqueductal gray matter, and dentate nucleus (grumose degeneration). palsy, pseudobulbar (see "disease, motor neuron.") related terms: acute pancreatitis; alcoholic pancreatitis; chronic (or chronic fibrosing) pancreatitis; interstitial pancreatitis. note: some causes of pancreatitis such as adverse drug reactions (e.g., due to azathioprine, furosemide, or estrogens) cannot be diagnosed at autopsy. possible associated conditions: acute fatty liver of pregnancy; apolipoprotein cli deficiency; cystic fibrosis; *hyperparathyroidism;* kidney transplantation. * status post endoscopic retrograde cholangiopancreatography. synonyms and related terms: calcifying panniculitis; histiocytic cytophagic panniculitis; mesenteric panniculitis (mes-enteric lipodystrophy); "sclerema neonatorum." note: the term weber-christian disease described systemic panniculitis with fever, bleeding, pulmonary and pancreatic lesions, and other abnormalities also involving lungs and pancreas, among others. however, this condition is not a specific entity and thus, the name has become obsolete. the term histiocytic cytophagic panniculitis is more descriptive and preferred. leukemia* and subcutaneous t-celllymphoma* may closely simulate panniculitis. possible associated conditions: alphaj-antitrypsin deficiency;* dermatomyositis;* rheumatoid arthritis;* scleroderma and morphea; sjogren's syndrome;* systemic lupus erythematosus.* external examination, skin, subcutaneous tissue, and breasts chest heart lungs liver and spleen record extent and character of skin lesions. record size, location, and gross appearance of subcutaneous nodules. submit tissue samples for histologic study of grossly unaffected skin, skin lesions, and subcutaneous nodules. request verhoeff-van gieson, gram, and grocott's methenamine silver stains. request s-l protein stain. ascertain that cell infiltrates are not leukemic or lymphomatous ( ) . dimpling of skin; necroses; fistulas. panniculitis with subcutaneous nodules in trunk, breasts, and thighs ( ) . calcification may occur in breast tissue but also at other sites (e.g., in kidney failure*). pancreatic enzymeinduced fat necroses associated with severe pancreatitis. widespread hardening of fat tissue with rupture of fat cells and crystal formation in "sclerema neonatorum." focal traumatic panniculitis also may occur in newborns. s-ioo stain negative in panniculitis but positive in rosai-dorfman disease (sinus histiocytosis with massive lymphadenopathy). mediastinal panniculitis. pericarditis;* interstitial myocarditis. * pneumonia;* pleuritis. fat embolism. * features of alphal-antitrypsin deficiency* ( ) . fatty changes of liver; splenitis. intestinal mucosal erosions and ulcers, with or without perforation. massive gastrointestinal hemorrhage in histiocytic cytophagic panniculitis. blind intestinal loop ( ). for sampling and specimen preparation, see chapter . prepare samples for electron microscopy. vacuolar myopathy. ( ) . external examination, skin, and mouth record extent of skin lesions; photograph; submit multiple samples for histologic study, preferably of areas that are free of secondary infection. prepare sections for immunofluorescent staining. bullous and other lesions of scalp, eyelids, nose, axillae, umbilicus, inframammary areas, back, hands, groins, genitalia, anus, knees, and feet. similar lesions may occur in the mouth. acantholysis is suprabasal or near granular layer. igg deposits on the surfaces of keratinocytes. note: (i) collect all tissues that appear to be infected. collect inguinal, popliteal, axillary, and supraclavicular lymph nodes for aerobic culture. photograph enlarged lymph nodes, and prepare smears of fresh cut surfaces. submit consolidated areas for microbiologic study (see above under "note"). perfuse both lungs with formalin. submit samples of pulmonary tissue and bronchial lymph nodes for histologic study. submit samples of grossly abnormal organs, exudates, or drainage fluids for culture and gram stain. hemorrhagic necrosis; variable suppuration. severe hemorrhagic edema; pneumonia with lobular to lobar features. large areas of necrosis teeming with organisms and minimal to suppurative inflammation. plasmacytoma (see "myeloma, multiple.") platybasia note: possible causes or associated conditions include arnold-chiari malformation;* basilar impression* orinvagination; fusion ofatlas to the foramen magnum; klippel-feil syndrome;* malpositioning of odontoid process; osteitis deformans (paget's disease of bone*); osteogenesis imperfecta;* osteomalacia;* rickets; syringobulbia, syringomyelia. * skull and spine prepare roentgenograms of skull and cervical spine. flattening of the base of the skull (angle formed bythe planeoftheclivus andtheplane of the anterior fossa exceeds °). pleuritis (see "effusion(s) and exudate(s), pleural.") pleurodynia, epidemic synonyms: bornholm disease; devil's grip; epidemic myalgia; epidemic myositis. blood heart and pericardium submit samples for viral culture. sample for histologic study and for viral cultures (coxsackievirus a and b; echoviruses). if pericardia! fluid can be obtained, submit for viral culture also. cultures may be negative and search for viral rna by in situ hybridization may be indicated. chest organs other organs dissect thoracic duct and its tributaries (see chapter ). perfuse one or both lungs with formalin. procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. submit samples of cystic lesions for histologic study. submit samples of cystic lesions for histologic study. gas in thoracic duct. emphysema* of lungs. necrotizing enterocolitis in premature infants. pyloric stenosis, colitis ( ), redundant sigmoid colon, ischemic bowel disease. mucosal pseudolipomatosis ( ) . usually, cysts are subserosal in adults and located between muscularis mucosae and muscularis propria in infants and children. mucosa may be inflamed. extraintestinal cysts, as listed above under "abdomen." cysts may also occur in vaginal mucosa. include bright-field and polarized light microscopy, transmission electron microscopy, scanning or transmission electron microscopy with energy dispersive x-ray analysis ( ), ion beam instrumentation, and secondary ion mass spectrometry. the last three methods are time-consuming, require much skill and expensive equipment, and do not lend themselves well to quantitation. ideally, bulk analysis and in situ microanalysis should be used in combination. analyses can be conducted in specialized laboratories at the following centers (for charges and other information, consult the appropriate laboratories): prepare chest roentgenogram. submit sample for microbiologic study. refrigerate sample for possible immunologic study. submit one lobe or samples of all lobes for bulk analysis and for in situ microanalysis (see above under "note"). microincineration may permit preliminary dust analysis. for demonstration of asbestos fibers, see chapter and ref. ( ) . submit one lobe or segment for microbiologic study. for pulmonary arteriography and bronchography, see chapter . prepare roentgenograms of entire lungs and slices. perfuse one lung (or both, if only routine studies are intended) with formalin. submit samples for histologic study of nodular dust lesions, diffuse fibrotic lesions, grossly uninvolved areas, bronchi, and bronchopulmonary lymph nodes. for preparation of paper-mounted sections, see chapter . prepare samples for transmission and scanning electron microscopic study. for removal, prosthetic repair, and specimen preparation, see chapter . alveolar rupture with dissection of air into the mediastinum in neonates with respiratory distress (see "syndrome, respiratory distress, of infant") and in adult patients ventilated on volume respirators. external examination prepare chest roentgenogram. photograph mediastinum. explore major veins and record compression in cases of tension pneumomediatinum. roentgenograms provide the best permanent record of a pneumomediastinum. air bubbles in mediastinal soft tissues. part ii / diseases and conditions abdomen and neck organs perfuse one lung with formalin. other procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. search for evidence of trauma of other lesions that may have allowed air to enter soft tissues. intubation injury (j); perforation or rupture of major bronchus, trachea, or esophagus. alveolar rupture, e.g., in asthma* ( ), may not be discernible. bronchiolitis obliterans ( ), interstitial pneumonia* ( ) and other lung conditions also may lead to pneumomediastinum. dissection of air from lesions in abdomen (e.g., retroperitoneal colonic perforation [ ] ) or in neck area. pneumonia, all types or type unspecified note: if the type of underlying infection is known, follow procedures suggested under the name of the infectious disease. if the etiologic agent of the pneumonia is unknown, proceed as follows: (l) collect all tissues that appear to be infected. and grocott's methenamine silver stains. ( ) special precautions may be required (see chapter ). ( ) serologic studies may be helpful once a specific etiologic agent is suspected. thus, collect serum at the time of autopsy or procure serum that was collected prior to death. ( ) this may be a reportable disease. related terms: acute interstitial pneumonia (hamman-rich syndrome); desquamative interstitial pneumonia (dip); idiopathic organizing pneumonia (or "bronchiolitis obliterans with patchy organizing pneumonia [boop]" or "cryptogenic organizing pneumonitis"); idiopathic pulmonary fibrosis; lymphoid interstitial pneumonitis (lip); nonspecific interstitial pneumonia (nsip) (or "cellular interstitial pneumonia" [eip]); usual interstitial pneumonia (uip); fibrosing alveolitis; pulmonary alveolitis; and many others. note: the conditions listed under "related terms" are histologic variants of idiopathic interstitial pneumonia. vip is synonymous with idiopathic pulmonary fibrosis (ipf) ( ). diffuse alveolar damage is the histologic finding in patients with the adult respiratory distress syndrome* (ards) causing interstitial and intra-alveolar fibrosis. this is an acute condition, referred to as acute interstitial pneumonia when it occurs as an idiopathic form of rapidly progressive interstitial pneumonia. possible associated conditions: acquired immunodeficiency syndrome* (aids) in patients with with lip or nonspecific interstitial pneumonia ( , ); trauma and shock in ards. secondary pulmonary fibrosis from inhalants (extrinsic allergic alveolitis or pneumonia; pneumoconiosis*), in drug-induced pneumonia, hypersensitivity pneumonitis (microgranulomatous hypersensitivity reaction of lung), rheumatoid arthritis,* sjogren's syndrome,* and other collagen-vascular diseases; primary biliary cirrhosis in patients with nonspecific interstitial pneumonia ( ). external examination postmortem chest roentgenograms provide the only reliable and permanent record of a pneumothorax and its main complication, mediastinal shift due to a tension pneumothorax (fig. - ) . if roentgenograms cannot be prepared, a reasonably reliable diagnosis still can be made if the prosector inserts a needle through the lateral chest wall. the needle should be connected to a water-filled flask. ifa pneumothorax is present, gas bubbles appear in the flask as shown in fig. - . one can also expose the intact parietal pleura and observe if the lung tissue is separated from the parietal pleura by gas. incising the thorax at the base of a water-filled skin pocket is the least reliable method. infants can be totally submerged under water before the chest cavity is incised. however, care must be taken not to make an accidental incision into the underlying lung tissue. poisoning, all types or type unspecified note: if a specific substance is suspected-for instance, arsenic or ethylene glycol-follow procedures described under the appropriate entry. similar entries can be found for poisons whose general character or source is known-for instance, gas or mushroom poisoning. for some substances, the appropriate entry can be found under "abuse,...," "death,...," or "dependence, ..." in all instances, routine sampling of toxicologic material should be done as described in chapter . if no specific substances can be incriminated, the toxicologist must be provided with all available clinical information, as shown in chapter . in most cases of fatal poisoning, the coroner or medical examiner must be notified. poisoning, alkaloid note: see under specific name of alkaloid-for instance, "dependence, cocaine," "poisoning, atropine," "poisoning, digitalis," or "poisoning, strychnine." only a fraction of this large group of plant poisons has been listed. whether the specific name of the alkaloid is known or unknown, complete toxicologic sampling is recommended. poisoning, antimony ote: toxicologic material should be submitted for anal ysis, as suggested under "poisoning, arsenic." iatrogenic antimony toxicity may occur after treatment with antimony compounds for conditions such as filariasis, fungal infections, and schistosomiasis.* external examination and eyes pharynx and gastrointestinal tract heart, liver, and kidneys record skin changes and eye abnormalities. for toxicologic sampling of contents, see chapter . submit tissue samples for histologic study. request frozen sections for sudan stain. if exposure was from du t in smelting work, dermatitis and conjunctivitis may be present. severe gastroenteritis in acute poisoning. if victim drank antimony trichloride, ulcerative pharyngitis and gastritis may be present. fatty changes of myocardium and hepatic and renal parenchyma. poisoning, arsenic note: toxicologic material will be contaminated by bringing it in contact with fluids. keratinized tissues take up arsenic from solutions. put plastic bags over hands of victim. if exhumed bodies are investigated for arsenic poisoning, include material from surrounding soil and coffin along with tissues submitted for chemical analysis. interpretation of toxicologic findings: after fatal poisoning, arsenic concentrations in the liver tend to exceed . - . mg/ioo g wet tissue. in acute poisoning, arsenic concentrations in hair may reach ! g/g ( ) and in nails ! g/g. organs collect all contents, particularly in accidental poisoning in children. body dry and warm after death. mydriasis. iatrogenic atrioventricular block ( ) . fruits of atropa belladonna or seeds of datura stramonium may be found. no characteristic findings. poisoning, barbiturate(s) note: this type of poisoning has become uncommon. barbiturates may cause sudden death. in all instances, concomitant alcohol intoxication* must be ruled out. standard toxicologic sampling is sufficient. blood bile urine esophagus and stomach liver and brain procedures submit samples of blood from portal vein and peripheral veins or heart for toxicologic study. refrigerate for possible toxicologic study. record total volume and ph value. request tests for protein, glucose, and ketones; request drug screen. submit all contents and record their character. analyze for barbiturates and alcohol. submit samples for toxicologic study. evidence of alcohol intoxication.* poisoning by other addictive drugs. gritty residues of unabsorbed tablets, powder, or capsules. mucosal corrosion, ulceration, and discoloration from capsules may occur. concentration of barbiturate in parenchyma important for interpretation. poisoning, bismuth note: accidental poisoning is common (industrial exposure or drugs with soluble bismuth compounds). search also for other heavy metals. acute kidney failure* may be the cause of death. external examination and oral cavity stomatitis with bluish black discoloration of gums; loose teeth; sticky white membranous patches in mouth and throat. jaundice. protein casts and tubular epithelial cells in sediment. gray or black mucosal membranes; swelling of mucosa; intestinal ulcers that may be perforated. hemosiderosis. fatty changes; hemosiderosis. fatty changes; renal tubular degeneration with amorphic basophilic deposits in epithelium of convoluted tubules. hemosiderosis. see above under "external examination." for removal and specimen preparation, see chapter . peripheral neuritis. synonyms: bromine poisoning; bromism. note: the lethal dose is about . g in children and i g in adults (ingested). after fatal methyl bromide poisoning, headspace gas chromatography revealed a subclavian blood concentration of . microgram/ml whereas inorganic bromide concentrations were micrograms/ml in the blood ( ). tissue concentrations were lower than those in the blood. toxicologic samples should include liver and kidneys. chemical bums on face and conjunctivitis indicate direct exposure; skin pustules over body and nodose bromoderma of the legs indicate bromism. blood is best suited for bromide determination. after ingestion of bromide, necrosis with brown discoloration of mucosa of upper gastrointestinal tract may be present. after inhalation of bromide, swelling and inflammation ofmucous membranes in upper and lower respiratory tracts may be present. there may be pulmonary edema. pneumonia occurs in bromism. organs to be analyzed for cd should have no contact with water or be contaminated with blood; they should be sealed in polyethylene bags. cd leaks into fixation fluid. postmortem blood concentrations are very high and no indicator of the antemortem values ( ). external examination and oral cavity blood urine gastrointestinal tract other organs procedures submit sample for toxicologic study. submit sample for determination of cadmium concentration. submit sample for toxicologic study and a portion of one lobe for microbiologic study. perfuse one lung with formalin. fix bowel as soon as possible. collect renal tissue for light microscopic and electron microscopic study. sample for toxicologic study. submit samples for histologic study also. poisoning, carbon monoxide note: if the victim had been in a fire, see also under "bums." carbon monoxide poisoning may rarely be responsible for automobile accidents. relatively low carboxyhemoglobin concentrations may contribute to death if there is concomitant poisoning-forinstance, with alcohol or drugs, particularly sedatives. anemia, atherosclerotic heart disease, and chronic pulmonary disease also increase sensitivity to carbon monoxide. ifblood had been withdrawn at time of hospital admissionfor instance, for crossmatching-submit this for carboxyhemoglobin determination. if no blood can be obtained, see under "heart, kidneys, and other organs." for quick-orienting qualitative tests, for quantitative methods of carbon monoxide determi-nation, and for interpretation of toxicologic findings, see below. request also determination of hemoglobin concentrations and of blood alcohol. request drug screen. for shipping of blood and tissues for carbon monoxide determination, see chapter . it should be noted that losses of up to % of the original saturation occurred when blood was kept in uncapped container at room temperature for yz wk or at °c for wk. external examination blood heart, kidneys, and other organs brain record color of fingernails, particularly in heavily pigmented persons in whom lividity is difficult to discern. record appearance of blood and submit sample of postmortem blood for toxicologic study. see also above under "note." if no blood can be obtained, prepare water extract of spleen, kidneys, or other organs. request determination of carbon monoxide content and of carbon monoxide-binding capacity of this mixture. submit tissue samples for histologic study. for removal and specimen preparation, see chapter . pink skin and fingernails; bullous edema of skin; decubital ulcers. blood tends to be cherry red. for interpretation of toxicologic findings, see below. necrosis of papillary muscles in the heart or myocardial infarction may occur. renal tubular degeneration may also be found. acute kidney failure* has been observed after rhabdomyolysis complicated by compartment syndrome ( ) . hemorrhagic necrosis of basal ganglia (lenticular nucleus in globus pallidus); diffuse petechial hemorrhages in white matter; cerebral edema. acute hydrocephalus in infants ( ) . many methods of carbon monoxide determination have been described. currently, carboxyhemoglobin is detected in most medical examiner toxicology laboratories by visible spectrophotometry or gas chromatography. in hospitals, carboxyhemoglobin is frequently detected and reported in the course of routine arterial blood gas analysis. pink discoloration of skin and organs usually indicates the presence of more than % carboxyhemoglobin (but rule out cyanide poisoning* and exposure to cold*). in a healthy, middle-aged person, a carboxyhemoglobin concentration greater than - % is usually fatal. if the victim was anemic or suffered from chronic lung disease, particularly emphysema* or atherosclerotic heart disease, the concentration may be lower. in association with alcohol, sedatives, and other drugs, carboxyhemoglobin levels may also be much lower and yet fatal. a heavy cigarette smoker may have a carboxyhemoglobin concentration of - %, and higher levels may occur in police officers and other persons exposed to automobile exhaust in dense traffic. if the victim survived the carbon monoxide poisoning for several hours, postmortem blood samples usually will fail to show the presence of carboxyhemoglobin. in these instances, blood taken at the time of admission to the hospital may still be available and of particular value. if the victim had spent h in fresh air before death, - % of the carbon monoxide will have been removed, and - % will have been removed during each subsequent hour. even though clearance may be complete, death may still occur-primarily from brain damage and infectious complications in prolonged coma. or delayed by only a few hours, particularly after inhalation of carbon tetrachloride. sudden death probably is caused by cardiac dysrrhythmia. alcohol concentrations should be determined in all cases or, if death was delayed, evidence of drinking at the time of exposure should be sought. alcohol considerably increases the hazards of carbon tetrachloride. note: see also under "bronchitis, acute chemical" and under "poisoning, gas." hydrochloric acid is sold by plumbing supply houses and pool supply companies as muriatic acid. it is a liquid. chlorine is a water-soluble gas. as supplied for pool sanitation, liquid chlorine is usually acidic. for convenience, chlorine and hydrochloric acid are discussed here together. prepare photographs of face. conjunctivitis and cyanosis in chlorine gas poisoning; burns of lips from hydrochloric acid. see also above under "larynx and trachea." photograph opened esophagus and stomach. sample for histologic study, particularly if there is doubt whether a perforation was antemortem or postmortem. for removal and specimen preparation, see chapter . severe pulmonary edema, bronchopneumonia, and swelling of mucous membranes in chlorine poisoning. arterial thrombosis may occur. pulmonary fibrosis may develop after prolonged survival. swelling and ulceration of mucous membranes in chlorine poisoning; acute laryngotracheitis. tracheoesophageal perforation. corrosion of mucosa with thickening, hemorrhage, and blackish discoloration after ingestion of hydrochloric acid. antemortem and postmortem perforation may occur. glomerular capillary thromboses. hemorrhages in white matter ( ). poisoning, cyanide synonym: hydrocyanic acid (hydrogen cyanide) poisoning. note: hydrocyanic acid (hydrogen cyanide, hcn) is a water-soluble gas. its salts, sodium cyanide and potassium cyanide are sold as "eggs" to the jewelry industry. hydrocyanic acid is formed when cyanide salts are dissolved in acidic solutions. containers from which the poison might have been ingested or inhaled should also be submitted for toxicologic examination. for cyanide screening tests in the autopsy room and for interpretation offindings, see below. caution: stomach may still contain cyanide gas, formed by acidic reaction of cyanide salt. it may be best to open the stomach under a hood ( ). the odor is quite characteristic for cyanide poisoning but most persons are unable to smell this odor. it is helpful to know in advance if any person in an office or laboratory can smell cyanide. forensic pathologists who can smell the compound state that it has its own specific odor, which differs from the often quoted smell of bitter almonds. autopsies also can be done in a negatively pressured isolation room ( ). for odor, see above under "note." bright red skin color is not always present. corrosion around mouth and in oral cavity may be found after ingestion of potassium or sodium cyanide. blood is fluid and sometimes bright red. if potassium or sodium cyanide was ingested, brown-red mucosal corrosion may be present in stomach or in upper digestive tract. pulmonary edema. for odor, see above under "note." if death was not instantaneous, there may be hyaline thrombi in small blood vessels, minute hemorrhages, and necroses of lenticular nuclei. this test can be used for blood and gastric contents ( ) . dip squares of filter paper in a small amount ofsaturated picric acid. let these squares dry until barely moist. place a drop ofthe material to be tested-e.g., blood or gastric contents--on a piece of paper. let material dry for a moment, and then place one drop of % sodium carbonate in the center of the material to be tested. ifcyanide is present, a reddish purple color will chromatograph out from the material. the higher the concentration of cyanide the more blue the color will be. it is possible to recognize whole blood because the blood turns a rather dark brown and the reddish to purple color is clearly visible. high concentrations of sulfide interfere by giving a false-positive test. another screening test is done as follows ( ) . dip filter paper into normal blood. then treat the paper with potassium chlorate, whereupon brown methemoglobin forms. place this preparation into the fluid suspected of containing cyanide (e.g., blood, gastric contents, pulmonary edema fluid). if bright red cyanmethemoglobin forms, the reaction is positive. if the concentration of cyanide in the stomach is high and the concentration in the lungs is low, cyanide was ingested. alternatively, if the pulmonary cyanide concentration is high and the concentrtaion in the gastric contents is low, hydrogen cyanide most likely was inhaled. occasionally, minimal cyanide levels will be present in decomposed bodies. related term: digoxin toxicity. note: certain drugs may interfere with correct digitalis determination. blood heart vitreous procedures submit sample of peripheral blood for digoxin radioimmunoassay. freeze fresh myocardium for digitalis extraction. submit sample for digoxin radioimmunoassay. poisoning, drug(s) (see "dependence, drug(s), all types or type unspecified" or under "poisoning,•••" followed by specific name of drug.) poisoning, ethanol (ethyl alcohol) (see "alcoholism and alcohol intoxication," "cardiomyopathy, alcoholic," "disease, alcoholic liver," "syndrome, fetal alcoholic," and syndrome, wernicke-korsakorr.") poisoning, ethylene glycol related term: antifreeze poisoning. note: pulmonary and cerebral manifestations are the main findings in acute poisoning, and renal tubular necrosis is the primary finding in chronic poisoning. for general toxicologic sampling, see chapter . calcium oxalate crystals can be demonstrated in routine histologic sections but also in scanning electron micrographs of thick deparaffinized sections. eyes blood urine for removal and specimen preparation, see chapter . in acute cases, submit sample for ethylene glycol determination; in chronic cases, request determination of calcium concentrations. prepare sediment. papilledema; optic nerve atrophy. ethylene glycol in serum (i) . protein casts; calcium oxalate crystals ( ) . crystals are light yellow and birefringent, arranged as sheaves, rhomboids, or prisms. poisoning, food related terms: bacillary dysentery* (shigella food poisoning); botulism;* clostridium perfringens food poisoning; favism; mushroom poisoning;* salmonella food poisoning; staphylococcal food poisoning. note: if cause of food poisoning is unknown, submit suspected food for aerobic and anaerobic cultures, gram stain of smears, and routine toxicologic study. this should include tests for heavy metals (antimony, cadmium, and lead) that may have leaked from old cooking utensils. test for the presence of staphylococcal enterotoxin are done only in specialized laboratories. if botulism is suspected, follow procedures described under that heading. mushroom poisoning also is listed as a separate entity. if salmonella food poisoning is suspected, see under "fever, typhoid." see also under "enteritis" or "enterocolitis" or under another specific heading such as "dysentery, bacillary." obtain sufficient material for microbiologic and histologic study to identify organisms such as chlamydia, clostridium (type f strains), salmonella, shigella, verotoxic e. coli, yersinia, and others. external examination gastrointestinal tract submit contents for aerobic and anaerobic cultures (see above under "note"); prepare smears of contents for gram stain. submit samples for histologic study. debilitated states; patients in extremes of life. enteritis or enterocolitis. poisoning, gas note: anesthesia-associated death, * carbon monoxide poisoning,* and sniffing and spray death* are presented under the appropriate headings. procedures discussed here deal with other volatile substances, including chemical irritants such as ammonia (nh ), chlorine or hydrochloric acid poisoning (see also under that heading); methylene chloride, phosgene (coci ), sulfurous acid (h s ), or sulfur dioxide (s ) ' gases from body cavities, heart chambers, or blood vessels can be removed as described under "embolism, air." gases can also be trapped with a rubber dam after cutting organs under water. samples from various organs should be shipped in hermetically sealed nonplastic containers or in analyzing solutions. external examination, and oral cavity blood larynx and trachea record extent of chemical bums. submit sample for gas analysis. in many instances, inhaled gases can be demonstrated chromatographically in gas from head space above sealed blood specimen. chemical bums in and around mouth or conjunctivas. chemical bums. other organs if gas was inhaled and is to be analyzed, submit intact lungs with bronchi ligated in airtight, nonplastic container to laboratory that can conduct gas analysis. if survival was short, formalin perfusion of lungs is not recommended; it may cause artifactual ballooning and internal ruptures of organ. submit samples of tissue for routine histologic study. see above under "note." chemical pneumonia; pulmonary edema. after longer survival, obliterating fibrous bronchiolitis, chronic bronchitis, and saccular bronchiectasis* may occur. poisoning, glycol (see "poisoning, ethylene glycol.") poisoning, halogen (see "fluorosis," "poisoning, bromide," "poisoning, chlorine or hydrochloric acid," "poisoning, gas?, and "poisoning, iodine!') poisoning, heavy metal (see "poisoning, antimony," "poisoning, arsenic," "poisoning, cadmium," "poisoning, lead," poisoning, mercury," "poisoning, thallium!') poisoning, insecticide (see "poisoning, organophosphate(s)*) poisoning, iodine related terms: lugol's solution; tincture of iodine. for toxicologic sampling, see chapter . external examination and eyes urine, blood, and parenchymal organs lungs and upper respiratory tract stomach record color of skin and extent of corrosive lesions. submit samples for toxicologic study. submit samples for histologic study. submit contents for toxicologic study; photograph mucosa; prepare histologic sections. see above under "stomach." perioral corrosive lesions; yellow discoloration of skin; conjunctivitis after exposure to vapors. acute inflammation of respiratory tract after inhalation of vapors. corrosive gastritis; if starch was used as antidote, gastric lining will be bluish. histologically, well-preserved mucosa is present because of in vivo fixation. mucosa may show same changes as stomach. swelling of tubular epithelium. synonyms: propanol; rubbing alcohol. procedures see under "alcoholism and alcohol intoxication." nonspecific autopsy findings: visceral congestion; pulmonary and cerebral edema. poisoning, lead note: lead-free syringes and lead-free polyethylene containers should be used. blood lead concentrations can be determined by inductively coupled plasma mass spectrometry ( ). for screening methods, see ref. ( ) . this is a reportable disease in some states. external examination, oral cavity, and hair bluish lead line at gingival margin in victims with poor oral hygiene. external examination, oral cavity, and hair for diagnosis of chronic plumbism, analysis of scalp hair may be useful. analysis is done by neutron activation (see also under "poisoning, arsenic"). remove samples with lead-free syringe (see above under "note") or -ml vacutainer tubes (becton, dickinson and company). do not add anti-coagulant or preservative. for coliection procedures, see above under "blood" and under "note." request also determination of coproporphyrin concentrations. submit sample for histologic study; submit remaining tissue for toxicologic analysis. submit with contents for toxicologic study, particularly in acute poisoning. submit sample from each kidney for histologic study; submit remaining tissue of both kidneys separately for toxicologic study. submit at least g of fresh bone for toxicologic study. for removal and specimen preparation, see chapter . poisoning, lsd (d-lysergic acid diethylamide) (see "abuse, hallucinogen(s).") poisoning, lye related terms: ammonium hydroxide poisoning; calcium oxide or quicklime poisoning; poisoning by alkaline corrosives; potassium hydroxide poisoning; sodium hydroxide poisoning. external examination and oral cavity blood neck organs, esophagus, trachea, and lungs record extent of oral, perioral, and other facial corrosive injuries. photograph lesions. prepare histologic sections of tissue from inside of lips or mouth. submit sample for toxicologic study. after removal of heart, remove neck organs with hypopharynx, esophagus, larynx, and trachea. leave stomach attached to esophagus. open pharynx and esophagus along posterior midline. in acute cases, formalin perfusion of lungs is not recommended; it may cause artifactual baliooning and internal ruptures of organ. lye bums on face and chest in acute cases; scars and manifestations of malnutrition* in chronic cases. sweliing, edema, and necrosis of mucous membranes in acute poisoning. fibrosis and strictures in chronic cases. bronchitis* and bronchopneumonia. submit specimen from pharynx for histologic study. for removal and specimen preparation, see chapter . submit sample of brain for toxicologic study. exfoliative dermatitis. blue line at gingival margin; hypertrophy of gum; acute and chronic gingivitis; exfoliation and loss of teeth ( ) . degeneration of myocardium. increased concentrations of mercury ( ) . congestion. erosive gastritis and colitis. increased concentrations of mercury ( ) . degeneration of proximal tubules; calcifications. chronic kidney failure* may be the cause of death. induration of mucosa. increased concentrations of mercury ( ) . cortical hemorrhages. cholinesterase activity will be low. pulmonary edema if poison was inhaled. airways may contain aspirated material. cholinesterase activity can be determined reliably even after decomposition and embalming. clinically, guillain-barre syndrome has been observed after poisoning with organophosphate. in acute poisoning, the cholinesterase levels may be % of the normal values (see below). the cholinesterase levels in the blood are not affected by the duration of the postmortem interval; measurement may be attempted even on decomposed or exhumed bodies. severe fatty changes; periportal necroses. fatty changes in myocardium, skeletal muscles, and other organs. for toxicologic sampling (gastric contents, urine, blood, brain, and other organs), see chapter . rigor mortis after fatal strychnine poisoning may occur very soon after death and may be very severe (opisthotonos); it may persist until decomposition sets in. congestion of viscera; no characteristic morphologic autopsy findings. acute pancreatitis has been observed ( ). high strychnine concentrations (also demonstrable in exhumed bodies [ ] ). poisoning, thallium note: for toxicologic sampling, see below and chapter . thallium is used as a rodenticide and pesticide, and has some industrial applications. retrobulbar neuritis. chapter and . submit brain for toxicologic study. submit sections of brain and optic nerves for histologic study. submit specimens for toxicologic study (take from lower extremity). for removal, prosthetic repair, and specimen osteomalacia;* osteomyelofibrosis. preparation of bones, see chapter . for preparation of sections and smears of bone marrow, see chapter . submit samples for toxicologic study. synonym: acute anterior poliomyelitis. note: the disease has been nearly eliminated in the usa but not in many other countries. ( ) collect all tissues that appear to be infected. ( for removal and specimen preparation, see chapter . in acute cases, submit portions of brain and spinal cord for viral culture. neurogenic atrophy of skeletal muscles in areas of paralysis. electrolyte disorder. * hypertensive heart disease; myocarditis.* aspiration or bronchopneumonia (or both); edema; atelectasis; embolism;* alveolar wall necrosis (acute or organizing diffuse alveolar damage) after oxygen toxicity. acute ulcers. acute gastric dilatation; acute gastroduodenal ulcers; gastrointestinal erosions and hemorrhages. dilatation of colon; perforation of cecum. urolithiasis and nephrolithiasis,* pyonephrosis and pyelonephritis. * phlebothrombosis of legs, most commonly on left side. necrosis of anterior hom cells of spinal cord, with neuronophagia and perivascular inflammatory reaction. old lesions show neuronal loss and gliosis. medulla ("bulbar polio") and other areas of brain stem, cerebellum, and cerebrum, particularly the motor cortex, may be affected in various degrees. part ii / diseases and conditions pregnancy note: in some instances, procedures described under "death, abortion-associated," "embolism, amniotic fluid," or "toxemia of pregnancy" may be indicated. synonym: werner syndrome. external examination and skin abdomen procedures record volume of intraperitoneal fluid; prepare smears; submit samples of peritoneum for histologic study. colloid carcinoma of stomach or colon. well-differentated adenocarcinoma of ovary or, rarely, of appendix; ruptured appendiceal mucinous adenoma is the most common cause of peritoneal adenomucinosis. pseudotumor cerebri synonym: benign intracranial hypertension; meningeal hydrops. note: this condition, which generally affects young, obese females, is characterized by symptoms and signs of increased intracranial pressure without a demonstrable cause. hence, intra-cranial mass lesions, infections, and related conditions should be excluded. such conditions include adrenal insufficiency;* guillain-barre syndrome* (increased colloid-osmotic pressure); hyperadrenalism; hypervitaminosis a* (e.g., after treatment of acne); hypoparathyroidism;* hypothroidism;* infectious mono-nucleosis;* lyme disease; pregnancy; sydenham's chorea; throm-bus ofthe lateral or superior sagittal sinus (otitic hydrocephalus); and wiskott-aldrich syndrome. submit samples of all accessible organs and tissues, with or without gross lesions. submit material for electron microscopy. for removal and specimen preparation, see chapter . for removal, prosthetic repair, and specimen preparation, see chapter . heart small bowel liver procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. fix the bowel as soon as possible. record weight. submit samples for histologic study. for removal, prosthetic repair, and specimen preparation, see chapter . aortitis involving ascending aorta and aortic valve with regurgitation; mitral valve and adjacent myocardium and conduction system also may be involved. sprue-like changes with loss of villi. fibrosis or cirrhosis and other abnormalities, particularly in patients who had been taking methotrexate. psoriatic arthritis and spondylitis. synonyms and related terms: allergic purpura; anaphylactoid purpura; hypersensitivity vasculitis. external examination and skin rabies note: (i) in most instances, an autopsy limited to the brain is sufficient to confirm the diagnosis. ( ) rabies virus is especially infectious and thus, universal precautions should be strictly followed (see chapter ) . avoid the use of scalpels whenever possible. the generation ofaerosols should be assidu- see above under "note." if a complete autopsy is done, sample heart, lungs, kidneys, pancreas, submaxillary salivary glands, and adrenal glands; freeze or refrigerate sampled tissues and submit for viral study (see below under "brain and spinal cord"). obtain serum for serologic studies. freeze or refrigerate cerebral tissue immediately after removal and submit frozen to special laboratory for fluorescent antibody staining. take these sections from hippocampus or brain stem. place the refrigerated tissues in % neutral glycerol saline solution for preservation. fix remaining brain and spinal cord tissue in % formalin and submit for histologic study. as an alternative to freezing of tissue, immunoperoxidase methods for detection of rabies viral antigens can now be applied to formalin-fixed tissue ( in almost all instances, the procedures described under "assault" and under "homicide" must also be followed. see also refs. ( ) and ( ) . for the evaluation of bite marks, photographs with and without scales and black and white film should be used. a forensic dentist is required to compare the victim's bite marks to the teeth of suspects. swabs of possible sperm or other fluids must be sent to the crime lab for proper evaluation. external examination other organs and tissues genital organs comb pubic hair over a towel and then pull sample; also, pull samples of hair from head. collect fingernail clippings and place in containers marked "right" and "left." collect blood, hair, fibers, and residues of urine or saliva and/or semen that may have stained the victim's clothing or that may be found on the skin of the victim. for study of stains and scrapings, see below. introduce sterile dry cotton swab into the posterior vault of the vagina or-preferablyinto the cervical canal. prepare smear on glass slide and send to crime laboratory for identification of sperm (see also above under "note" using a sterile cotton swab moistened with sterile saline, lift suspected dried spermatic fluid from hair or skin of external genitalia, perineum, buttocks, or other sites. stains on clothing will be extracted by forensic laboratory personnel. let specimens air dry in absence of sunlight and then place in paper bags or envelopes and seal. smears are made for the demonstration of spermatozoa, cytologic changes, bacteria, and other possible findings (some crime labs prefer to make their own smears from the swabs). other samples are used for the determination of acid phosphatase, as described below. usually, the survival time of morphologically recognizable spermatozoa in the vagina is less than h, but on occasion this may be much longer. thus, within the first h after coitus, % of cervical smears have been found to be positive for spermatozoa. at day , spermatozoa have been found in % of the smears. obviously, if the assailant had had a vasectomy, spermatozoa will not be found at any time. spermatozoa have been found in the vagina of some women several days or weeks after death. dried stains (see above) may give positive results after longer intervals. acid phosphatase activities greater than bodansky units (for method, see above) indicate that probably less than h have elapsed since the time of intercourse (i) . in another study, vaginal swab specimens showed acid phosphatase activities of more than , king if there is evidence of parotid involvement, other salivary glands should also be studied. parotid gland can be biopsied from scalp incision. submaxillary gland can be removed with floor of mouth. for sampling and specimen preparation, see chapter . for removal, prosthetic repair, and specimen preparation, see chapter . chronic meningitis; space-occupying lesions (submeningeal nodular granulomas). granulomatosis. iridocyclitis; chorioretinitis; papilledema; posterior uveitis; keratoconjunctivitis sicca; conjunctival follicles; cataracts. involvement of lacrimal glands and other orbital tissues ( ) . sarcoidosis of parotid gland is common. sarcoid neuropathy and sarcoid myopathy. cystic changes, primarily of small bones of hands and feet. sarcoidosis of joints ( ). synonyms and related terms: bilharziasis; schistosoma haematobium infection; schistosoma japonicum infection; schis-tosoma mansoni infection. note: unless specifically stated, the changes listed below refer to chronic schistosomiasis mansoni and japonica. ( ) collect all tissues that appear to be infected. ( ) request direct examination for schistosoma. the following procedures have been described and recommended ( ). for demonstration of schistosoma eggs, compress -mm tissue fragments-for instance, mucosa of the urinary bladder-between glass slides. if this gives negative results, digest a -g portion of tissue in potassium hydroxide. for the recovery of adult worms of schistosoma mansoni and schistosoma haematobium, remove the viscera en bloc and rinse with water. separate the intestines from the mesentery. subsequently, perfuse the portal vein system, the liver, and one lung with saline ( ) . then pass the perfusion fluid through a monofilament nylon cloth with an aperture size of m. submerge the cloth in water and examine with a dissecting microscope. fix worms in formalin solution. examine the intestinal mucosa directly. from the urinary bladder, ureters, and surrounding connective tissue, worms can be recovered as follows. inject water into the tissue until the tissue increases or times in thickness. then compress the tissue gently with a glass plate. cut slices . - . cm in thickness. compress these slices between the glass plate and the stage of a dissecting microscope and examine for the presence ofadult worms. many worms also will be present in the fluid expressed from the tissue as it is cut. for the counting of eggs in tissues, urine, and feces, see ref. ( ) . immunodiagnostic methods (elisa and imrnunoblot) also have been developed prepare skeletal roentgenograms. there are no diagnostic laboratory tests but it still is advisable to save a blood sample. record heart weight. test competence of valves (chapter ). open heart in line of blood flow. photograph and measure valvular lesions. prepare sections of valves, myocardium, conduction system (if there was a history of heart block), and ascending aorta. perfuse at least one lung with formalin. submit any consolidated area for microbiologic study. fix intestines as soon as possible. abnormalities as listed in right-hand column. sample for histologic study and request amyloid stains or renal parenchyma. prepare roentgenograms of spine, sacroiliac joints, symphysis ossium pubis, and manubriosternal, sternoclavicular, and humeroscapular joints. if spine cannot be removed in its entirety, it can be split in midline and one half can be removed, with costovertebral and costotransversal joints. hip joints should be exposed. histologic sections should include synovia and periarticular tissue. secondary and peripheral osteoarthritis* may be present. leukemia* developed in some patients who had had radiation treatment ( or earlier). acute anterior uveitis. ( ) request fungal culture. ( ) request grocott's methenamine silver stain. ( ) record weight of all organs (for expected weights, see part iii). submit samples of all major organs, including endocrine glands, lymphatic and fat tissue, bone, and bone marrow for histologic study. hunger edema; skin changes secondary to vitamin deficiencies. hypoproteinemia. atrophy; hemosiderosis of spleen. degree of atrophy varies from organ to organ; atrophy of fat tissue, lymphoid tissue, and gonads usually is most pronounced. severe infections, such as tuberculosis,* may be present without having been apparent clinically. steatohepatitis, nonalcoholic (nash) note: the morphologic findings in the liver are indistinguishable from those in alcoholic liver disease* ( ). follow autopsy procedures described under "disease, alcoholic liver." if the patient had received a liver transplant, procedures described under "transplantation, liver" should be followed also. possible associated conditions: celiac sprue (rare); diabetes mellitus* (type ); hyperlipidemia; malnutrition* from bulemia (rare); morbid obesity with liver failure particularly after episodes of rapid weight loss (e.g., after recent gastroplasty); lipodystrophy; mild obesity;* short bowel syndrome (rare); total parenteral nutrition. * stenosis, congenital valvular pulmonary related terms: isolated (pure, simple, or dome-shaped) pulmonary stenosis. note: the pulmonary valve is usually acommissural in isolated congenital pulmonary stenosis. with other coexistant congenital heart disease (such as tetralogy), the pulmonary valve is usually bicuspid and hypoplastic, but may be unicommissural or dys-plastic and tricuspid. heart and great vessels; peripheral pulmonary arteries brain procedures culture any grossly infected valve. for general dissection techniques, see chapter . record weight of heart, thickness of ventricles, and annular circumferences. for removal and specimen preparation, see chapter . infective endocarditis* of pulmonary valve and tricuspid valve; infective endarteritis at bifurcation of pulmonary trunk. it is preferable to have autopsies of fetuses and stillborns performed by pathologists experienced in perinatal pathology. if such personnel are not immediately available, the attending pathologist may nevertheless collect important information. if attempts to induce abortion appear to have caused the death of the mother, see "death, abortion-associated." the placenta should be immediately procured from the delivery room should it not arrive in the laboratory with the fetus. this will avoid the possibility of the placenta being discarded by the delivery room staff. no fetal autopsy is complete without a careful examination of the placenta (see part i, chapter ). pathologic changes of placenta that are causative of stillbirth are summarized in ref. . fascia lata or other aseptically obtained tissue should be collected for tissue culture for karyotype analysis. if the fetus is at all autolyzed, then the fascia lata cells may not grow in tissue culture. therefore, if the fetus shows any evidence of autolysis, tissue should be taken aseptically, from placenta immediately beneath the chorionic plate. a portion ofplacenta and liver should also be snap-frozen for possible molecular analysis. the initial stage of the autopsy should include photography and radiography, taking anterior-posterior and lateral views. the photographs will record the degree of maceration, which can be roughly correlated with the duration of fetal demise before delivery [ ] external measurements should include body weight, circumference ofhead, chest and abdomen, crown-rump length, crown-heel length, and foot length. these measurements are compared with tables of standards for normal fetuses (see part iii of this book). assessment of growth retardation may be based on these data. a careful external examination should search for abnormalities such as jaundice, bulging fontanel, cranial bone softening, hyper-or hypotelorism, choanal atresia, external ear anomalies, cleft lip, cleft palate, macroglossia, micrognathia, colobomata, cystic hygroma, shortened neck, contractures, omphalocele, gastroschisis, abnormal external genitalia, anal atresia, absent vagina, sacral pits, open neural tube defects, hemihypertrophy, syndactyly, clinodactyly, transverse palmar creases, or incomplete descent of testes. the organ bloc may be removed in the manner similar to the adult, using the technique of letulle (see chapter ) . if the thyroid gland is noted to be in its usual location and if it appears normal, then the tongue may be left in the body. in macerated fetuses, it is suggested that the organ bloc be fixed overnight in formalin solution prior to dissection. to aid adequate fixation, the following simple steps may be done: i) wash the organ bloc thoroughly prior to fixation; ) place multiple transverse cuts through the liver and lungs; ) dissect the posterior leaves of the diaphragm away from the adrenal glands and kidneys; ) bivalve the adrenal glands and kidneys in the coronal plane; ) instill formalin in the lumen of the intestine, using a syringe; and ) gently instill formalin into both ventricles of the heart, being careful to avoid the ventricular septum. the procedure for dissection of the organs is similar to that of the adult except: ) the venous and arterial connections of the heart, including the patency of the ductus arteriosus must be determined before the heart is removed; ) the esophagus must be opened posteriorly prior to its complete removal so that esophageal atresia or tracheo-esophageal fistula may be recognized and photographed prior to further dissection; ) the location of the appendix and of the testes should be recorded. until the intestine has been examined for stenosis or atresia, the mesentery should be left attached. the degree of autolysis as seen grossly and with histologic examination of both the fetus and the placenta can be used to estimate the duration of time between fetal death and delivery ( , , ) . trichrome stain is useful for better visualizing histologic features in severely autolyzed tissue. to remove the brain, benecke's technique of one of its modifications may be used. stillbirth vs livebirth. a decision has to be made whether the infant was born alive or was stillborn. the hydrostatic lung test, described in the previous edition, appears unreliable. the presence of gas in the lungs does not rule out stillbirth. after death, air can be introduced into the lungs, or putrefaction gases might be present. however, air artificially introduced after death will not distend the alveoli and can be squeezed out, whereas this does not seem to be the case after active ventilation. the distribution of fat in the fetal zone of the adrenal cortex may indicate whether intrauterine death was acute, more prolonged, or chronic ( ). this is particularly helpful if the stillborn baby is macerated. if the mother died also, see under "death, abortion-associated" strangulation note: in many instances, procedures described under "hom-icide" must also be followed. if a rope or some other material had been used (see also under "hanging"), leave ligature in place until autopsy can be done. see also under "hypoxia." toxicologic sampling, particularly for alcohol, should be done in all instances (chapter ). results than body fluids. ( ) universal precautions should be strictly followed. the generation of aerosols should be minimized. to sterilize tissue surfaces in preparation for culture, swab the surface with povidone iodine. avoid searing the tissue surface since this will produce an aerosol. keep no more than one scalpel in the dissecting area at anyone time. have an assistant available with clean, gloved hands to receive specimens in containers, so as to minimize the degree of contamination on the outside of the containers. for cleaning procedures and related information, see chapter . ( ) hiv-l in tissue may be demonstrated using polymerase chain reaction (pcr), immunohistochemistry, in situ hybridization, or immunofluorescence. ( ) for immunocytochemical and molecular studies, fix tissue in ethanol or carnoy's solution. the virus can be identified using pcr in most tissues, whether fresh, frozen, or fixed in ethanol. ( ) serologic tests as well as direct fluorescent antibody tests are avail-able for many of the expected infections. ( ) this is not a reportable disease. external examination and skin record body weight and evidence of lipodystrophy following aids medications. record and photograph any skin lesions. examine oral cavity. if the diagnosis is in doubt, samples can be submitted for enzyme immunoassay. cachexia. severe loss of subcutaneous fat in face and extremities, associated with large fat deposits on upper back and upper abdomen ("protease paunch"). cutaneous kaposi's sarcoma (l) . test may be positive for many weeks after death ( ) . syndrome, myelodysplastic synonyms and related terms: chronic myelomonocytic leukemia;* refractory anemia; refractory anemia with excess of blasts; refractory anemia with excess of blasts in transformation; refractory anemia with ringed sideroblasts; refractory dysmyelopoietic anemias. note: the myelodysplastic syndromes are represented by a heterogeneous group of normocytic anemias, often with neutropenia, thrombocytopenia, and monocytosis. for expected bone marrow changes, see above under "synonyms and related terms." autopsy procedures are similar to those recommended for most cases of leukemia, with particular attention paid to intercurrent infections and thrombocytopenic hemorrhages. in all instances, material should be collected using aseptic technique for tissue culture for chromosome analysis. common findings in these conditions are deletion of the long arm ofchromosome , deletion of chromosome or , or trisomy . syndrome, nephrotic note: see under name of suspected underlying condition, such as amyloidosis,* anaphylactoid purpura,*diabetes mellitus,* glomerulonephritis,* goodpasture's syndrome,* heavy metal poisoning, hemolytic uremic syndrome,* infective endo-carditis,* polyarteritis nodosa,* syphilis, * or systemic lupus erythematosus. * if accelerated hypertension or constrictive pericarditis is the suspected underlying condition, see under "hypertension (arterial), all types or type unspecified" or "pericarditis," respectively. in all instances, the renal veins and the inferior vena cava should be opened in situ. "en masse" removal of organs is recommended for this purpose. if thrombosis is found, record exact location and size of clot and submit sample of clot with wall of veins for histologic study. see also under "thrombosis, venous." coronary atherosclerosis and its complications seem to be increased in patients with the nephrotic syndrome. submit sample for bacteriologic, viral, and serologic study. record weight and submit samples for histologic study. if heart block was present, submit samples of conduction system for histologic study (chapter ). submit consolidated areas for microbiologic study. perfuse at least one lung with formalin. for removal of urethra, see chapter . procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. for removal and specimen preparation, see chapter . consult roentgenograms (see above under "external examination, skin, and oral cavity"). keratoderma (keratosis blennorrhagica) with vasculitis ( ) of sole of feet, of palms, and of circumcised glans penis. arthritis* of knees, ankles, and metatarsal and midtarsal joints, with or without ankylosis. osteoporosis.* usually, sterile culture. pericarditis; * myocarditis.* aortic valve lesions. pleuritis and pneumonia.* pulmonary fibrosis involving upper lobes. erosions, papules, and plaques in urethral or bladder mucosa. enteritis. thrombophlebitis. conjunctivitis; multifocal choroiditis; acute anterior uveitis; iritis; keratitis. arthritis* (see above under "external examination, skin, and oral cavity") resembling rheumatoid arthritis. * nonspecific synovitis. for removal and specimen preparation, see chapter . syndrome, reye's note: hypoglycemia* may have been present, but that condition is difficult or impossible to confirm after death. an immediate autopsy is indicated (see below under "liver" and "pancreas"). vitreous blood urine heart lungs submit sample for sodium, chloride, and urea nitrogen determination. submit sample for microbiologic (viral) and serologic study, for ammonia and bilirubin determination, and for determination of salicylate levels if there is a history of treatment with this drug. obtain sample for biochemical and toxicologic analysis. record weight. request frozen sections for sudan stain. submit fresh tissue for bacterial and viral culture. request paraffin sections and frozen sections for fat stain (see above under "heart"). influenza;* varicella.* manifestations of liver failure. evidence of salicylate administration. assay for organic acids should rule out acylcoenzyme a dehydrogenase deficiency, and toxicity, e.g., of acetaminophen and valproic acid (l) . cardiomegaly; fatty changes of myocardium and patchy myocytolysis. viral pneumonia rarely present. acute interstitial pneumonia;* bronchitis;* hemorrhages. lipid-laden histiocytes in alveoli. record body weight and length, stature, and distribution of head, axillary, and pubic hair. record and prepare photographs of features of face and neck. prepare skeletal roentgenograms. submit samples of breast tissues for histologic study. these specimens should be collected using aseptic technique for tissue culture for chromosome analysis (see chapter ) . procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. submit for histologic study. submit samples of all portions for histologic study. if biliary atresia is suspected, follow procedures described under that heading. dissect kidneys and ureters in situ and record findings. submit samples of gonads or-if gonads cannot be identified-equivalent ridges on mesosalpinx for histologic study. also submit samples of endometrium, cervix, and vagina. record weight and submit sample for histologic study. procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. for removal and specimen preparation, see chapter . procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. for sampling and specimen preparation, see chapter . peroxidase-labeled gastrin antibodies seem to react with cells in all gastrinomas. aberrant gastrinoma may occur at hilus of spleen. metastases are found in regional lymph nodes and liver. there may be manifestations of multiple endocrine neoplasia. * secondary syphilis. levaditi's stain or warthin-starry stain is recommended for paraffin sections, and labeled fluorescent antibody techniques are recommended for frozen sections. india ink preparations or the fontana-masson silver stain has been used for the study of fresh lesions, and electron microscopy has also been employed. ( ) in adult autopsies, no special precautions are indicated (see below under "liver"). ( ) serologic studies are available from local and state health department laboratories. ( ) this is a reportable disease. external examination record and prepare photographs of all abnormalities listed in right-hand column. prepare smears and sections of acute lesions; prepare sections of older skin lesions or anogenital mucosal lesions. hunterian chancre in primary syphilis; condylomata lata. noduloulcerative gummas and scarring in later stages. cerebrospinal fluid lymph nodes heart and aorta other organs and tissues prior to wk gestation, the destructive effects of syphilis may not be seen. gummata are rare in neonates. tabes dorsalis is also uncommon. serologic diagnosis is difficult in the neonate because of transplacental transfer of maternal igg antibodies. acquired syphilis (syphilis in adulthood) is presented above under a separate heading. ( ) collect all tissues that appear to be infected. ( ) culture methods are not available, but animal inoculation can be performed. consultation with a microbiology laboratory is recommended. ( ) special stains for treponema pallidum rarely are positive except with material from fresh lesions of primary or secondary syphilis and of syphilitic hepatitis of the newborn. levaditi's stain or warthin-starry stain is recommended for paraffin sections, and labeled fluorescent antibody tech-niques are recommended for frozen sections. india ink preparations or the fontana-masson silver stain has been used for the study of fresh lesions, and electron microscopy has also been employed. ( ) in neonates, special precautions are indicated (see below under "liver") ( ) serologic studies are available from local and state health department laboratories. ( ) this is a report· able disease. (i) collect all tissues that appear infected. ( ) request aerobic and anaerobic cultures. however, the presence of tetanus bacilli established in culture is not diagnostic, since spores of c. tetani frequently contaminate wounds. record body weight and length and appearance of wound(s); photograph and excise wound(s) for histologic study. record evidence of parenteral drug abuse, especially subcutaneous injection (i.e., "skin popping"). prepare chest roentgenogram. evidence of weight loss; subcutaneous abscesses. tetanus may occur in drug addicts. tension pneumothorax* after mechanical ventilation. submit sample for microbiologic study. submit any consolidated area for microbiologic study. bacterial meningitis must be ruled out. aspiration and bronchopneumonia; embolism;* atelectasis. tetany note: see under name ofsuspected underlying conditions, such as hyperparathyroidism,* malabsorption syndrome,* or vitamin deficiency. * respiratory or metabolic alkalosis* cannot be confirmed after death; determination of blood ph is not helpful because acidity increases rapidly after death. the concentration of serum phosphates also increases after death. synonym: large ventricular septal defect with pulmonary stenosis* or atresia.* note: the basic anomaly consists of subpulmonary stenosis, ventricular septal defect, overriding aorta, and secondary right ventricular hypertrophy. for general dissection techniques, see chapter . surgical interventions include modified blalock-taus-sig subclavian-to-pulmonary arterial shunt; complete repair with patch closure of ventricular septal defect, and reconstruction of right ventricular outflow tract (with a patch or with an extracardial conduit). possible associated conditions: origin of left pulmonary artery from aorta; minor abnormalities of the tricuspid valve; absent ductal artery ( %); atrial septal defect* (in %; pentalogy of fallot); bicuspid pulmonary valve;* dextroposition of aorta; double aortic arch; hypoplastic pulmonary arteries; patent ductal artery;* patent oval foramen; complete atrioven-tricular septal defect* (usually with down's syndrome*); persis-tent left superior vena cava; pulmonary valve atresia (see "atresia, pulmonary valve, with ventricular septal defect"); right aortic arch ( %); second ventricular septal defect; origin ofleft anterior descending (lad) or right coronary artery (rca) from contralateral aortic sinus or coronary artery ( %); syndrome with absent pulmonary valve and massively dilated pulmonary arteries (rare). chest cavity heart lungs other organs record course of superior vena cava and of its tributaries. record course of thoracic aorta and of its main branches. record origin of pulmonary arteries. if infective endocarditis is suspected, follow procedures described under that heading (chapter ). for coronary arteriography, see chapter . perfuse both lungs with formalin. request verhoeff-van gieson stain. procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. persistent left superior vena cava. right aortic arch with or without right-sided ductal artery; double aortic arch; absent ductal artery. origin of the left pulmonary artery from descending aorta. for possible additional findings, see above under "note" and under "possible associated conditions." infective endocarditis* (usually of pulmonary or aortic valve). see also above under "possible associated conditions." marked right ventricular hypertrophy. right ventricular dilatation and fibrosis with late postoperative right-sided heart failure or sudden death due to arrhythmia. old in situ thrombosis of small pulmonary artery branches. paradoxic embolism; cerebral abscess. * thalassemia synonyms and related terms: congenital hemolytic anemia; alpha-thalassemia; beta-thalassemia major (cooley's anemia); beta thalassemia minor (beta-thalassemia trait). note: the changes described below are observed primarily in beta-thalassemia major. unless the cause of the renal vein thrombosis and the nature of the complicating or underlying renal disease are known, follow procedures described under "glomerulonephritis." procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. in infants, manifestations of dehydration. * pulmonary embolism. * tumor of the kidney* (renal cell carcinoma); aortic aneurysm; lymphadenopathy; other mass lesions. inferior vena cava thrombosis involving orifice or whole length of renal veins. tumor thrombus (e.g., from renal cell carcinoma). femoral vein thrombosis. rare venous malformations also may cause renal vein thrombosis (l) . hemorrhagic infartion. parenchymal renal disease with nephrotic syndrome,* including amyloidosis* and vasculitis* (these last conditions may be associated with renal vein thrombosis). acute gastroenteritis in infancy. hyperparathyroidism,* pregnancy,* trauma, and other conditions. thrombosis, venous note: for peripheral venous thrombosis, the autopsy procedures are essentially similar to those described under "phlebitis." see also under "hypertension, portal," "syndrome, budd-chiari," "thrombosis, cerebral venous sinus," "thrombosis, lateral sinus," and "thrombosis, renal vein." thymoma (see "'fumor of the thymus.") thyroiditis synonymsand related terms: chronic fibrosing thyroiditis (riedel's struma); chronic thyroiditis with transient thyrotoxicosis; hashimoto's thyroiditis; pyogenic thyroiditis; subacute (granulomatous, giant cell, de quervain's) thyroiditis. possibleassociatedconditions: withhashimoto's thyroiditisautoimmune (chronic) hepatitis,*megaloblastic anemia,*rheumatoid arthritis,*sjogren's syndrome,*and systemic lupus erythematosus.* with riedel's struma-retroperitoneal fibrosis,* sclerosing cholangitis,* sclerosing mediastinitis,*and other conditions with idiopathic fibrosis. with subacute thyroiditis-viral infection. submit sample for determination of protein concentration; record appearance of sediment. for removal and specimen preparation, see chapter . thromboses of small vessels. glomerulonephritis.* hemorrhagic necroses. proteinuria; abnormal sediment. abruptio placentae, small placenta with accelerated maturation of villi; infarcts; fibrinoid necrosis of decidual arterioles. thrombotic occlusion of small vessels with hemorrhagic necroses; anterior lobe of pituitary gland may contain necroses (see also "syndrome, sheehan's"). submit samples for histologic study. if infection is expected, submit material for microbiologic study. other procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. transposition, complete, of the great arteries note: the basic anomaly is the origin of the aorta from the right ventricle, and of the pulmonary artery from the left ventricle, with a shunt, and usually with a right anterior aorta. there are four major types: ( ) with an intact ventricular septum ( %), ( ) with a ventricular septal defect* ( %), ( ) with a ventricular septal defect and subvalvular pulmonary stenosis* ( %), and ( ) with an intact ventricular septum and subvalvular pulmonary stenosis* ( %). for general dissection techniques, see chapter . interventions include atrial septostomy or septectomy; mustard or senning atrial switch procedure; rastelli-type repair with a valved extracardiac conduit; and jatene arterial switch procedure with lecompte maneuver. possible associated conditions: abnormal origin ofcoronary arteries ( %); atrial septal defect* ( %); coarctation of the aorta;* interrution of the aortic arch;* juxtaposition of atrial appendages ( %); overriding aorta ( %); overriding pulmonary artery ( %); patent ductal artery;* patent oval foramen; subvalvular pulmonary stenosis* ( %); tubular hypoplasia ofthe aortic arch;* ventricular septal defect* ( %), often mal-alignment type ( %). synonyms: atrioventricular and ventriculoarterial discordance; l-transposition. note: the basic anomaly is a mirror-image ventricular inversion, with a left anterior aorta, and with blood flow from right atrium to left ventricle to pulmonary artery, and from left atrium to right ventricle to aorta. for general dissection techniques, see chapter . interventions include patch closure of the ventricular septal defect; relief of pulmonary stenosis; relief of tricuspid insufficiency; and insertion of pacemakers. possible associated conditions: anterior and posterior av nodes ( %), prone to develop complete heart block; dysplasia or epstein's malformation* ofleft-sided tricuspid valve ( %); mirror-image epicardial coronary artery distribution ( %); right-sided mitral valve anomalies; subvalvular pulmonary stenosis* ( %); ventricular septal defect* ( %). kinyoun's, or other acid-fast stains. polymerase chain reaction for mycobacterial dna may be helpful in the differential diagnosis ofgranulomas ( ). ( ) universal precautions should be strictly followed and aerolization should be avoided. ( ) reliable serologic studies are not available. a definite diagnosis requires isolation of the organism. ( ) this is a reportable disease. cavitary, fibrocalcific, miliary, bronchial, and other types of pulmonary tuberculosis; granulomas in hilar lymph nodes. most organs and tissues may be involved, including liver, pancreas, spleen, kidneys, adrenal glands and other endocrine glands, gonads, and lymph nodes. tuberculous meningitis; tuberculoma. iridocyclitis or panophthalmitis. tuberculous arthritis and synovitis (hips, spine, knee); tuberculous osteomyelitis (anterior aspect of vertebrae; metaphysis of long bones). part ii / diseases and conditions procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. chronic ulcerative colitis may be associated with carcinoma of bile ducts, typically arising in psc.* metastases common in regional lymph nodes and lungs. i for removal, prosthetic repair, and specimen preparation and decalcification procedures, see chapter . if osteoblastic metastases appear to be present, search for primary tumor in prostate and breast, carcinoid tumors and hodgkin's lymphoma. procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. cachexia. evidence of hypercalcemia (particularly in presence of osteolytic metastases). metastases to bone (e.g., from carcinoma of prostate, breast, bronchi, thyroid gland, kidney, or urinary bladder) usually involve red bone marrow. therefore, distal extremities are rarely involved by metastatic tumors in adults. metastases, commonly in lungs. metastatic calcification in patients with hypercalcemia. eosinophilia. myopathy.* metastases in liver and regional lymph nodes. see also above under "possible associated conditions." malakoplakia (rare) ( ) . part ii / diseases and conditions other organs and peripheral arteries tumor(s). if tumor is within a heart chamber (usually a myxoma), record extent of mobility and capability of tumor to obstruct a valvular orifice. submit samples of tumor(s) for histologic study and, particularly if the tumor is of unknown type, for immunohistochemical study and electron microscopy (chapter ). procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. metastatic carcinoma or melanoma. secondary cardiac effects by tumors include carcinoid heart disease, amyloid heart disease in multiple myeloma, and lymphocytic myocarditis in pheochromocytoma. treatment effects such as adriamycin cardiotoxicity may be noted also. tumor (myxoma) emboli in pulmonary or peripheral vessels ( ) . multiple aneurysms* of cerebral and other arteries may rarely be associated with atrial myxomas. procedures depend on expected findings or grossly identified abnormalities as listed above and in right-hand column. part ii / diseases and conditions if there was evidence of endocrine activity (see above), snap-freeze portion of fresh tumor for hormone assay. perfuse lungs with formalin. sample neoplastic and non-neoplastic tissue for histologic study. procedures depend on expected findings or grossly identified abnormalities as listed above and in right-hand column. for removal and specimen preparation, see chapter . for removal and specimen preparation, see chapter . for removal and specimen preparation, see chapter . for removal and specimen preparation, see chapter . for removal and specimen preparation, see chapter . see above under "possible associated conditions." note that hypoglycemia* generally cannot be confirmed at autopsy. nonbacterial thrombotic endocarditis.* carcinoma may be associated with asbestosis or other types of pneumoconiosis,* chronic bronchitis,* emphysema,* interstitial pneumonia,* and many other bronchopulmonary diseases. see above under "possible associated conditions." metastases (regional lymph nodes, liver, bones, brain, and many other sites) and metastatic calcification. migratory thrombophlebitis.* encephalomyelitis;* cerebellar corticoid degeneration;* subacute spinocerebellar degeneration. * crooke cell hyperplasia. myasthenic syndrome (eaton-lambert syndrome); myopathy;* dermatomyositis.* peripheral neuropathy. see above under "external examination and skin." bones (with red marrow) are common sites of metastases. record presence or absence of testes. procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. thmor of the soft tissues the possible sites and characteristics of soft tissue tumors vary so much that no universally applicable autopsy techniques can be presented. in all instances, the size, weight, and location of the tumor(s) must be recorded and tissue must be sampled for histologic study. if the tumor had not been classified prior to death, samples should be snap-frozen for immunohistochemical study. other samples should be prepared for electron microscopic study. evidence of paraneoplastic syndromes (see below) may require additional procedures. possible associated conditions: only a few paraneoplastic syndromes or systemic complications can be presented here. for the type of soft tissue tumor that was associated with each condition, see title of reference. kasabach-merritt syndrome ( ) (thrombocytopenia, microangiopathic hemolytic anemia, and acute or chronic coagulopathy associated with a rapidly enlarging hemangioma); liver function abnormalities ( ); neurofibromatosis ( ); osteomalacia ( ). heart esophagus, stomach, and duodenum other organs and tissues t in most instances, detennination of vitreous sugar concentration and of blood group is not indicated. inspect valves and prepare photographs and sections of vegetations. leave esophagus and part of duodenum attached to stomach. submit samples of tumor and of grossly uninvolved stomach for histologic study. request pas stain and warthin-starry stain for identification of h. pylori. portions of endocrine gastric tumor should be snap-frozen for immunohistochemical study and possible hormone assay. record location of tumor metastases. other organs procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. for dissection of the thoracic duct (for search of tumor cell clusters), see chapter . tumor of the uterus (with cervix) possible associated conditions: acquired immunodeficiency syndrome* (aids) ( ) if peritonitis is present, submit exudate for bacteriologic study. record volume of exudate and location of perforation. see "disease, ischemic heart." other procedures depend on grossly identified abnormalities as listed in right-hand column. procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. perfuse at least one lung with formalin. for celiac arteriography, see chapter . open stomach and duodenum in situ and record site of perforation or penetration. record measured or estimated volume of blood in gastrointestinal tract. rinse ulcer with saline to locate eroded vessel(s). pin stomach and duodenum on corckboard (serosa toward board) and fix specimen in formalin before sectioning. prepare histologic sections of ulcer(s) and of remainder of stomach. request warthin-starry stain for h. pylori. procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. free air in abdomen suggests perforation of ulcer. peritonitis. * perforation of ulcer. coronary atherosclerosis and manifestations of coronary insufficiency are commonly associated with peptic ulcer disease. in rare instances, pericardial fistula may be present from ulcer in hiatus hernia. peptic ulcers may be associated with emphysema,* tuberculosis,* and other chronic pulmonary diseases. vasculitis (see "aortitis," "arteritis, .•.," "phlebitis," and "purpura,•••") ventricle, double inlet left ( ) synonyms: single functional ventricle; univentricular heart; univentricular atrioventricular connection; holmes heart. note: the basic anomaly is the connection of both atrioventricular valves to the left ventricle, often with transposed great arteries and a restrictive ventricular septal defect. there are four major types, based on the ventriculoarterial connection: ( ) with congenitally corrected transposition ( %); ( ) with complete transposition ( %); ( ) with normally related great arteries (holmes heart; %); and ( ) double outlet, persistent truncal artery, or pulmonary atresia ( %). for general dissection techniques, see chapter . possible associated conditions: bicuspid pulmonary valve; bilateral mirror-image mitral valves (without tricuspid morphology); subvalvular aortic stenosis,* often with hypoplasia, coarctation, or interruption of the aortic arch; subvalvular pulmonary stenosis;* dual av nodes and progressive heart block in patients with congenitally corrected transposition. ventricle, double outlet right synonym: origin of both great arteries from right ventricle; taussig-bing anomaly. note: the basic anomaly is the origin of the aorta and pulmonary artery primarily from the right ventricle, usually with a ventricular septal defect, and often with subpulmonary stenosis. for general dissection techniques, see chapter . possible associated conditions: complete atrioventricular septal defect (often with asplenia syndrome); muscular discontinuity between aortic and mitral valves; right ventricular infundibular stenosis; ventricular septal defect* that may be subaortic, subpulmonary, doubly committed, or remote. examine histologically with h&e and oil red a stains. hepatomegaly and splenomegaly, jaundice. deficiency of acid lipase ( ). foam cells in the lamina propria. hepatomegaly, severe steatosis with cholesterol clefts in hepatocytes and kupffer cells. fibrosis. splenomegaly with lipid-laden foam cells. symmetric enlargement with dystrophic calcifications, giving the adrenals a gritty texture. vacuolated cells of the inner zona fasciculata and entire zona reticularis with cholesterol clefts. foam cells may be found in the lungs, bone marrow, lymph nodes, vessel walls, as well as schwann and ganglion cells. new insights into lipoprotein assembly and vitamin e metabolism from a rare genetic disease angioid streaks associated with abetalipoproteinemia familial hypobetalipoproteinemia: genetics and metabolism aviation pathology scuba diving accidents: decompression sickness, air embolism aetiology and occurrence of diving injuries. a review of diving safety scuba decompression illness and diving fatalities in an overseas military community diving-related emergencies bodies found in water achalasia and squamous cell carcinoma of the esophagus: analysis of patients esophageal achalasia and adenocarcinoma in barrett's esophagus: a report of two cases and a review of the literature a technique for necropsy evaluation of stenosis of the foramen magnum and rostral spinal canal in osteochondrodysplasia mutation analysis of the men i gene in multiple endocrine neoplasia type i, familial acromegaly and familial isolated hyperparathyroidism. i increased incidence of neoplasia in females with acromegaly benign and malignant tumors in patients with acromegaly pathology of acromegaly ethanol in sequestered hematomas stages of acute alcoholic influence/intoxication electrolyte imbalance in alcoholic liver disease collection and storage of specimens for alcohol analysis analysis for alcohol in postmortem specimens disposition of alcohol in man medicolegal aspects of alcohol. garriott jc blood, urine and other tissue specimens for alcohol analysis relationship between postmortem blood and vitreous humor ethanol levels larson cpo alcohol: fact and fallacy gradwohl's legal medicine left ventricular hypertrophy is more prominent in patients with primary aldosteronism than in patients with other types of secondary hypertension intracranial aneurysm and hemorrhagic stroke in glucocorticoid-remediable aldosteronism association of adrenal medullae and cortical nodular hyperplasia: a report of two cases with clinical and morpho-functional consideration amyloidosis: recognition, confirmation, prognosis, and therapy electron microscopy of primary and secondary cutaneous amyloidosis and systemic amyloidosis ocular amyloidosis current status review: cerebral amyloid spinal cord compression by amyloid deposits the burden of "sticky" amyloid: typing challenges quantification of cerebral amyloid angiopathy and parenchymal amyloid plaques with congo red histochemical stain kidney involvement in takayasu arteritis pathogenesis of rheumatoid arthritis splenic involvement in rheumatic diseases aspergillus sinusitis in patients with aids: report of three cases and review invasive aspergillosis in systemic lupus erythematosus aspiration (see "obstruction, acute airway aspergillus endocarditis, myocarditis and pericarditis complicating necrotizing fasciitis. case report and subject review aspergillus-related lung disease biliary atresia and the polysplenia syndrome: its impact on final outcome biliary atresia nephromegaly and elevated hepatocyte growth factor in children with biliary atresia coccidiomycosis pneumonia in a nonendemic area associated with inftiximab codeine (see "dependence, drug[s], all types or type unspecified all types or type unspecified (see "enterocolitis, other types or type undetermined chronic ulcerative (see "disease, inflammatory bowei cytologic diagnosis of cryp -tococcus neofonnans in hiv-positive patients cryptococcosis as an opportunistic infection in immunodeficiency secondary to paracoccidioidomycosis hypereosinophilia in disseminated cryptococcal disease cryptococcosis-a review of autopsy cases from a -year period in a large hospital cryptosporidiosis in persons with hiv infection broncho-pulmonary cryptosporidiosis in four hiv-infected patients cryptosporidiosis and inflammatory bowel disease sclerosing cholangitis associated with chronic cryptosporidiosis in a child with a congenital immunodeficiency disorder cyanide (see "poisoning, cyanide cyst(s), choledochal choledochal cyst-dinical features and classification cyst(s), liver (see "disease, fibropolycystic, of the liver and biliary tract pulmonary related terms: congenital cystic adenomatoid malformation; congenital pulmonary lymphangiectasis; intralobular bronchopulmonary sequestration. references infection or calcification of cysts; pyelonephritis;* perinephric abscess. obstructive uropathy in recessive polycycstic renal disease, congenital hepatic fibrosis is present and cystic bile ducts may be present in dominant cases ( ). see above under "possible associated conditions the pathology ofhuman renal cystic disease cystic kidney: genetics, pathologic anatomy, clinical picture, and prenatal diagnosis hepatic fibrosis associated with hereditary cystinosis: a novel form of noncirrhotic portal hypertension ophthalmic manifestations and histopathology of infantile nephropathic cyctinosis: report of a case and review of the literature pathological investigations of deaths following surgery, anaesthesia and medical procedures clinical outcome versus post-mortem finding in thoracic surgery: a lo-year experience order from american registry of pathology sales office intra-alveolar pulmonary siderophages in sudden infant death: a marker for previous imposed suffocation a contribution to a possible differentiation between sids and asphyxiation sudden infantdeath syndrome (sids)-standardized investigations and classification: recommendations investigation of the sudden deth of infants: a multicenter analysis alpha l-antitrypsin deficiency-associated panniculitis: resolution with intravenous alpha i-antitrypsin administration and liver transplantation clinical manifestations of alpha l-antitrypsin deficiency risk of hepatobiliary disease in adults with severe alpha i-antitrypsin deficiency (pizz): is chronic viral hepatitis b or c an additional risk factor for cirrhosis and hepatocellular carcinoma? alpha i-antitrypsin deficiency and inflammatory bowel disease severe alphal-antitrypsin deficiency (piz homozygosity) with membranoproliferative glomerulonephritis and nephrotic syndrome, reversi-bleafterorthotopic livertransplantation klippel-feil syndrome associated with malfonned larynx. case report klippel-feil syndrome in association with a craniocervical dennoid cyst presenting as aseptic meningitis in an adult: case report klippel-feil syndrome accompanied by an aneurysm of the non-coronary sinus of valsalva the hereditary ataxias clinical and genetic characterizations of q-linked autosomal dominant spinocerebellar ataxia (ad-sca) and frequency analysis of ad-sca in the japanese population cocain-related medical problems. consecutive series of cases direct cocaine cardiotoxicity demonstrated by endomyocardial biopsy the pathology and etiology of cocaineinduced heart disease ischemic colitis related to cocaine abuse hepatic dysfunction accompanying acute cocaine intoxication high incidence of malignancies in patients with dennatomyositis and polymyositis: an li-year analysis partial lipodystrophy associated with juvenile dennatomyositis: report of two cases chronic pneumomediastinum and subcutaneous emphysema: association with dennatomyositis spontaneous esophageal rupture in adult dennatomyositis polyneuropathy in juvenile dermatomyositis combined glucose and lactate values in vitreous humor for postmortem diagnosis of diabetes mellitus diabetes is related to cerebral infarction but not to ad pathology in older persons hepatic lesions resembling alcoholic liver disease relationship between cardiomyopathy and liver disease in chronic alcoholism cyanamide-associated alcoholic liver disease: a sequential histologic evaluation caroli's disease: - experiences caroli 's disease and autosomal dominant polycystic kidney disease: a rare asso-ciation? spontaneous rupture of a bile duct and its endoscopic management in a patient with caroli's syndrome bartonella henselae endocarditis in an immunocompetent adult cat-scratch disease and bacillary angiomatosis attack, transient cerebral ischemic" and "infarction, cerebral!') cholesteryl ester storage disease: hepatopathology and effects of therapy with lovestatin clinical, biochemical and histological analysis of seven patients with cholesterol ester storage disease cutaneous manifestations of chronic granulomatous disease. a report of four cases and review of the literature aspergillus endocarditis in chronic granulomatous disease colitis complicating chronic granulomatous disease. a clinicopathological case report hypertrophic cranial pachymeningitis with spinal epidural granulomatous lesion ocular pathologic findings of chronic granulomatous disease of childhood review: creutzfeldt-jakob disease survival of creutzfeldt-jakob disease virus in formol-fixed brain tissue guidelines for high risk autopsy cases: special precautions for creutzfeldt-jakob disease tissue handling in suspected creutzfeldt-jakob disease and other human spongiforme encephalopathies (prion diseases) cerebrospinal fluid concentration of neuron-specific enolase in diagnosis of creutzfeldt-jakob disease autopsy case of sporadic creutzfeldt-jakob disease presenting with signs suggestive of brainstem and spinal cord involvement metastatic crohn's disease: a rare cutaneous manifestation crohn's disease with pulmonary involvement in a year old boy mesenteric fibromatosis asso-ciated with crohn's disease cholangiocarcinoma and crohn's disease the pancreas as a site of granulomatous inflammation in crohn's disease dermatomyositis associated with crohn's disease cardiocyte storage and hypertrophy as a sole manifestation of fabry's disease. report on a case simulating hypertrophic non-obstructive cardiomyopathy an atypical variant of fabry's disease with manifestations confined to the myocardium pulmonary involvement in fabry disease cerebrovascular complications offabry's disease high incidence of thrombosis in fabry's disease oral involvement in chronic graft versus host disease after allogenic bone marrow transplantation massive pericardial effusion complicating the course of chronic graft-versus-host disease (cgvhd) in a child with acute lymphoblastic leukemia following allogeneic bone marrow transplantation the histological spectrum of pulmonary graft-versushost disease in bone marrow transplant recipients bronchiectasis in bone marow transplantation oncocytic metaplasia of bile duct epithelium in hepatic gvhd immune-mediated myelopathy after allogeneic marrow transplantation gunther's (see ''porphyria, congenital erythropoietic heavy-chain synonyms and related terms: gamma heavy-chain inflammatory bowel disease (first of two parts) takayasu's arteritis associated with idiopathic ulcerative colitis cerebrovascular complications ofinflammatory bowel disease pathology of inflammatory bowel disease legionnaires' disease source of infection for sporadic legionnaires' disease: a review legionnaire's disease associated with macular rash; two cases severe skin loss after meningococcal septicemia: complications in treatment osteonecrosis following meningococcemia and disseminated intravascular coagulation in an adult: case report and review rhabdomyolysis during the subacute stage of meningococcal sepsis primary meningococcal arthritis in a child: case report and literature review chondrosarcoma as a complicating factor in paget's disease of bone lymphoma arising in paget's disease osteoarthritis and paget's disease sickle cell disease sickle cell disease and sudden death: a retrospective/prospective study of autopsy cases and literature review vascular lesions of the liver in sickle cell disease. a clinicopathologic study in living patients whipple's disease and "tropheryma whippelii periodic acid-schiff-negative granulomatous lymphadenopathy in patient with whipple's disease. localization of the whipple bacillus to noncaseating granulomas by electron microscopy serial imaging studies of cerebral whipple's disease: from onset to end the wilson's disease gene is a putative copper transporting p-type atpase similar to menke's gene the liver biopsy diagnosis of wilson's disease. methods in pathology hepatolenticular degeneration (wilson's disease) venous air embolism in homicidal blunt impact head trauma: case reports a practical device for demonstrating air embolism amniotic fluid . kulka w. laboratory methods and technical notes: a practical device for demonstrating air embolism proof of fatal air embolism venous air embolism from head and neck wounds hantavirus pulmonary syndrome: a clinical description of patients with a newly recognized disease liver involvement in hemorrhagic fever with renal syndrome hantavirus infection induces a typical myocarditis that may be responsible for myocardial depression and shock in hanta virus pulmonary syndrome lassa fever in the united states. investigation ofa case and new guidelines for management lassa fever: review of virology, immunopathogenesis, and algorithms for control and therapy early diagnosis of lassa fever by reverse transcription-pcr rheumatic pneumonia: reappearance of a previously recognized complication of rheumatic fever acute rheumatic fever and poststreptococcal acute glomerulonephritis caused by t serotype streptococcus scleritis, uveitis, and glaucoma in a patient with rheumatic fever comparison of clinical features and pathologic findings in fatal cases of typhoid fever during the initial and later stages of the disease typhoid fever complicated by acute renal failure and hepatitis: case reports and review hemophagocytosis and granulomas in the bone marrow of a child with down syndrome diseases involving intrahepatic bile ducts idiopathic eosinophilic esophagitis with stricture formation in a patient with longstanding eosinophilic gastroenteritis eosinophilic gastroenteritis presenting with colitis and cholangitis eosinophilic gastroenteritis presenting with acute pancreatitis idiopathic midline destructive disease: does it . mendenhallwmetai.lethalmidlinegranoloma-nasalnaturalkillerff-celi exist? nasal cocain abuse mimicking midline granuloma pulmonary lymphomatoid granulomatosis in acquired immunodeficiency syndrome: lesions with epstein-barr virus infection recurrent bilateral spontaneous pneumothoraces associated with pulmonary angiocentric immunoproliferative lesion lymphomatoid granulomatosis: pathogenesis, pathology and clinical implications wegener's granulomatosis: histological documentation of common and uncommon manifestations in patients necrotizing granulomatosis of the breast wegener's granulomatosis with gangrene of toes pathology of pulmonary granulomatous vasculitis. sarcoidosis gunshot (see "injury, firearm splenic involvement in rheumatic diseases wegener's granulomatosis, acute laryngotracheal airway obstruction and death in a -year-old female: case report and review of the literature antiendothelial antibodies in patients with wegener's granulomatosis: prevalence and correlation with disease activity and manifestations. i rheumatoi transfusion-transmitted disease references flattening, broadening, and possible fusion of villi. phlegmonous inflammation and edema, mainly in ileocecal region encephalitis.* leukopenia; thrombocytopenia; aplastic anemia ( ) (pancytopenia*) synovitis (arthritis*) hepatitis g virus infection in hepatitis c virus-positive patients co-infected or not with hepatitis b virus and/or human immunodeficiency virus hepatitis g and c coinfection in children tumor of the liver diaphragmatic related terms: congenital diaphragmatic hernia fulminant hepatitis in patients undergoing liver transplantation: evidence for anon-a, non-b, non-c marrow transplantation for hepatitis-associated aplastic anemia: a follow up oflong-term survivors langerhans cell histiocytosis research. past, present, and future an update on c onality, cytokines, and viral etiology in langerhans cell histiocytosis langerhans cell histiocytosis in adults pulmonary langerhans cell granulomatosis central nervous system disease in langerhans cell histiocytosis immunohistochemical analysis oflangerin in langerhans cell histocytosis and pulmonary inflammatory and infectious diseases pathological approach to the diagnosis of hydrocephalus thoracic lipomeningocele associated with diastematomyelia, tethered spinal cord, and hydrocephalus. case report infectious causes ofhydrops fetalis human parvovirus b infection associated with hydrops fetalis cardiac abnormalities associated with hydrops fetal is gmi-gangliosidosis presenting as nonimmune hydrops fetalis: a case report a case of recurrent infantile polycystic kidney associated with hydrops fetalis the pathologist and the hydropic placenta, fetus, or infant chronic diarrhea associated with thymoma and hypogammaglobulinemia (good's syndrome) systemic amyloidosis in a patient with hypogammaglobulinemia. i hcv infection in patients with primary defects of immunoglobulin production encepahomyelitis in primary hypogammaglobulinemia retinitis pigmentosaand hypogammaglobulinemia intestinal obstruction in the newborn with congenital syphilis meconium ileus and its equivalent as a risk factor for the development of cirrhosis: an autopsy study in cystic fibrosis biliary atresia and meconium ileus associated with nieman-pick disease postmortem cranial mri and autopsy correlation in suspected child abuse cytomegalovirus-associated pseudotumor simulating gastric malignancy in acquired immuno-deficiency syndrome: a case report with review of literature bone marrow fibrin ring granulomas and cytomegalovirus infection cutaneous reactions following herpes zoster infections: report of three cases and a review of the literature herpes zoster and internal malignancy posteriorhorn varicella-zoster virus myelitis death or injury caused by electrocution myocardial hemorrhagic necrosis in delayed death from electrocution safety in bullet recovery procedures: a study of the black talon bullet lightning injuries: prognostic signs of death lightning injuries adrenal insufficiency, recurrent bacteremia, and disseminated abscesses caused by nocardia asteroides in a patient with acquired immunodficiency syndrome ards and adrenal insufficiencyassociated with the antiphospholipid antibody syndrome adrenal insufficiency from bilateral adrenal hemorrhage after total knee replacement surgery non-hodgkin's lymphoma presenting with adrenal insufficiency and hypothyroidism: an autopsy case report a case ofprimary unilateral adrenal burkitt-like large cell lymphoma presenting as adrenal insufficiency effect of growth hormone on fatty liver in panhypopituitarism increased fracture frequency in adult patients with hypopituitarism and gh deficiency pituitary abscess the liver: an atlas and text of ultrastructural pathology intubation (see "injury, intubation iodine (see "poisoning, iodine attack, transient cerebral ischemic" and "infarction, cerebral heart (see "disease, ischemic heart isomorism (see "syndrome, polysplenia and asplenia a study on performance of two serological assays for diagnosis of leprosy detection of mycobacterium leprae infection by pcr pathology of eye in leprosy references intervillous abscesses containing grampositive, non-acid-fast bacilli bacteria are predominantly intracellular. enterocolitis. listeria monocytogenes can be cultured from meconium. generalized lymphadenitis. disseminated abscesses and/or granulomas, particularly after transplacental infection listeria monocytogenes empyema in an hiv infected patient fatal listeria meningitis, endocarditis and pericarditis in a patient with haemochromatosis liver abscesses due to listeria monocytogenes yust . brainstem abscess and meningitis due to listeria monocytogenes in an adult with juvenile chronic arthritis pulmonary hypertension associated with systemic lupus erythematosus: predominantly thrombotic arteriopathy accompanied by plexiform lesions the liver in systemic lupus erythematosus: pathologic analysis of cases and review of japanese autopsy registry data chronic pancreatitis: a complication of systemic lupus erythematosus disseminated perivenous necrotizing encephalomyelitis in systemic lupus erythematosus: report of an autopsy case skeletal muscle lymphocytic vasculitis in systemic lupus erythematosus: relation to disease activity clinically occult avascular necrosis of the hip in systemic lupus erythematosus storage histiocytes and hemophagocytosis: a common finding in the bone marrow of patients with active systemic lupus erythematosus extensive medium vessel vasculitis with sle: an unsual association cutaneous manifestations of sexually transmitted ing cause of proctitis in men who have sex with men lymphogranuloma venereum as a cause cutaneous malakoplakia in a patient with acquired immunodeficiency syndrome (aids) extensive malakoplakia ofthe nasopharynx: management of a rare disease malakoplakia and colorectal adenocarcinoma coinfection is common in measles associated pneumonia giant cell pneumonia: light microscopy, immunohistochemical, and ultrastructural study of an autopsy case subacute sclerosing panencephalitis manifesting as viral retinitis: clinical and histopathologic findings intestinal neuronal dysplasia thoracic lipomeningocele associated with diastematomyelia, tethered spinal cord, and hydrocephalus. case report disease, meningococcal encephalitis, all types or type unspecified" and "meningitis mercury (see "poisoning, mercury with myelofibrosis synonym: idiopathic myelofibrosis acute cardiac tamponade associated with pericardial extramedullary hematopoiesis in agnogenic myeloid metaplasia methyl alcohol) (see "poisoning, methanol (methyl alcohol) thrombotic thrombocytopenic (see "purpura, thrombotic thrombocytopenic epstein-barr virus in inflammatory diseases of the liver and liver allografts: an in situ hybridization study fulminant hepatitis due to epstein-barr virus infection the mucopolysaccharidoses: a clinical review and guide to management biochemical diagnosis of mucopolysaccharidoses: experience of diagnoses in a -year period ( - ) mucormycosis synonym: phycomycosis; zygomycosis. note: diseases that may be complicated by mucormycosis include bums,* diabetes mellitus,* leukemia,* lymphoma,* and tuberculosis cardiac involvement in mucopolysaccharidosis: effects of allogeneic bone marrow transplantation mitral and aortic regurgitation in patients with mucopolysaccharidosis toxic epidermal necrolysis possibly linked to hyperacute graft-versus-host disease after allogeneic bone marrow transplantation pulmonary complications in toxic epidermal necro-iysis: a prospective clinical study references possible or expected findings pyelonephritis;* nephrocalcinosis; granulomas; tumor infiltrates; manifestations of the conditions listed below under "other organs renal stones in patients with rheumatoid arthritis nephrolithiasis as a presenting feature ofchronic sarcoidosis: a prospective study nephrolithiasis and risk of hypertension posterior lens opacities; retinal hamartomas. peripheral neuropathy with focal schwannomatous changes or onion-bulb-like schwann cell or perineural cell proliferation neurofibromatosis type i. in: pathology and genetics of tumours of the nervous system neurofibromatosis type . in: pathology and genetics oftumours ofthe nervous system neurological complications involving the central nervous system in neurofibromatosis type i primary cutaneous nocardia asteroides infection after heart transplantation native valve endocarditis due to nocardia-like organisms hepatobiliary complications of total parenteral nutrition total parenteral nutrition: a histopathologic analysis of the liver changes in children obesity and breast cancer risk nonalcoholic steatohepatitis obesity cardiomyopathy: pathogenesis and pathophysiology bennett cpo pachydennoperiostitis in childhood unilateral hypertrophic osteoarthropathy associated with aortofemoral graft infection gastroesophageal reflux and esophagitis-associated hypertrophic osteoarthropathy hypertrophicosteoarthropathy caused by lipoid pneumonia achondroplasia" and dyschondroplasia, oilier's osteogenesis imperfecta synonyms and related terms: lobstein's syndrome osteogenesis imperfecta congenita; type i, ii, and iii; osteogenesis imperfecta cystica hypercalcemia in osteogenesis imperfecta treated with pamidronate gastrointestinal problems in patients who have type-iii osteogenesis imperfecta osteomalacia related terms: osteoporosis;* renal osteodystrophy; rickets. note: many possible causes of osteomalacia may not be apparent at autopsy, e.g., sodium fluoride or diphosphonate toxicity or use of anticonvulsant drugs osteomalacia associated with adult fanconi syndrome: clinical and diagnostic features epstein-barr virus in b-celllymphomas associated with chronic suppurative inflammation osteomyelitis and osteonecrosis in inflammatory bowel disease references hyperlipidemia; hyperuricemia. fracture or traumatic dislocation of hip see above under "note osteomyelitis and osteonecrosis in inflammatory bowel disease osteonecrosis and human immunodeficiency virus infection report of fifteen cases. therapeutic recommendations malignant infantile osteopetrosis: otolaryngological complications and management renal tubular acidosis and osteopetrosis with carbonic anhydrase ii deficiency: pathogenesis of impaired acidification spondylolysis in children who have osteopetrosis osteopetrosis with arnold chiari malformation type and brain stem compression osteoporosis and atherosclerosis in chronic renal failure endocrine disorders and osteoporosis otosclerosis: a measles virus associated inflammatory disease correlations between pathologic changes in the stapes and conductive hearing loss in otosclerosis the aetiology of otosclerosis: a review of the literature etiologic links between chronic pancreatitis and pancreatic cancer hyperparathyroidism and pancreatitis during pregnancy pancytopenia related terms: agranulocytosis;* aplastic anemia; fanconi's anemia.* note: some causes of pancytopenia such as adverse drug reactions (e.g., due to antimetabolites, sulfa drugs thrombotic thrombocytopenic purpura/hemolytic uremic syndrome secondary to pancreatitis retinopathy as asys-temic complication of acute pancreatitis references mesenteric panniculitis necrotizing pancreatitis. * vasculitis with thrombi; adrenocortical infarctions. vasculitis with thrombi and infarctions. relapsing polychondritis ( ). see also above under "possible associated conditions subcutaneous panniculitic t-cell lymphoma is a tumor of cytotoxic t lymphocytes abecassism. alpha i-antitrypsindefi-ciencyassociated panniculitis: resolution with intravenous alpha l-anti-trypsin administration and liver transplantation blind loop syndrome: an unusual cause of panniculitis. j am acad paracoccidioidomycosis synonyms: paracoccidioides brasiliensis infection cutaneous panniculitis and relapsing polychondritis: two cases normal subcutaneous fat, necrosis of adipocytes and classification of the panniculitides paraneoplastic pemphigus a case of pemphigus vulgaris with esophageal involvement rheumatoid constrictive pericarditis (clinical conference) benign paroxysmal (see "fever, familial mediterranean pneumatosis intestinalis in pediatric acquired immunodeficiency syndrome pneumatosis cystoides intestinalis: an unusual complication of systemic amyloidosis pneumatosis intestinalis in children with primary combined immunodeficiency pneumatosis cystoides intestinalis with abdominal free air in a -year-old girl after allogeneic bone marrow transplantation pneumatosis intestinalis: a review pneumatosis coli: a proposed pathogenesis based on study of cases and review of the literature collagenous inorganic dust pneumoconiosis, diffuse type: aluminosis; asbestosis; chronic pulmonary berylliosis; talcosis; collagenous inorganic dust pneumoconiosis, nodular type: silicosis; noncollagenous inorganic dust pneumoconiosis (includes mixed-dust fibrosis): baritosis; china clay pneumoconiosis; chromite pneumoconiosis; coal worker's pneumoconiosis; fuller's earth pneumoconiosis; hematite or magnetite miner's lung; siderosis or welder's lung; stannosis; organic dust pneumoconiosis: byssinosis. note: in addition to the aforementioned classic forms of pneumoconiosis, many other airborne substances have been impli-cated in recent years, for example, cerium uncommon causes ofoccupational interstitial lung diseases rare earth (cerium oxide) pneumoconiosis: analytical scanning electron microscopy and literature review diagnostic criteria for chronic beryl ium disease (cbd) based on the uk registry - pneumocystis carinii (see "infection, pneumocystis carinii this condition is diagnosed by inspection, palpation, and roentgenography. tension pneumomediastinum may autopsy evaluation of asbestos exposure: retrospective study of cases with quantitation of ferruginous bodies in digested lung tissue atypical mycobacteriosis as a complication of talc pneumoconiosis silicosis, mixed dust pneumoconiosis, and lung cancer complications associated with the use ofthe esophageal-tracheal combitube pneumomediastinum: an unusual complication of asthma in a young man spontaneous pneumomediastinum in a patient with bronchiolitis obliterans after bone marrow references i. katzenstein a-l, myers j. idiopathic pulmonary fibrosis: clinical relevance of pathologic classification update on lymphoid interstitial pneumonitis lymphocytic interstitial pneumonitis and nonspecific interstitial pneumonitis histologic diagnosis of extrinsic allergic alveolitis role of electron microscopy in interstitial lung disease detennination of arsenic in hair using neutron activation arsenic intoxication associated with tubulointerstitial nephritis atrioventricular block after administration of atropine in patients follow-ing cardiac transplantation death following intentional methyl bromide poisoning: toxicological data and literature review pulmonary edema, alveolar wall damage, and interstitial pneumonia after acute inhalation. severe pulmonary fibrosis may develop in chronic cases. gastroenteritis after nonlethal food poisoning. degeneration of proximal tubules and proteinuria in acute poisoning degenerative changes of liver and myocardium problems in the analysis of cadmium in autopsied tissues elevated urinary cadmium concentrations in a patient with acute cadmium pneumonitis cadmium-associated renal disease the effects of storage conditions on the stability of carbon monoxide in postmortem blood acute renal failure, poisoning, carbon tetrachloride synonym: tetrachloromethane poisoning. note: toxicologic sampling of body fluids and organs should be done routinely in all cases. in many instances, however, death occurs i wk to d after exposure, and by this time no carbon tetrachloride is demonstrable. death may be sudden compartment syndrome, and systemic capillary leak syndrome complicating carbon monoxide poisoning southern je acute hydrocephalus following carbon monoxide poisoning prevention of occupational cyanide exposure in autopsy prosectors gradwohl's legal medicine references possible or expected findings digoxin values in digitalis toxicity are greater than ng/ml ( ) digoxin concentration may be higher or lower than concentration in serum, depending on how long before death drug was taken (i) digoxin concentrations in postmortem specimens after overdose and therapeutic use a fluorimetric determination ofmyocardial digoxin at autopsy, with identification of digitalis leaf, digitoxin and gitonin ethylene glycol poisoning: toxicokinetic and analytical factors affecting laboratory diagnosis ethylene glycol poisoning: case report of a record-high level and a review lead concentrations in human plasma, urine and whole blood trace metals (lead and cadmium screening) an emg case report oflead neuropathy years after a gunshot injury aminoaciduria and glycosuria following severe childhood lead poisoning risk of nervous system cancer among workers exposed to lead demonstration of mercury in the human brain and other organs years after metallic mercury exposure spontaneous exfoliation of teeth following severe elemental mercury poisoning: case report and histological investigation for mechanism. oral surg oral med oral pathoi acute chemical pancreatitis associated with nonfatal strychnine poisoning homicide by strychnine poisoning coronary arteritis, with or without aneurysms and infarctions, primarily in childhood. myocardial hypertrophy secondary to hypertension. * minimal or no involvement by polyarteritis nodosa; considerable involvement in other types ofnecrotizing vasculitis (see "arteritis, all types or type unspecified") with or without formation of aneurysms and infarctions, may occur in all organs. the liver may show bile duct injury and rarely, nodular regenerative hyperplasia ( ) more frequently involved in giant cell elastic arteritis.* polyarteritis of small muscular arteries, including vasa nervorum. arthritis* may rarely be present with swollen joints. infarctions;* subarachnoid hemorrhage; arteritis of cerebral arteries. papillitis; retinal hemorrhages polyarteritis nodosa-like vasculitis in human immunodeficiency virus infection the coexistence of familial mediterranian fever and polyarteritis nodosa: report of a case microscopic polyarteritis during polymyalgia rheumatica remission development of polyarteritis nodosa in the course of inactive systemic lupus erythematosus bone marrow pathology in relapsing polychonditis: high frequency of myelodysplastic syndrome relapsing polychondritis associated with subclinical sjo-gren's syndrome and phlegmon of the neck giant cell arteritis and polymyalgia rheumatica posterior scleritis and polymyalgia rheumatica polymyalgia rheumatica in patients with ankylosing spondylitis: a report of cases inflammatory changes of hip synovial structures in polymyalgia rheumatica polymyositis (see "dermatomyositis polyneuritis (see "syndrome, guillain-barre familial, and related syndromes related terms: cowden's disease (multiple hamartoma syndrome); familial colonic polyposis gardner's syndrome; non-polyposis syndrome (hereditary nonpolyposis colorectal cancer syndrome) sampling for histologic study depends on expected findings or grossly identified abnormalities as listed in right-hand column. (see also below under "soft tissues syndrome; mucocutaneous pigmentations (buccal, perioral, priorbital, distal extremities) in peutz-jeghers syndrome;* papules in face and oral mucosa in cowden's disease; tumors of skin and subcutis (see below under "soft tissues gardner's syndrome. osteomas or exostoses (mandible, calvara desmoid in familial adenomatous polyposis malignant potential in intestinal juvenile polyposis syndromes ampullary carcinoma in familial adenomatous polyposis adenoma of the common bile duct in gardner's syndrome may cause relapsing acute pancreatitis orbital osteoma in gardner's syndrome myelopathylmyelitis," and "syndrome, guillain-barre.") references possible or expected findings hypertrichosis; erythrodontia; scarring and mutilation of hands and face. hemolytic anemia. erythrocytes contain large amounts of uroporphyrin i; nonnoblasts and reticulocytes exhibit intense red fluorescence uroporphyrin i and coproporphyrin i in high concentrations. splenomegaly (may have been treated by splenectomy) correction of congenital erythropoietic porphyria by bone marrow transplantation successful cord blood stem cell transplantation for congenital erythropoietic porphyria (gunther's disease) congenital erythropoietic porphyria associated with nephrotic syndrome atypical red cell inclusions in congenital erythropoietic porphyria possible associated conditions: adverse drug reaction; chronic alcoholism;* chronic hepatitis c ( ); human immunodeficiency virus infection porphyria cutanea tarda and human immunodeficiency virus: two cases associated with hepatitis c porphyria cutanea tarda and antibodies to hepatitis c virus an unhappy triad: hemochromatosis, porphyria cutanea tarda and hepatocel ularcarcinoma-a case report variegate porphyria possible associated conditions: ebstein's malformation of tricuspid valve. references possible or expected findings chronic eczematous skin lesions or superficial scarring; nail changes. perivascular pas-positive hyaline ( ) brown protoporphyrin deposits with red to yellow birefringence with a maltese cross configuration in hepatocytes, kupffer cells, and bile canaliculi erythropoietic protoporphyria cytofluorometry as a diagnosis of protoporphyria recessive inheritance of erythropoietic protoporphyria with liver failure pseudogout synonym: calcium pyrophosphate dihydrate (cppd) deposition disease puncture grossly affected joints and submit sample of synovial fluid for crystal analysis under compensated polarized light ( ) references possible or expected findings punctate calcium deposits in kneejoints and, less commonly, in joints of hips, ankles, shoulders, or wrists or in symphysis ossium pubis and intervertebral disks. arthritis* with synovitis. crystals of calcium pyrophosphate dihydrate in periarticular tissue ( ) and synovial fluid. cartilage contains calcium salts of pyrophosphate, hydroxyapatite, and orthophosphate. alkaptonuria;* gout calcium pyrophosphatedihydratedeposition disease presenting as tumoral calcinosis (periarticularpseudogout) calcifications in dermis; calcifications of blood vessels. diaphragmatic hernia.* hypertensive cardiomegaly. characteristic plaques in pericardium and endocardium, with or without mitral valve involvement. coronary atherosclerosis may have been a causeofangina( ).coronarythrombosisand myocardial infarction may be present also. accelerated atherosclerosis; calcification of peripheral vessels. gastrointestinal hemorrhage;* peptic ulcer degeneration of bruch's membrane with retinal hemorrhages; sclerosis of choroid vessels calcification of elastic fibers in pseudoxanthoma elasticum new report of severe coronary artery disease in an eighteen-year-old girl with pseudoxanthoma e asticum. case report and review of the literature hivrelated thrombotic thrombocytopenic purpura: report of cases and a review of the literature beham-pyelonephritis synonyms and related terms: acute ascending pyelone angiotropic large cell lymphoma presenting as thrombotic micro-angiopathy (thrombotic thrombocytopenic purpura) splenic pathology in thrombotic thrombocytopenic purpura emphysematous pyelonephritis in diabetic patients nephrobronchialfistulaandlungabscessresulting from nephrolithiasis and pyelonephritis a case ofan enterorenal pyothorax (see "empyema, pleurai.") fistula and pyelonephritis with airin renal pelvis systemic amyloidosis secondary to pyonephrosis. resolution after nephrectomy medicolegal investigation of death the laboratory's role in detecting sexual assault toluidine blue in the detection at autopsy of perineal and anal lacerations in victimes of sexual abuse sarcoidosis presenting as heart disease sarcoidosis and its ocular manifestations rheumatic features of sarcoidosis cutaneous sarcoidosis: differential diagnosis schistosoma mansoni and s. haematobium infections in egypt. i. evaluation of techniques for recovery of worms and eggs at necropsy. am i a quantitative post-mortem study of schistosomiasis mansoni in man immunodiagnosis of schistosomiasis. screen with fast-elisa and confinn with imrnunoblot hepatic connective tissue changes in hepatosplenic schistosomiasis schistosomiasis in women: manifestations in the upper reproductive tract sclerosis, systemic amyotrophic lateral (see "disease, motor neuron diffuse (see "sclerosis, schilder's cerebrai multiple synonyms and related terms: acute and subacute variants: acute multiple sclerosis (marburg type), acute necrotizing myelopathy; concentric sclerosis (ba type); concentric lacunar leukoencephalopathy; encephalitis periaxialis diffusa (schilder's type; see also next entry silicone ganuloma in acral skin in a patient with silicone-gel implants and systemic sclerosis organizing diffuse alveolar damage associated with progressive systemic sclerosis bayle , fiessinger in. brain involvement in scleroderma: two autopsy cases skeletal muscle pathology in systemic sclerosis mechanisms of scleroderma-induced lung disease thberous sclerosis complex and subependymal giant cell astrocytoma bilateral temporal arachnoid cysts associated with tuberous sclerosis poisoning, alkaloid accident, diving [skin or scuba scurvy (see "deficiency, vitamin c shigellosis (see "dysentery, bacillary shock related terms: anaphylactic shock; bacteremic shock; many others. note: see also under name of suspected underlying condition, such as "bums intestinal histological and ultrastructura inflammatorychanges in spondyloarthropathy and rheumatoid arthritis the sacroiliac joint in the spondyloarthropathies granulomatous ileitis in a patient with ankylosing spondylitis sporotrichosis synonym: sporothrix (sporotrichum) schenckii infection. note: ( ) mckiernan . partial lipodystrophy in coeliac disease coeliac disease and lymphoma: current status review: nonalcoholic steatohepatitis alcoholic and non-alcoholic wernicke's encephalopathy. be alert to the preventable and treatable disease non-alcoholic fatty liver disease in the metabolic syndrome the placenta in stillbirth estimating the time of death in stillborn fetuses: i. histologic examination of fetal organs: an autopsy study of stillborns estimating the time of death in stillborn fetuses: ii. histologic evaluation ofthe placenta; a study of stillborns estimating the time ofdeath in stillborn fetuses: iii. external fetal examination; a study of stillborns fat distribution in the adrenal cortex as an indication of the mode of intrauterine death stimulant(s) (see "dependence, drug(s), all types or type undetermined cutaneous manifestations of the acquired immunodeficiency syndrome aids: cardiac findings from autopsies the liver in hiv infection neuropathology of the spinal cord in the acquired immunodeficiency syndrome postmortem enzyme immunoassay for human immunodeficiency virus bk virus-associated renal failure among hiv patients - or write to american registry ofpathology sales office pathology of acquired immunodeficiency syndrome (aids) in children thymus involution in the acquired immunodeficiency syndrome micrencephaly in children congenitally infected with human immunodeficiency virus-a gross-anatomical morphometric study a rapid method for detecting the predominant ashkenazi jewish mutation in the bloom's syndrome gene bloom syndrome: multiple retinopathies in a chromosome breakage disorder severe eczema in a patient with di-george's syndrome digeorge's syndrome orthe iii -iv pharyngeal pouch syndrome: pathology and a theory of pathogenesis basal ganglia calcification and psychosis in q . deletion syndrome down's synonyms and related terms: mongolism possible associated conditions: acute lymphocytic, my references epicanthal folds; cleft lip/palate; high arched palate; furrowed tongue; rhagades. depressed nasal bridge small, broad or fiat nose; slanted palpebral fissures; fiat occiput; brachycephaly; palmar "simian crease"; short fifth middle finger ventricular septal defect;* complete atrioventricular septal defect* ( ). duodenal stenosis and atresia; imperforate anus. microcephaly; poorly developed secondary gyri hypoplastic brain stem, medulla and cerebellum; polymicrogyria; neurofibrillary tangles; meningomyelocoele. acute lymphocytic leukemia; acute myelocytic leukemia; acute megakaryocytic leukemia cardiovascular disease in down syndrome possible or expected findings hyperelasticity of skin, bruises or scars, hemorrhages, and hyperpigmentation. lipomatous pseudotumors that may be calcified or ossified. normal or abnormal amounts and fragmentation of elastic tissue. abnormalities of collagen. dislocation of hip, shoulder, patella, radius, or clavicle. loose-end clavicles; spondylolisthesis mitral valve prolapse; aortic insufficiency.* aortic dissection, aneurysm, ectasia, occlusion ( ). various congenital anomalies. congenital anomalies of gastrointestinal and genitourinary tracts. bleeding from various organ sites vascular ehlers-danlos syndrome: imaging findings note: this syndrome belongs to a large family (with more photograph abnormal features as listed in right-hand column. record appearance of external genitalia. prepare skeletal roentgenograms. procure long bones and vertebral bodies short-limb dwarfism* with narrowing of the rib cage; polydactyly; dysplasia of fingernails; thin and sparse hair; premature eruption of teeth; defective dentition; eye abnormalities; upper lip bound down by multiple frenula. cryptorchidism; epispadias; hypospadias. chondrodysplasia with acromelic micromelia (shortening of the distal segment of the limb); fusion of capitate and hamate bones of wrist; defects of lateral aspect of proximal tibia see lesions listed above under "external examination empty sella synonyms and related terms: primary empty sella syndrome; secondary empty sella syndrome (e.g., after surgical removal or spontaneous infarction of pituitary adenoma) chorea, philic pneumonia: histopathologic features in nine cases. am rev resp hereditary ascariasis and hookworm chronic eosinophilic pneupophosphatemic vitamin d-resistant rickets; galactosemia;transport defect; tyrosinemia. * excludes fan- weight and external measurements; record and photograph abnormalities as listed in right-hand column. prepare skeletal roentgenograms. radiographs of long bones. submit for tissue culture for possible enzyme analysis. possible or expected findings redlblond hair, fair skin (diminished pigmentation); dehydration;* stigmata of hypothyroidism;* delay of sexual maturation. rickets; osteomalacia.* positive rheumatoid factor. various types of pneumonia after splenectomy, presence of accessory spleen may account for treatment failure. manifestations of portal hypertension.* lymphadenopathy of mediastinal and paraaortic lymph nodes. anemia;* neutropenia sinusoidal lymphocytosis of the liver in felty's syndrome with a review of the liver involvement in felty's syndrome splenic involvement in rheumatic diseases felty's and pseudo-felty's syndrome nodular regenerative hyperplasia of the liver in rheu-matic diseases: report of seven cases and review of the literature fetal alcohol syndrome: craniofacial and central nervous system manifestations goodpasture's syndrome anti-glomerular basement membrane glomerulonephritis: a morphologic study of cases gronblad-strandberg (see "pseudoxanthoma elasticum acute inflammatory polyradiculoneuropathy; guillain-barre-strohl syndrome hemolytic uremic syndrome/thrombotic thrombocytopenic purpura: pathophysiology and treatment cyclosporin-induced hemolytic uremic syndrome: factors that obscure its diagnosis enterohemorrhagic escherichia coli :h : an emerging pathogen hemolytic uremic syndrome as a presenting form of hiv infection hemolytic uremic syndrome in prostatic carcinoma diabetes secondary to genetic disorders. baillieres suprasellar tumors of maldevelopmental origin in klinefelter's syndrome. a report of two cases klippel-feu synonym: congenital fusion of cervical vertebrae primary yolk sac tumor of the prostate in a patient with klinefelter's syndrome extragonodal germ cell tumors are often associated with klinefelter syndrome organs and tissues procedures possible or expected findings external examination prepare roentgenograms of chest, neck, and head skull, spine, brain, and spinal cord myasthenia gravis disorders with dysraphia (see below), fusion of cervical vertebrae. congenital elevation of the scapula (sprengel's deformity) chiari malformation;* basilar impression;* meningomyelocele; platybasia;* spinal cord compression; weight and length; record and photograph abnormalities as listed in right-hand column. record weight and sample for histologic study. if renal transplantation ( ) had been carried out, see also under that heading. follow procedures described under "glomerulonephritis retinal dystrophy ( ) and other retinal changes the cardinal manifestations of bardet-biedl syndrome, a form of laurence-moon-biedl syndrome laurence-moon-biedl syndrome accompanied by congenital hepatic fibrosis pediatric renal transplantation in laurence-moon-biedl syn-drome possible or expected findings typical skeletal proportions. arachnodactyly; pectus excavatum genu recurvatum; dislocation of joints diaphragmatic hernia. * infective endocarditis. * atrial septal defect. * myxomatous transformation of mitral ring. mitral valve prolapse. aortic and pulmonary dilatation and valvular insufficiency.* aortic dissection* with dissection of adjacent vessels. ascending aortic aneurysm multiple cysts (see "cyst(s), pulmonary") aortopulmonary fistula: an uncommon complication in dystrophic aortic aneurysm peripartum acute myocardial infarction in marfan's syndrome aneurysm of the cervical internal carotid artery associated with marfan's syndrome--ease report noonan's syndrome in association with acute leukemia lymphatic dysplasia in a neonate with noonan's syndrome noonan syndrome: a clinical description emphasizing the cardiac findings cerebral arteriovenous malformation in noonan's syndrome clinical variability in a noonan syndrome family with a new ptpnii gene mutation peutz-jeghers syndrome bilateral large-cell calcifying sertoli cell tumor of the testes in peutz-jeghers syndrome: a case report ovarian tumors associated with the peutz-jeghers syndrome robin sequence and a deficiency of the left forearm in a girl with a deletion of chromosome g -gter congenital heart disease in the pierre robin syndrome glossoptosis-apnea syndrome chiari i malformation, caudal regression syndrome, and pierre robin syndrome: previously unreported combination risk factors for squamous cell carcinoma of the esophagus heterotaxy syndrome; ivemark's syndrome possible associated conditions: with asplenia: right isomerism (bilateral mirror-image right-sided symmetry) of heart, lungs, and abdominal viscera; common atrium; total anomalous pulmonary venous connection; absent coronary sinus; complete atrioventricular septal defect;* subpulmonary stenosis;* pulmonary valve atresia;* midline symmetric liver; malrotation of bowel; absent spleen. with polysplenia: left isomerism (bilateral mirror-image left-sided symmetry) ofheart and lungs, with variable sidedness ofabdominal viscera primary immunodeficiencies reiter's syndrome-like patternin aids-associated psoriasiformdermatitis reference pneumothorax;* pneumomediastinum;* pneumoperitoneum. septicemia. right ventricular hypertrophy and/or dilatation. intubation trauma and mural edema and hemorrhage in trachea; hyaline membrane disease intubation trauma. hemorrhages or other evidence of birth trauma; germinal matrix hemorrhages in premature infants; infarctions of subcortical white matter in term infants coalson . pathology of bronchopulmonary dysplasia pathology of arrested acinar development in postsurfactant bronchopulmonary dysplasia inborn errors of metabolism and reye's syndrome: differential diagnosis prevalence and non-specificity of microvesicular fatty changes liver histopathology in clinical reye syndrome ultrastructural study of intranuclear inclusions in the exo-crine pancreas in reye's syndrome neuropathologic findings in reye syndrome sanfilippo's (see "mucopolysaccharidosis scheie's (see "mucopolysaccharidosis segawa's (see "syndrome, dystonia severe acute respiratory (sars) note: this highly contagious illness, caused by a coronavirus (sars-cov)details, see biosafety level laboratory for autopsies of patients with severe acute respiratory syndrome: principles, practices, and prospects pathology and pathogenesis of severe acute respiratory syndrome pulmonary involvement in primary sjogren's syndrome polymyositis and sjogren's syndrome associated with bronchiolitis obliterans organizing pneumonia the gastrointestinal manifestations of sjogren's syndrome clinicopathological factors relating malignant lymphoma with sjogren's syndrome acute transverse myelopathy and cutaneous vasculopathy in primary sjogren's syndrome primary localized cutaneous amyloidosis with unusual clinical features in a patient with sjogren's syndrome disease, parkinson's erythema multiforme man related terms: armadillo disease; continuous muscle fiber activity; neuromyotonia autoimmune central nervous system paraneoplastic disorders: mechanisms, diagnosis, and therapeutic options sudden infant death (sids) (see "death, sudden unexpected, of infant.") syndrome, superior vena cava related term: superior vena cava obstruction continue dissection of veins into neck and axillas. record and photograph site of thrombosis or of compression by surrounding pathologic conditions. submit samples for histologic study. benign or malignant tumors; fibrosing mediastinitis;* postradiation fibrosis; infectious disease (tuberculosis,* histoplasmosis*); thoracic aortic aneurysm;* chronic constrictive pericarditis;* chest trauma; arteriovenous fistula between ascending aorta and superior vena cava toxic shock synonyms and related terms: staphylococcal scarlet fever. note: ( ) collect all tissues this is a reportable disease. premature aging; increased number of pigmented nevi; infantile sex organs; webbed neck; broad chest with wide spacing of nipples. short fourth metacarpal; abnormal epiphyseal fusions; osteochondrosis-like changes of spine bicuspid aortic valve;* coarctation of aorta. * rarely other anomalies ( ) decreased/absent follicles. intestinal telangiectases. biliary atresia. * horseshoe kidney; double ureters. streak gonads without germ cells or follicles hashimoto's thyroiditis. * manifestations of diabetes mellitus,* hypertension,* or thyrotoxicosis. neuroblastoma and related tumors ( ). keratoconus acute aortic dissection, aortic insufficiency, and a single coronary artery in a patient with turner's syndrome neuroblastoma and related tumors in turner's syndrome turner's syndrome associated with bilateral retinal detachments weil's (see "leptospirosis related terms: alcoholic wernicke's encephalopathy; korsakoff's psychosis; nonalcoholic wernicke's encephalopathy ( , ); and specimen preparation, see chapter . for selection of histologic samples, see right-hand column melissas . wernicke's encephalopathy after vertical banded gastroplasty for morbid obesity syndrome, respiratory distress, of infant.") syndrome, wiskott-aldrich (see "syndrome, primary immunodeficiency malformation, ebstein's" and "preexcitation, ventricular alcoholic and non-alcoholic wernicke's encephalopathy. be alert to the preventable and treatable disease cerebro-hepato-renal syndrome; infantile refsum's disease. * note: this congenital familial cholestatic syndrome results from impaired assembly of peroxisomes and has its main manifestations in the brain, liver, and kidneys postmortem findings and prenatal diagnosis ofzellwegersyndrome. case report alcoholism and alcohol intoxication" and "disease, alcoholic liver possible associated condition: multiple endocrine neoplasia primary cardiac gastrinoma causing zollinger-ellison syndrome localization, syphilis, acquired synonym: treponema pallidum infection. note: congenital syphilis is presented below under a separate heading and surgical management of gastrinoma fundic argyrophil carcinoid tumor in a patient with sporadic-type zollinger-ellison syndrome nonimmune hydrops fetalis due to congenital syphilis associated with negative intrapartum mater-nal serology screening syringomyelia synonyms and related term: hydromyelia; idiopathic syringomyelia; secondary syringomyelia; syringobulbia. possible associated conditions: with idiopathic syringomyelia-arnold chiari malformation, type i;* basilar impression;* . oppenheimer eh, dahms bb. congenital syphilis in the fetus and neonate congenital syphilis: detection of treponema pallidum in stillborns with secondary syringomyelia-intramedullary gliomas (ependymoma, pilocytic astrocytoma) and vascular tumors; spinal arachnoiditis and pachymeningitislisted in right-hand column. prepare roentgenograms of spine and joints. for removal and specimen preparation, see chapter . for histologic sampling, see right-hand column. hypertrophy of body parts. muscle atrophy of upper extremities and hands cervical spinal cord is swollen and tense, with cavitation (syrinx) containing clear fluid. wall of cavity consists of degenerated glial and neural elements, with marked gliosis. spinal cord parenchyma is markedly compressed. see also above under hydrops fetalis caused by alpha-thalassemia: an emerging health care problem limb defects in homozygous alpha-thalassemia: report of three cases poisoning, thallium thirst (see "dehydration thromboangiitis obliterans (see "disease, buerger's purpura, thrombotic thrombocytopenic" and "syndrome, hemolytic uremic the pathological manifestations of pulmonary toxoplasmosis in the acquired immunodeficiency syndrome a spectrum in the pathology of toxoplasmosis in patients with acquired immu transfusion (see "reaction to transfusion bone marrow note: if the patient had symptoms of acute or chronic graft-versus-host disease (gvhd), see "disease, graftversus-host morphology and diagnostics of human toxoplasmosis fatal myocardial coinfection by toxoplasma gondii and parvovirus b in an hiv patient if there is evidence of infection, submit material for microbiologic studies. if there is evidence of recurrent leukemia or lymphoma, sample as described under those headings. if the patient had symptoms of thrombotic thrombocytopenic purpura (tip) or hemolytic uremic syndrome (hus), see these headings. record average size. fix specimens in b-plus®. make touch preparations. request giemsa or wright stain. for preparation of sections and smears (imprints), see chapter . manifestations of graft-versus-host disease (e.g., scleroderma-like changes).* septicemia. interstitial pneumonia* and cytomegalovirus infection* or human herpesvirus infection ( ). bacterial, fungal bacterial or fungal infections in gvhd.* other manifestations of gvhd. * recurrent leukemia,* lymphoma* (including posttransplant, epstein-barr virus associated lymphoproliferative disorder). recurrent solid tumor, e.g., carcinoma of breast, lung (small cell carcinoma), ovary lymphomatous or leukemic infiltrates myeloid cells may be absent in marrow graft rejection or nonimmunologic marrow graft failure. references hematomas; hemorrhagic necroses; infarcts; bacterial or fungal infections leukoencephal-opathy; vascular siderocalcinosis; neuro-axonal spheroids ( ) human herpesvirus infection and associated pathogenesis following bone marrow transplantation pulmonary veno-occlusive disease in an adult following bone marrow transplantation. case report and review of the literature acute renal failure following bone mar-row transplantation transplantation-associated thrombotic thrombocytopenic purpura and hemolytic uremic syndrome thrombotic microangiopathies associated with drugs and bone marrow transplantation record status of all vascular anstomoses. if there are hemorrhages, record volume and site. record weight, ventricular thickness, and valve circumferences. take multiple sections from all areas of myocardium. cut coronary arteries in cross sections; request verhoeff-van gieson stain. procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. references cushingoid features (see "syndrome, cushing's") after steroid therapy. suture dehiscence. hemothorax or hemomediastinum in recent cases. left ventricular hypertrophy, from cyclosporine-related hypertension. acute transplant rejection (for grading, see ref. ). chronic transplant vasculopathy with coronary artery stenosis ( ) opportunistic infection ( ). pneumonia a working formulation for the standardization of nomenclature in the diagnosis of heart and lung rejection: heart and lung rejection study group autopsy findings in cardiac transplant patients: a lo-year experience fulminant toxoplasmosis following heart transplantation banff schema for grading liver allograft rejection: an international consensus document record status of all vascular anstomoses. if there are hemorrhages, record volume and site. record lung weights separately. if lung infection is suspected, submit material for microbiologic possible or expected findings cushingoid features (see "syndrome, cushing's") after steroid therapy. suture dehiscence. hemothorax or hemomediastinum in recent cases. opportunistic infection (in patients with a single lung transplant, infections may involve references the nontransplant lung, as well). chronic bronchitis* and bronchiectasis.* acute rejection (rare); chronic airway rejection (obliterative bronchiolitis); chronic vascular rejection. (for grading of rejection, see ref .) post-transplant lymphoprolijerative disorder a working formulation for the standardization of nomenclature in the diagnosis of heart and lung rejection: heart and lung rejection study group recurrent salmonella enteritidis and hepatic tuberculosis tularemia synonyms and related terms: francisella tularensis infection; pasteurella tularensis infection cerebral abscesses complicating tularemia meningitis endocrine (see "neoplasia, multiple endocrine any type note: ifthe tumor had been treated by surgery, irradiation, chemotherapy, or other means (the most common situation possible associated conditions: with adrenocortical adenoma-conn's syndrome with adrenocortical carci-noma-cushing's syndrome;* hypoglycemia;* virilization. with pheochromocytoma-cerebellarhemangioblastoma ery-throcytosis; hypercalcemia; hypertension,* multiple enarteries record body weight and abnormal features. photograph skin changes. record heart weight and thickness of ventricles. procure multiple sections of myocardium possible associated conditions.") focal myocarditis. * hypertensive cardiovascular disease (see "hypertension myocardial infarction without severe coronary artery disease (with pheochromocytoma) asystematicreviewoftheassociationbetween barrett's esophagus and colon neoplasm malakoplakia and colorectal adenocarcinoma an unusual case of colonic mixed adenoendocrine carcinoma: collision vs composite tumor. a case report and review of the literature submit lymph nodes for histologic study. remove neck organs with tongue together with esophagus. open pharynx and esophagus in posterior midline. if fistulas and abscesses are suspected, dissect esophagus but leave attached to mediastinum, stomach and diaphragm. record width of lumen of esophagus at various levels. photograph and record size and location of tumor; sample tumor and uninvolved esophagus for histologic study. request pas stain of uninvolved esophagus (sample from all levels). procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. references cachexia. eczematoid dermatitis with adult renal cell carcinoma. cushing's syndrome,* galactorrhea or feminization or masculinization in some cases of renal cell carcinoma. evidence of hyperalcemia. pulmonary tumor embolism after invasion of renal vein and inferior vena cava. acquired renal cystic disease prolactin, renin, and prostaglandins in some renal cell carcinomas. see above under "possible associated conditions wilms tumor associated with polycythemia: case report and review of the literature renal medullary carcinoma: a potential sickle cell nephropathy of children and adolescents acquired cystic kidney disease byler's disease, carcinoid syndrome,*cirrhosis* of any type (mostly nonbiliary), congenital hepatic fibrosis, * cushing's syndrome,* erythrocytosis, genetic hemochromatosis, * glycogen storage disease (type ),* hepatitis b or c virus infection, hereditary tyrosinemia (type i),* hypercalcemia, hypercholesterolemia, hypoglycemia,* neurofibromatosis,* osler-rendu-weber disease* (hereditary hemorrhagic telangiectasia), polycythemia,* porphyria (acute intermittent and porphyria cutanea tarda), * pseudohyperparathyroidism,* and thorium (thorotrast) deposition. with bile duct carcinoma (cholangiocarcinoma}-clonorchis sinensis or opisthorchis viverrini infection, fibropolycystic liver and biliary tract disease,* hepatolithiasis, hypercalcemia, pri-mary sclerosing cholangitis,* and thorium (thorotrast) deposition. with hepatoblastoma-alpha-fetoproteinemia, cardiac and renal malformations, cleft palate, diaphragmatic hernia,* down's syndrome,* familial colonic polyposis,* hemihypertrophy, nephroblastoma. with angiosarcoma-thorium (thorotrast) deposition. see also below under "possible or expected findings describe and photograph cut surfaces. sample tumor and nonneoplastic liver for histologic study. if thorium deposition is suspected, prepare roentgenograms of liver slices and submit samples for energy-dispersive x-ray microanalysis of paraffin sections. procedures depend on expected findings or grossly identified abnormalities as listed above and in right-hand column. cachexia. precocious puberty. feminization and gynecomastia in rare cases of hcc. spider angiomas; clubbing of fingers. see above under "possible associated conditions thorotrast storage may cause cirrhosis, hcc, bile duct carcinoma, or angiosarcoma. see above under "possible associated conditions tumor of the lung or bronchus possible associated conditions: abnormal concentrations of hormons or other metabolites in blood and tumor tissue (adrenocorticotropic, antidiuretic, growth hormone, parathyroid-like substances, or -hydroxyindolacetic acid); acanthosis nigricans s syndrome;*dermal hyperpigmentation; feminization; hyperglycemia; hypercalcemia; hypoglycemia;* hypokalemia; hyponatremia; precocious puberty. for syndromes affecting the brain, peripheral nerves or muscles, see below under "possible or expected findings organs and tissues procedures possible or expected findings external examination and skin record body weight. record abnormal features as listed in right-hand column; prepare photographs. prepare histologic sections of normal and grossly abnormal skin clubbing of fingers; spider angiomas. for other rare tumor-related changes, see above under "possible associated conditions thmor of the pancreas possible associated conditions: with carcinoma of the exocrine pancreas--cushing's syndrome;* diabetes mellitus* (rare); hypercalcemia; hyperglycemia with islet cell tumor-abnormal concentrations of hormons in blood and tumor tissue (see below under "pancreas"); features. dermal hyperpigmentation. for other rare tumor-related changes, see above under "possible associated conditions biliary obstruction caused by tumor in head of pancreas. islet cell tumor with adrenocorticotropic hormone, gastrin, glucagon, insulin, or other peptide hormones. see above under "possible associated conditions paraneoplastic pemphigus associated with pancreatic carcinoma pancreatic cystadenocarcinoma in peutz-jeghers syndrome testes may have been surgically removed to achieve androgen deprivation. urinary obstruction and hydronephrosis. * osteoblastic metastases hemolytic uremic syndrome in prostatic carcinoma thmor of the small intestine possible associated conditions: acquired immunode oncogenic osteomalacia associated with prostatic cancer gardner's syndrome) submit sample of non-neoplastic small bowel for histologic study. procedures depend on expected findings or grossly identified abnormalities as listed above. cachexia. mucocutaneous pigmentations associated with peutz-jeghers syndrome. * carcinoid tumor. lymphoma (see also above under "possible associated conditions"). familial polyposis or related syndrome. * celiac sprue. * crohn's disease. * see above under "possible associated conditions kasabach merritt syndrome in infants paraneoplastic hepatopathy associated with soft tissue sarcoma neurofibro-thmor of the spinal cord possible associated conditions: with angioma-cerebellar hemangioblastoma; segmental cutaneous vascular nevi; with matosis in children with soft tissue sarcoma tissues record abnormal features; photograph and submit nevi for histologic study. for removal and specimen preparation, see chapter . see also below under "vertebral column bone erosion and calcification of spinal canal. vertebral hemangiomas may be associated with arteriovenous malformation of spinal cord. manifestations of von hippel-lindau disease. * thmor of the stomach possible associated conditions: hypoglycemia;* megaloblastic anemia;* skin changes (as listed under "possible or expected findings)weight. record abnormal features; photograph and submit samples of normal and abnormal skin for histologic study. possible or expected findings cachexia; lower leg lymphoedema ( ) metastases common in liver, retroperitoneal and supraclavicular lymph nodes (virchow's node), ovaries (krukenberg tumor), and peritoneal cui de sac (blumer's shelf') carcinoma of the stomach with hyperkeratosis palmaris and plantaris and acanthosis of the esophagus gastric lymphoma ofmucosa-associated lymphoid tissue and helicobacter pylori gastric signet-ring cell carcinoma: unilateral lower extremity lymphoedema as the presenting feature thmor of the testis possible associated conditions: demyelinating neuropathy record location, size, and weight of both testes and of testicular tumor. submit samples of tumor and of ininvolved testis and epididymis for histologic study. snap-freeze tumor tissue for hormone assay. possible or expected findings cachexia. cryptorchism; hypospadia. gynecomastia. increased concentrations of alphafetoprotein and human chorionic gonadotropin testicular microlithiasis. secondary testicular tumors (metastases to the testes) are rare, except in association with leukemia* in children. references pulmonari embolism.* paraneoplastic diseases as listed above under "possible associated conditions chronic inflammatory demyelinating polyneuropathy (clop) associated with seminoma dermatomyositis associated with intratubular germ cell tumor and metastatic germ cell cancer testicular cancer in association with developmental renal anomalies and hypospadias submit samples of lymph nodes, spleen, peyer's plaques, and bone marrow for histologic study. other procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. references cachexia. dermal hyperpigmentation. pemphigus foliaceus (rare). hypogammaglobulinemia and other immunoglobulin abnormalities. usually, thymoma presents as an infiltrating, anterior mediastinal mass that rarely metastasizes. carcinoid tumor, malignant lymphoma* (hodgkin's disease), and metastases from carcinomaofthe breast* and other tumors also may occur in this location herpes simplex*) and fungal infections (e.g., candidiasis*) due to thymoma-related immunodeficiency ( ). manifestations of paraneoplastic diseases and conditions as listed above under "possible associated conditions thrombotic thrombocytopenic purpura accompanied by transient pure red cell aplasia and thymoma glomerulonephritis associated with myasthenia gravis thymoma and cellular immune deficiency in an adolescent possible associated conditions: with papillary carcinoma-familial adenomatous polyposis thyroid carcinoma associated with familial adenomatous polyposis an association between acromegaly and thyroid carcinoma thyroid carcinoma causing fatal laryngeal obstruction prostate cancer metastasis to thyroid gland schistosomiasis and the risk of bladder cancer in human papilloma virus infection ( ) or herpesvirus infection ( ) with invasive cervical carcinoma. erythropoietin may be found in some tumors. thrombosis. * myopathy* may be associated with carcinoma of the cervix. cerebellar cortical degeneration* may be associated with carcinoma of the uterus. manifestations of uremia (see "failure, kidney") invasive cervical cancer in human immunodeficiency virus-infected and uninfected hospital patients association of risk factors for cervical cancer and human papilloma viruses in invasive cervical cancer association of herpesvirus infection with the development of genital cancer tyrosinemia type ii: a challenge for ophthalmologists and dermatologists hereditary tyrosinemia type i (chronic form): pathologic findings in the liver richner-hanhart syndrome (tyrosinemia ii): early diagnosis of an incomplete presentation with unusual findings acute pancreatitis-associated acute gastrointestinal mucosal lesions: incidence, characteristics, and clinical significance uncinariasis (see "ancylostomiasis uremia (see "failure, kidney from: handbook of autopsy practice urticaria pigmentosa of childhood (see ''mastocytosis, systemic acute aortic dissection;* aneurysma(s) of cerebral arteries; aortic insufficiency;* calcific aortic stenosis,* coarctation of the aorta;* infective endocarditis;* shone's syndrome; turner's syndrome. * organs and tissues procedures possible or expected findings heart if infective endocarditis is suspected, see chapter . open heart in cross-sections (see chapter ). photograph aortic valve and test valvular competence note: reye's syndrome* is a possible postviral complication of varicella that must be distinguished from varicella encephalitis. varicella may also cause exacerbation of tuberculosis.* (i) collect all tissues that appear infected bicuspid aortic valves are associated with aortic dilatation out of proportion to coexistent valvular lesions varicella-zoster virus infection in children with underlying immunodeficiency virus infection group a beta-hemolytic streptococcal epiglottitis as a complication of varicella infection optic neuritis: a rare complication of primary varicella infection hsv-l-induced acute retinal necrosis syndrome presenting with severe inflammatory orbitopathy, proptosis, and optic nerve involvement varicella with acute motor axonal neuropathy descending necrotizing mediastinitis in a child with chicken pox double outlet right ventricle with intact ventricular septum respiratory syncytial (see "pneumonia, all types or type unspecified vitamin a (see ''deficiency, vitamin a" and "hypervitaminosis a vitamin bt (thiamine) (see "syndrome, wernicke-korsakott vitamin bt (see "anemia, megaloblastic deficiency deficiency, vitamin d" and "hypervitaminosis d waldenstrom's macroglobulinemia (see ''macroglobulinemia, waldenstrom's.) waterhouse-friderichsen syndrome (see "disease, meningococcal wegener's granulomatosis (see "granulomatosis, wegener's werdnig-hottman disease (see "disease, motor neuron acid esterase activity in cultured skin fibroblasts and amniotic fluid cells using -methylumbelliferyl palmitate which may be secondarily infected by bacteria. dark field microscopy will identify the organism expected findings mucosal ulcers, resembling those of typhoid fever, primarily of distal ileum and colon. villous shortening, crypt hyperplasia with mucosal microabscesses. microabscesses in regional mesenteric lymph nodes zellweger's syndrome (see "syndrome mucormycosis" and procedures under "diabetes mellitus abscesses and granulomas may occur in all organs and tissues. granulomatous lesions in central nervous system ( ) and eyes ( ). fungal osteomyelitis* that may be multifocal, including sites such as metacarpals and metatarsals. external examination other procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. osteosclerosis of vertebrae, ribs, clavicles, pelvic bones, scapulae, skull, and metaphyseal ends of femur and humerus. osteomyelofibrosis or osteoreticulosis; rarely, panhyperplasia of bone marrow; increase of megakaryocytes.changes associated with chronic granulocytic leukemia* or with polycythemia vera* may imitate idiopathic myelofibrosis. widespread extramedullary hematopoiesis ( ) with splenomegaly. infectious diseases, including tuberculosis;* gouty arthritis; ascites and other manifestations of portal hypertension;* or hepatic vein thrombosis (budd-chiari syndrome*). cardiac tamponade ( ) . synonyms and related terms: hepatic porphyria; protoporphyrinogen oxidase deficiency ( ) .note: the manifestations of the disease closely resemble those in porphyria cutanea tarda and hereditary coproporphyria. measurements of porphyrins and porphyrin precursors are the only clearly distinguishing features. for autopsy procedures, see under "porphyria cutanea tarda." fibrin and platelet thrombi, with or without microaneurysms and purpura in kidneys, adrenal glands, pancreas, heart, and brain; lesions may also be present in liver; spleen ( ), lymph nodes, muscle, bone marrow, and synovium. fibrin thrombi can be demonstrated with labeled antihuman fibrin antibodies. lesions in precapillary arterioles. hypertrophic osteoarthropathy* (with pleural mesothelioma).pleural effusions. * asbestosis may be complicated by pleural mesothelioma. other organs and tissue sample bladder tumor and uninvolved urinary bladder for histologic study.cachexia. hydronephrosis* (obstructive uropathy) and hydroureter, usually caused by distal ureteral obstruction. recurrent nephrolithiasis* or pyelitis may have been present and is a risk factor for urinary bladder carcinoma.chronic urocystitis; infestation with schistosoma haematobium ( ) (uncommon in north america). manifestations of uremia (see "failure, kidney"). key: cord- -bb h w authors: brann, david h.; tsukahara, tatsuya; weinreb, caleb; lipovsek, marcela; van den berge, koen; gong, boying; chance, rebecca; macaulay, iain c.; chou, hsin-jung; fletcher, russell; das, diya; street, kelly; de bezieux, hector roux; choi, yoon-gi; risso, davide; dudoit, sandrine; purdom, elizabeth; mill, jonathan s.; hachem, ralph abi; matsunami, hiroaki; logan, darren w.; goldstein, bradley j.; grubb, matthew s.; ngai, john; datta, sandeep robert title: non-neuronal expression of sars-cov- entry genes in the olfactory system suggests mechanisms underlying covid- -associated anosmia date: - - journal: biorxiv doi: . / . . . sha: doc_id: cord_uid: bb h w altered olfactory function is a common symptom of covid- , but its etiology is unknown. a key question is whether sars-cov- (cov- ) – the causal agent in covid- – affects olfaction directly by infecting olfactory sensory neurons or their targets in the olfactory bulb, or indirectly, through perturbation of supporting cells. here we identify cell types in the olfactory epithelium and olfactory bulb that express sars-cov- cell entry molecules. bulk sequencing revealed that mouse, non-human primate and human olfactory mucosa expresses two key genes involved in cov- entry, ace and tmprss . however, single cell sequencing and immunostaining demonstrated ace expression in support cells, stem cells, and perivascular cells; in contrast, neurons in both the olfactory epithelium and bulb did not express ace message or protein. these findings suggest that cov- infection of non-neuronal cell types leads to anosmia and related disturbances in odor perception in covid- patients. sars-cov- (cov- ) is a pandemic coronavirus that causes the covid- syndrome, which can include upper respiratory infection (uri) symptoms, severe respiratory distress, acute cardiac injury and death ( - ). cov- is closely related to other beta-coronaviruses, including the causal agents in pandemic sars and mers (sars-cov and mers-cov, respectively) and endemic viruses typically associated with mild uri syndromes (hcov-oc and hcov- e) ( ) ( ) ( ) . clinical reports suggest that infection with cov- is associated with high rates of disturbances in smell and taste perception, including anosmia ( ) ( ) ( ) ( ) ( ) . while many viruses (including coronaviruses) induce transient changes in odor perception due to inflammatory responses, in at least some cases covid-related anosmia has been reported to occur in the absence of significant nasal inflammation or coryzal symptoms ( , ( ) ( ) ( ) . this observation suggests that cov- might directly target odor processing mechanisms, although the specific means through which cov- alters odor perception remains unknown. cov- -like sars-cov -infects cells through interactions between its spike (s) protein and the ace protein on target cells. this interaction requires cleavage of the s protein, likely by the cell surface protease tmprss , although other proteases (such as cathepsin b and l, ctsb/ctsl) may also be involved ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . other coronaviruses use different cell surface receptors and proteases to facilitate cellular entry, including dpp , furin and hspa for mers-cov, anpep for hcov- e, tmprss d for sars-cov (in addition to ace and tmprss ), and st gal and st gal for hcov-oc and hcov-hku ( , ( ) ( ) ( ) . we hypothesized that identifying the specific olfactory cell types susceptible to direct cov- infection (due to e.g., ace and tmprss expression) would provide insight into possible mechanisms through which covid- causes altered smell perception. the nasal epithelium is divided into a respiratory epithelium (re) and olfactory epithelium (oe), whose functions and cell types differ. the nasal re is continuous with the epithelium that lines much of the respiratory tract and is thought to humidify air as it enters the nose; main cell types include basal cells, ciliated cells, secretory cells (including goblet cells), and brush/microvillar cells ( , ) (figure ). the oe, in contrast, is responsible for odor detection, as it houses mature olfactory sensory neurons (osns) that interact with odors via receptors localized on their dendritic cilia. osns are supported by sustentacular cells, which act to structurally support sensory neurons, phagocytose and/or detoxify potentially damaging agents, and maintain local salt and water balance ( ) ( ) ( ) ; microvillar cells and mucus-secreting bowman's gland cells also play important roles in maintaining oe homeostasis and function ( , ) (figure ). in addition, the oe contains globose basal cells (gbcs), which are primarily responsible for regenerating osns during normal epithelial turnover, and horizontal basal cells (hbcs), which act as reserve stem cells activated upon tissue damage ( ) ( ) ( ) . osns elaborate axons that puncture the cribriform plate at the base of the skull and terminate in the olfactory bulb, whose local circuits process olfactory information before sending it to higher brain centers (figure ). it has recently been demonstrated through single cell rna sequencing analysis (referred to herein as scseq) that cells from the human upper airway -including nasal re goblet, basal and ciliated cells -express high levels of ace and tmprss , suggesting that these re cell types may serve as a viral reservoir during cov- infection ( ) . however, analyzed samples in that dataset did not include any osns or sustentacular cells, indicating that tissue sampling in these experiments did not include the oe ( , ) . here we query both new and previously published bulk rna-seq and scseq datasets from the olfactory system for expression of ace , tmrpss and other genes implicated in coronavirus entry. we find that non-neuronal cells in the oe and olfactory bulb, including support, stem and perivascular cells, express cov- entryassociated transcripts and their associated proteins, suggesting that infection of these non-neuronal cell types contributes to anosmia in covid- patients. schematic of a sagittal view of the human nasal cavity, in which respiratory and olfactory epithelium are colored (left). for each type of epithelium, a schematic of the anatomy and known major cell types are shown (right). in the olfactory bulb in the brain (tan) the axons from olfactory sensory neurons coalesce into glomeruli, and mitral/tufted cells innervate these glomeruli and send olfactory projections to downstream olfactory areas. glomeruli are also innervated by juxtaglomerular cells, a subset of which are dopaminergic. to determine whether genes relevant to cov- entry are expressed in osns or other cell types in the human oe, we queried previously published bulk rna-seq data derived from the whole olfactory mucosa (wom) of macaque, marmoset and human ( ) , and found expression of almost all cov-entry-related genes in all wom samples ( figure s a ). to identify the specific cell types in human oe that express ace , we quantified gene expression in scseq derived from four human nasal biopsy samples recently reported by durante et al ( ) . neither ace nor tmprss were detected in mature osns, whereas these genes were detected in both sustentacular cells and hbcs (figures a-e) . in contrast, genes relevant to cell entry of other covs were expressed in osns, as well as in other oe cell types. we confirmed the expression of ace proteins via immunostaining of human olfactory epithelium biopsy tissue, which revealed expression in sustentacular and basal cells, and an absence of ace protein in osns ( figures f and s e ). together, these results demonstrate that sustentacular and olfactory stem cells, but not mature osns, are potentially direct targets of cov- in the human oe. given that the nasopharynx is a major site of infection for cov- ( ), we compared the frequency of ace and tmprss expression among the cell types in the human re and oe ( ) . sustentacular cells exhibited the highest frequency of ace expression in the oe ( . % of cells) although this frequency was slightly lower than that observed in respiratory ciliated and secretory cells ( . % and . %, respectively). while all hbc subtypes expressed ace , the frequency of expression of ace was lower in olfactory hbcs ( . % of cells) compared to respiratory hbcs ( . % of cells) ( figure b ). in addition, all other re cell subtypes showed higher frequencies of ace and tmprss expression than was apparent in oe cells. these results demonstrate the presence of key cov- entry-related genes in specific cell types in the oe, but at lower levels of expression than in re isolated from the nasal mucosa. we wondered whether these lower levels of expression might nonetheless be sufficient for infection of cov- . it was recently reported that nasal re has higher expression of cov- entry genes than re of the trachea or lungs ( ) , and we therefore asked where the oe fell within this previously established spectrum of expression. to address this question, we developed a two step alignment procedure in which we first sought to identify cell types that were common across the oe and re, and then leveraged gene expression patterns in these common cell types to normalize gene expression levels across all cell types in the oe and re ( figure s ). this approach revealed a correspondences between goblet cells in the re and bowman's gland cells in the oe ( % mapping probability, see methods), and between pulmonary ionocytes in the re and a subset of microvillar cells in the oe ( % mapping probability, see methods); after alignment, human oe sustentacular cells were found to express ace and tmprss at levels similar to those observed in the remainder of the non-nasal respiratory tract ( figure g) ( ) . these results are consistent with the possibility that specific cell types in the human olfactory epithelium express ace at a level that is permissive for direct infection. ( ) . each dot represents an individual cell, colored by cell type (hbc = horizontal basal cell, osn = olfactory sensory neuron, sus = sustentacular cell, mv: microvillar cell, resp.: respiratory, oec = olfactory ensheathing cell, smc=smooth muscle cell). (b) percent of cells expressing ace and tmprss . ace was not detected in any osns, but was observed in sus cells and hbcs, among other olfactory and respiratory epithelial cell types. olfactory and respiratory cell types are shown separately. ace and tmprss were also co-expressed above chance levels (odds ratio . , p-value . e- , fisher's exact test). (c) umap representations of detected immature (gng ) and mature (gng ) osns. neither ace nor tmprss are detected in either population of osns. the color represents the normalized expression level for each gene (number of umis for a given gene divided by the total number of umis for each cell). (d) umap representations of all cells, depicting the normalized expression of cov- related genes ace and tmprss , as well as several cell type markers. ace and tmprss are expressed in respiratory and olfactory cell types, but not in osns. ace and tmprss are detected in hbc (krt ) and sustentacular (cyp a ) cells, as well as other respiratory epithelial cell types, including respiratory ciliated (foxj ) cells. (e) various cov related genes including ace and tmprss , are expressed in respiratory and olfactory cell types, but not in osns. gene expression for ace and tmprss as well as marker genes for olfactory and respiratory epithelial cell types are shown normalized by their maximum expression across cell types. mhv, mouse hepatitis virus. (f) ace immunostaining of human olfactory mucosal biopsy samples. ace protein (green) is detected in sustentacular cells and krt -positive basal cells (red; white arrowhead). nuclei were stained with dapi (blue). bar = µm. the ace and krt channels from the box on the left are shown individually on the right (g) gene expression across cell types and tissues in durante figure s ). the tissues correspond to progressive positions along the airway from nasal to distal lung. ace expression in olfactory hbc and sustentacular cells is comparable to that observed in other cell types in the respiratory tract. to further explore the distribution of cov- cell entry genes in the olfactory system we turned to the mouse, which enables interrogative experiments not possible in humans. to evaluate whether that expression patterns observed in the mouse correspond to those observed in the human oe, we examined published datasets in which rna-seq was independently performed on mouse wom and on purified populations of mature osns ( ) ( ) ( ) . the cov- receptor ace and the protease tmprss were expressed in wom, as were the cathepsins ctsb and ctsl (figures a and s a) ( ) . however, expression of these genes (with the exception of ctsb) was much lower and ace expression was nearly absent in purified osn samples (figures a and s a, see legend for counts). genes used for cell entry by other covs (except st gal ) were also expressed in wom, and de-enriched in purified osns. the deenrichment of ace and tmprss in osns relative to wom was also observed in two other mouse rna-seq datasets ( , ) ( figure s b ). these data demonstrate that, as in humans, ace and other cov- entry-related genes are expressed in the mouse olfactory epithelium. the presence of ace and tmprss transcripts in mouse wom and their (near total) absence in purified osns suggest that the molecular components that enable cov- entry into cells are expressed in non-neuronal cell types in the mouse nasal epithelium. to identify the specific cell types that express ace and tmprss , we performed scseq (via drop-seq, see methods) on mouse wom ( figure b ). these results were consistent with observations made in the human epithelium: ace and tmprss were expressed in a fraction of sustentacular and bowman's gland cells, and a very small fraction of stem cells, but not in osns (zero of , identified mature osns, figures c and s c-d) . of note, only dorsally-located sustentacular cells, which express the markers sult c and acsm , were positive for ace ( figures d and s d-e) . indeed, reanalysis of the ace + subset of human sustentacular cells revealed that all positive cells expressed genetic markers associated with the dorsal epithelium ( figure s d ). an independent mouse scseq data set (obtained using the x chromium platform, see methods) revealed that olfactory sensory neurons did not express ace ( of mature osns were positive for ace ), while expression was observed in a fraction of bowman's gland cells and hbcs ( figure s , see methods). expression in sustentacular cells was not observed in this dataset, which included relatively few dorsal sustentacular cells (a possible consequence of the specific cell isolation procedure associated with the x platform, which distinguishes it from drop-seq; compare figures s c and d) . staining of the mouse wom with anti-ace antibodies confirmed that ace protein is expressed in sustentacular cells and is specifically localized to the sustentacular cell microvilli ( figure e -k). ace + sustentacular cells were identified exclusively within the dorsal subregion of the oe; critically, within that region many (and possibly all) sustentacular cells expressed ace ( figure f -g). this observation is consistent with the possibility that ace protein can be broadly expressed in cell populations that exhibit sparse expression when characterized by scseq. staining was also observed in bowman's gland cells but not in osns ( figure h -j). taken together, these data demonstrate that ace is expressed by sustentacular cells that specifically reside in the dorsal epithelium in both mouse and human. considered positive if any transcripts (umis) were expressed for a given gene. sustentacular cells (sus) from dorsal and ventral zones are quantified separately. ace is detected in dorsal sustentacular, bowman's gland, hbcs, as well as respiratory cell types. (d) umap representation of sustentacular cells, with expression of cov- related genes ace and tmprss , as well as marker genes for sus (both pan-sus marker cbr and dorsal specific marker sult c ) indicated. each point represents an individual sustentacular cell, and the color represents the normalized expression level for each gene (number of umis for a given gene divided by the total number of umis for each cell; in this plot ace expression is binarized for visualization purposes). ace -positive sustentacular cells are found within the dorsal sult c positive subset. umap plots for other cell types are shown in figure s . (e) ace immunostaining of mouse main olfactory epithelium. as shown in this epithelial hemisection, ace protein is detected in the dorsal zone and respiratory epithelium. note that the punctate ace staining beneath the epithelial layer is likely associated with vasculature (see figure f ). bar = µm. arrowheads depict the edges of ace expression, corresponding to the presumptive dorsal zone (confirmed in g). viral injury can lead to broad changes in oe physiology that are accompanied by recruitment of stem cell populations tasked with regenerating the epithelium ( , ) . to characterize the distribution of ace expression under similar circumstances, we injured the oe by treating mice with methimazole (which specifically ablates osns), and then employed a previously established lineage tracing protocol to perform scseq on hbcs and their descendants during subsequent regeneration (see methods) ( ) . this analysis revealed that after injury ace and tmprss are expressed in subsets of sustentacular cells and hbcs, as well as in the activated hbcs that serve to regenerate the epithelium (figures a-c and s ; note that activated hbcs express ace at higher levels than resting hbcs). analysis of the ace + sustentacular cell population revealed expression of dorsal epithelial markers ( figure d ). to validate these results, we re-analyzed a similar lineage tracing dataset in which identified hbcs and their progeny were subject to smart-seq -based deep sequencing, which is more sensitive than scseq ( ) . in this dataset, ace was detected in more than . % of gbcs, nearly % of activated hbcs and nearly % of sustentacular cells but was not detected in osns ( figures s b) . furthermore, larger percentages of hbcs, gbcs and sustentacular cells expressed tmprss . immunostaining with anti-ace antibodies confirmed that ace protein was present in activated stem cells under these regeneration conditions ( figure e ). these results demonstrate that activated stem cells recruited during injury express ace , and do so at higher levels than those in resting stem cells. given the potential for the re and oe in the nasal cavity to be directly infected with cov- , we assessed the expression of ace and other cov entry genes in the mouse olfactory bulb (ob), which is directly connected to osns via cranial nerve i (cn i); in principle, alterations in bulb function could cause anosmia independently of functional changes in the oe. to do so, we performed scseq (using drop-seq, see methods) on the mouse ob, and merged these data with a previously published ob scseq analysis, yielding a dataset with nearly , single cells (see methods) ( ) . this analysis revealed that ace expression was absent from ob neurons and instead was observed only in vascular cells, predominantly in pericytes, which are involved in blood pressure regulation, maintenance of the blood-brain barrier, and inflammatory responses (figures a-d and s - ) ( ) . although other potential cov proteases were expressed in the ob, tmprss was not expressed. we also performed smart-seq -based deep sequencing of single ob dopaminergic juxtaglomerular neurons, a population of local interneurons in the ob glomerular layer that (like tufted cells) can receive direct monosynaptic input from nose osns (figures e and s , see methods); these experiments confirmed the absence of ace and tmprss expression in this cell type. immunostaining in the ob revealed that blood vessels expressed high levels of ace protein, particularly in pericytes; consistent with the scseq results, staining was not observed in any neuronal cell type ( figure f ). these observations may also hold true for other brain regions, as re-analysis of deeply sequenced scseq datasets from different regions of the nervous system demonstrated that ace and tmprss expression is absent from neurons, consistent with prior immunostaining results ( figure s ) ( ) . given the extensive similarities detailed above in expression patterns for ace and tmprss in the mouse and human, these findings (performed in mouse) suggest that ob neurons are likely not a primary site of infection, but that vascular pericytes may be sensitive to cov- infection in the ob. here we show that subsets of oe sustentacular cells, hbcs, and bowman's gland cells in both mouse and human samples express the cov- receptor ace and the spike protein protease tmprss . human oe sustentacular cells express these genes at levels comparable to those observed in lung cells. in contrast, we failed to detect ace expression in mature osns at either the transcript or protein levels. these observations suggest that cov- does not directly enter osns, but instead may target oe support and stem cells. similarly, neurons in the ob do not express ace , whereas vascular pericytes do. thus primary infection of non-neuronal cell types -rather than sensory or bulb neurons -may be responsible for anosmia and related disturbances in odor perception in covid- patients. the identification of non-neuronal cell types in the oe and bulb susceptible to cov- infection suggests four possible, non-mutually-exclusive mechanisms for the acute loss of smell reported in covid- patients. first, local infection of support and vascular cells in the nose and bulb could cause significant inflammatory responses whose downstream effects could block effective odor conduction, or alter the function of osns or bulb neurons ( ) ( ). second, damage to support cells (which are responsible for local water and ion balance) could indirectly influence signaling from osns to the brain ( ) . third, damage to sustentacular cells and bowman's gland cells in mouse models can lead to diffuse architectural damage to the entire oe, which in turn could abrogate smell perception ( ) . finally, vascular damage could lead to hypoperfusion and inflammation leading to changes in ob function. immunostaining in the mouse suggests that ace protein is (nearly) ubiquitously expressed in sustentacular cells in the dorsal oe, despite sparse detection of ace transcripts using scseq. similarly, nearly all vascular cells positive for a pericyte marker also expressed ace protein, although only a fraction of ob pericytes were positive for ace message when assessed using scseq. although ace transcripts were more rarely detected than protein, there was a clear concordance at the cell type level: expression of ace mrna in a particular cell type accurately predicted the presence of ace protein, while ace transcript-negative cell types (including osns) did not express ace protein. if humans also exhibit a similar relationship between mrna and protein (a reasonable possibility given the precise match in olfactory cell types that express cov- cell entry genes between the two species), then ace protein is likely to be broadly expressed in human dorsal sustentacular cells. thus, in the there may be many sustentacular cells available for cov- infection in the human epithelium (which in turn could recruit a diffuse inflammatory process). that said, it remains possible that damage to the oe could be caused by more limited cell infection. for example, infection of subsets of sustentacular cells by the sdav coronavirus in rats ultimately leads to disruption of the global architecture of the oe, suggesting that focal coronavirus infection may be sufficient to cause diffuse epithelial damage ( ) . the natural history of cov -induced anosmia is only now being defined; while recovery of smell has been reported, it remains unclear whether in a subset of patients smell disturbances will be long-lasting or permanent ( ) ( ) ( ) ( ) ( ) ) . we observe that activated hbcs, which are recruited after injury, express ace at higher levels than those apparent in resting stem cells. while on its own it is likely that infection of stem cells would not cause acute smell deficits, in the context of infection the dual challenge of loss of sustentacular cells, together with the inability to effectively renew the oe over time, could result in persistent anosmia. many viruses, including coronaviruses, have been shown to propagate from the nasal epithelium past the cribriform plate to infect the ob; this form of central infection has been suggested to mediate olfactory deficits, even in the absence of lasting oe damage ( , ( ) ( ) ( ) ( ) ( ) . the rodent coronavirus mhv passes from the nose to the bulb, even though rodent osns do not express ceacam , the main mhv receptor ( , ) ( figures s c, s e, s a) , suggesting that covs in the nasal mucosa can reach the brain through mechanisms independent of axonal transport by sensory nerves; interestingly, ob dopaminergic juxtaglomerular cells express ceacam ( figure e ), which likely supports the ability of mhv to target the bulb and change odor perception. one speculative possibility is that local seeding of the oe with cov- -infected cells can result in osn-independent transfer of virions from the nose to the bulb, perhaps via the vascular supply shared between the ob and the osn axons that comprise cn i. although cn i was not directly queried in our datasets, it is reasonable to infer that vascular pericytes in cn i also express ace , which suggests a possible route of entry for cov- from the nose into the brain. given the absence of ace in ob neurons, we speculate that any central olfactory dysfunction in covid- is the secondary consequence of pericyte-mediated vascular inflammation ( ) . we note several caveats that temper our conclusions. although current data suggest that ace is the most likely receptor for cov- in vivo, it is possible (although it has not yet been demonstrated) that other molecules such as bsg may enable cov- entry independently of ace ( figures e, s c , s e, s a) ( , ) . in addition, it has recently been reported that low level expression of ace can support cov- cell entry ( ); it is possible, therefore, that ace expression beneath the level of detection in our assays may yet enable cov- infection of apparently ace negative cell types. we also propose that damage to the olfactory system is either due to primary infection or secondary inflammation; it is possible (although has not yet been demonstrated) that cells infected with cov- can form syncytia with cells that do not express ace . such a mechanism could damage neurons adjacent to infected cells. any reasonable pathophysiological mechanism for covid- -associated anosmia must account for the high penetrance of smell disorders relative to endemic viruses, the apparent suddenness of smell loss (which can precede the development of other symptoms), and the transient nature of dysfunction in many patients ( ) ( ) ( ) ( ) ( ) ( , ( ) ( ) ( ) ; definitive identification of the disease mechanisms underlying covid- -mediated anosmia will require additional research. nonetheless, our identification of cells in the oe and ob expressing molecules known to be involved in cov- entry illuminates a path forward for future studies. human scseq data from durante et al. ( ) was downloaded from the geo at accession gse . x genomics mtx files were filtered to remove any cells with fewer than total counts. additional preprocessing was performed as described above, including total counts normalization and filtering for highly variable genes using the spring gene filtering function "filter_genes" with parameters ( , , ) . the resulting data were visualized in spring and partitioned using louvain clustering on the spring k-nearest-neighbor graph. four clusters were removed for quality control, including two with low total counts (likely background) and two with high mitochondrial counts (likely stressed or dying cells). putative doublets were also identified using scrublet and removed ( % of cells). the remaining cells were projected to dimensions using pca. pca-batch-correction was performed using patient as a reference, as previously described ( ) . the filtered data were then re-partitioned using louvain clustering on the spring graph and each cluster was annotated using known marker genes, as described in ( ) . for example, immature and mature osns were identified via their expression of gng and gng , respectively. hbcs were identified via the expression of krt and tp and olfactory hbcs were distinguished from respiratory hbcs via the expression of cxcl and meg . identification of sus cells (cyp a , cyp j ), bowman's gland (sox , gpx ), and mv ionocytes-like cells (ascl , cftr, foxi ) was also performed using known marker genes. for visualization, the top principal components were reduced to two dimensions using umap with parameters (n_neighbors= , min_dist= . ). the filtered human scseq dataset contained cells. each of the samples contained cells from both the olfactory and respiratory epithelium, although the frequency of osns and respiratory cells varied across patients, as previously described ( ) . cells expressed ace and cells expressed tmprss . of the identified osns, including both immature and mature cells, none of the cells express ace and only ( . %) expressed tmprss . in contrast, ace was reliably detected in at least % and tmprss was expressed in close to % of multiple respiratory epithelial subtypes. the expression of both known cell type markers and known cov-related genes was also examined across respiratory and olfactory epithelial cell types. for these gene sets, the mean expression in each cell type was calculated and normalized by the maximum across cell types. data from deprez et al. ( ) were downloaded from the human cell atlas website (https://www.genomique.eu/cellbrowser/hca/; "single-cell atlas of the airway epithelium (grch human genome)"). a subset of these data was combined with a subset of the durante data for mapping between cell types. for the deprez data, the subset consisted of samples from the nasal re that belonged to a cell type with > cells, including basal, cycling basal, suprabasal, secretory, mucous multiciliated cells, multiciliated, sms goblet and ionocyte. we observed two distinct subpopulations of basal cells, with one of the two populations distinguished by expression of cxcl . the cells in this population were manually identified using spring and defined for downstream analysis as a separate cell type annotation called "basal (cxcl +)". for the durante data, the subset consisted of cells from cell types that had some putative similarity to cells in the deprez dataset, including olfactory hbc, cycling respiratory hbc, respiratory hbc, early respiratory secretory cells, respiratory secretory cells, sustentacular cells, bowman's gland, olfactory microvillar cells. to establish a cell type mapping: ) durante ( ) and deprez ( ) data were combined and gene expression values were linearly scaled so that all cells across datasets had the same total counts. pca was then performed using highly variable genes (n= genes) and pcabatch-correction ( ) with the durante data as a reference set. ) the table of votes t was z-scored against a null distribution, generated by repeating the procedure above times with shuffled cell type labels. the resulting z-scores were similar between the two possible mapping directions (durante -> deprez vs. deprez -> durante; r= . pearson correlation of mapping zscores). the mapping z-scores were also highly robust upon varying the number of votes-cast per cell (r> . correlation of mapping z-scores upon changing the vote numbers to or as opposed to ). only cell-type correspondences with a high zscore in both mapping directions (z-score > ) were used for downstream analysis. to establish a common scale of gene expression between datasets, we restricted to cell type correspondences that were supported both by bioinformatic mapping and shared a nominal cell type designation based on marker genes. these included: basal/suprabasal cells = "respiratory hbcs" from durante et al., and "basal" and "suprabasal" cells from deprez we next sought a transformation of the durante data so that it would agree with the deprez data within the corresponding cell types identified above to account for differing normalization strategies applied to each dataset prior to download (log normalization and rescaling with cell-specific factors for deprez et al. but not for durante et al.), we used the following ansatz for the transformation, where the pseudocount p is a global latent parameter and the rescaling factors ! are fit to each gene separately. in the equation below, t denotes the transformation and !" represents a gene expression value for cell i and gene j in the durante data: the parameter p was fit by maximizing the correlation of average gene expression across all genes between each of the cell type correspondences listed above. the rescaling factors ! were then fitted separately for each gene by taking the quotient of average gene expression between the deprez data and the log-transformed durante data, again across the cell type correspondences above. normalized gene expression tables were obtained from previous published datasets ( , ( ) ( ) ( ) . for the mouse data sets, the means of the replicates from wom or osn were used to calculate log fold changes. for the mouse data from saraiva et al. and the primate data sets ( , ) , the normalized counts of the genes of interest from individual replicates were plotted. below is a table with detailed sample information. sample information for the bulk rna-seq data analyzed in this study a new dataset of whole olfactory mucosa scseq was generated from adult male mice ( - weeks-old). all mouse husbandry and experiments were performed following institutional and federal guidelines and approved by harvard medical school's institutional animal care and use committee (iacuc). briefly, dissected main olfactory epithelium were cleaned up in µl of ebss (worthington) and epithelium tissues were isolated in µl of papain ( u/ml in ebss) and µl of dnase i ( u/ml). tissue pieces were transferred to a ml round-bottom tube (bd) and . ml of papain and µl of dnase i were added. after - . hour incubation with rocking at °c, the suspension was triturated with a ml pipette times and passed through µm cell strainer (bd) and strainer was washed with ml of dmem + % fbs (invitrogen). the cell suspension was centrifuged at g for min. cells were resuspended with ml of dmem + % fbs and centrifuged at g for min. cells were suspended with pbs + . % bsa and concentration was measured by hemocytometer. drop-seq experiments were performed as previously described ( ) . microfluidics devices were obtained from flowjem and barcode beads were obtained from chemgenes. of min drop-seq runs were collected in total, which were obtained from mice. replicates of drop-seq samples were sequenced across runs on an illumina nextseq platform. paired end reads from the fastq files were trimmed, aligned, and tagged via the drop-seq tools (v . ) pipeline, using star (v . . a) with genomic indices from ensembl release . the digital gene expression matrix was generated for , cells for _ , , cells for , _ , _ds , _ds , _ds , , cells for _ds , and , cells for . processing of the wom drop-seq samples was performed in seurat (v . . ). cells with less than umis or more than , umis, or higher than % mitochondrial genes were removed. potential doublets were removed using scrublet. cells were initially preprocessed using the seurat pipeline. variable genes "findvariablegenes" (y.cutoff = . ) were scaled (regressing out effects due to numi, the percent of mitochondrial genes, and replicate ids) and the data was clustered using pcs with the louvain algorithm (resolution= . ). in a fraction of sustentacular cells, we observed co-expression of markers for sustentacular cells and other cell types (e.g. osns). re-clustering of sustentacular cells alone separately out these presumed doublets from the rest of the sustentacular cells, and the presumed doublets were removed for the analyses described below. the filtered cells from the preprocessing steps were reanalyzed in python using scanpy and spring. in brief, the raw gene counts in each cell were total counts normalized and variable genes were identified using the spring gene filtering function "filter_genes" with parameters ( , , ); mitochondrial and olfactory receptor genes were excluded from the variable gene lists. the resulting variable genes were zscored and the dimensionality of the data was reduced to via principal component analysis. the k-nearest neighbor graph (n_neighbors= ) of these pcs was clustered using the leiden algorithm (resolution= . ) and was reduced to two dimensions for visualization via the umap method (min_dist= . ). clusters were manually annotated on the basis of known marker genes and those sharing markers (e.g. olfactory sensory neurons) were merged. the mouse wom drop-seq dataset contained cells that passed the above filtering. each of the clusters identified contained cells from all replicates in roughly equal proportions. of the mature osns and the immature osns, none of the cells express ace . in contrast, in the olfactory epithelial cells, ace expression was observed in the bowman's gland, olfactory hbcs, dorsal sustentacular cells. mice were sacrificed with a lethal dose of xylazine and nasal epithelium with attached olfactory bulbs were dissected and fixed in % paraformaldehyde (electron microscope sciences, ) in phosphate-buffered saline (pbs) for overnight at °c or for hours at room temperature. tissues were washed in pbs for times ( min each) and incubated in . m edta in pbs overnight at °c. the following day, tissues were rinsed by pbs and incubated in % sucrose in pbs for at least min, transferred to tissue freezing medium (vwr, - ) for at least min and frozen on crushed dry ice and stored at - °c until sectioning. tissue sections ( µm thick for the olfactory bulb and µm thick for nasal epithelium) were collected on superfrost plus glass slides (vwr, ) and stored at - °c until immunostaining. for methimazole treated samples, adult c bl/ j mice ( - weeks old, jax stock no. ) were given intraperitoneal injections with methimazole (sigma m ) at µg/g body weight and sacrificed at , , and -hour timepoints. sections were permeabilized with . % triton x- in pbs for min then rinsed times in pbs. sections were then incubated for - min in blocking solution that consisted of pbs containing % bovine serum albumin (jackson immunoresearch, - - ) and % donkey serum (jackson immunoresearch, - - ) at room temperature, followed by overnight incubation at °c with primary antibodies diluted in the same blocking solution. primary antibodies used are as follows. after secondary antibody incubation, sections were washed twice for - min in pbs, incubated with nm dapi in pbs for min and then rinsed with pbs. slides were mounted with glass coverslips using vectashield mounting medium (vector laboratories, h- ) or prolong diamond antifade mountant (invitrogen, p ). for co-staining of ace and nqo , slides were first stained with ace primary antibody and donkey anti-goat igg alexa secondary. after washes of secondary antibody, tissues were incubated with unconjugated donkey anti-goat igg fab fragments (jackson immunoresearch, - - ) at µg/ml diluted in blocking solution for hour at room temperature. tissues were washed twice with pbs, once in blocking solution, and incubated in blocking solution for - min at room temperature, followed by a second round of staining with the nqo primary antibody and donkey anti-goat igg alexa secondary antibody. confocal images were acquired using a leica spe microscope (harvard medical school neurobiology imaging facility) with nm, nm, nm, and nm laser lines. multi-slice z-stack images were acquired, and their maximal intensity projections are shown. for figure e , tiled images were acquired and stitched by the leica las x software. images were processed using fiji imagej software ( ) , and noisy images were median-smoothed using the remove outliers function built into fiji. sult c rna was detected by fluorescent rnascope assay (advanced cell diagnostics, kit ) using probe -c , following the manufacturer's protocol (rnascope fluorescent multiplex kit user manual, -um date ) for paraformaldehyde-fixed tissue. prior to initiating the hybridization protocol, the tissue was pre-treated with two successive incubations (first min, then min long) in rnascope protease iii (advanced cell diagnostics, ) at °c, then washed in distilled water. at the end of protocol, the tissue was washed in pbs and subjected to the -day immunostaining protocol described above. human olfactory mucosa biopsies were obtained via irb-approved protocol at duke university school of medicine, from nasal septum or superior turbinate during endoscopic sinus surgery. tissue was fixed with % paraformaldehyde and cryosectioned at µm and sections were processed for immunostaining, as previously described ( ) . sections from a female nasal septum biopsy were stained for ace ( figure f ) using the same goat anti-ace (thermo fisher, pa - , : ) and the protocol described above for mouse tissue. the human sections were co-stained with rabbit antikeratin (abcam, ab ; ab_ , : ) and were detected with alexafluor donkey anti-goat (jackson immunoresearch, - - ) and alexafluor donkey anti-rabbit (jackson immunoresearch, - - ) secondary antibodies ( : ). as further validation of ace expression and to confirm the lack of ace expression in human olfactory sensory neurons ( figure s e ), sections were stained with a rabbit anti-ace (abcam, ab ; rrid:ab_ , used at : ) antibody immunogenized against human ace and a mouse tuj antibody against neuronspecific tubulin (biolegend, ; rrid:ab_ ). anti-ace was raised against a c-terminal synthetic peptide for human ace and was validated by the manufacturer to not cross-react with ace for immunohistochemical labeling of ace in fruit bat nasal tissue as well as in human lower airway. recombinant human ace abolished labeling with this antibody in a previous study in human tissue, further demonstrating its specificity ( ) . the tuj antibody was validated, as previously described ( ) . biotinylated secondary antibodies (vector labs), avidin-biotinylated horseradish peroxidase kit (vector) followed by fluorescein tyramide signal amplification (perkin elmer) were applied per manufacturer's instructions. for dual staining, tuj was visualized using alexafluor goat anti-mouse (jackson immunoresearch, - - ; rrid: ab_ ). human sections were counterstained with ', -diamidino- -phenylindole (dapi) and coverslips were mounted using prolong gold (invitrogen) for imaging, using a leica dmi microscope system. images were processed using fiji imagej software (nih). scale bars were applied directly from the leica acquisition software metadata in imagej tools. unsharp mask was applied in imagej, and brightness/contrast was adjusted globally. month-old and month-old wild type c bl/ j mice were obtained from the national institute on aging aged rodent colony and used for the wom experiments; each experimental condition consisted of one male and one female mouse to aid doublet detection. mice containing the transgenic krt -creer(t ) driver ( ) and rosa -yfp reporter allele ( ) were used for the hbc lineage tracing dataset. all mice were assumed to be of normal immune status. animals were maintained and treated according to federal guidelines under iacuc oversight at the university of california, berkeley. the olfactory epithelium was surgically removed, and the dorsal, sensory portion was dissected and dissociated, as previously described ( ) . for wom experiments, dissociated cells were subjected to fluorescence-activated cell sorting (facs) using propidium iodide to identify and select against dead or dying cells; , cells/sample were collected in % fbs. for the hbc lineage tracing experiments krt -creer; rosa yfp/yfp mice were injected once with tamoxifen ( . mg tamoxifen/g body weight) at p - days of age and sacrificed at hours, hours, hours, days and days post-injury, as previously described ( , ) . for each experimental time point, yfp+ cells were isolated by facs based on yfp expression and negative for propidium iodide, a vital dye. cells isolated by facs were subjected to single-cell rna-seq. three replicates (defined here as a facs collection run) per age were analyzed for the wom experiment; at least two biological replicates were collected for each experimental condition for the hbc lineage tracing experiment. single cell cdna libraries from the isolated cells were prepared using the chromium single cell ' system according to the manufacturer's instructions. the wom preparation employed v chemistry with the following modification: the cell suspension was directly added to the reverse transcription master mix, along with the appropriate volume of water to achieve the approximate cell capture target. the hbc lineage tracing experiments were performed using v chemistry. the . % weight/volume bsa washing step was omitted to minimize cell loss. completed libraries were sequenced on illumina hiseq to produce paired-end nt reads. sequence data were processed with the x genomics cell ranger pipeline ( . . for v chemistry), resulting in the initial starting number before filtering of , wom cells and , hbc lineage traced cells. the scone r/bioconductor package ( ) was used to filter out lowly-expressed genes (fewer than umi's in fewer than cells) and low-quality libraries (using the metric_sample_filter function with arguments hard_nreads = , zcut = ). cells with co-expression of male (ddx y, eif s y, kdm d, and uty) and female marker genes (xist) were removed as potential doublets from the wom dataset. for both datasets, doublet cell detection was performed per sample using doubletfinder ( ) and scrublet ( ) . genes with at least umis in at least cells were used for downstream clustering and cell type identification. for the hbc lineage tracing dataset, the bioconductor package scone was used to pick the top normalization ("none,fq,ruv_k= ,no_bio,batch"), corresponding to full quantile normalization, batch correction and removing one factor of unwanted variation using ruv ( ) . a range of cluster labels were created by clustering using the partitioning around medoids (pam) algorithm and hierarchical clustering in the clusterexperiment bioconductor package ( ) , with parameters k s= ( , , , , , ) and alpha=(na, . , . , . ). clusters that did not show differential expression were merged (using the function mergeclusters with arguments mergemethod = 'adjp', cutoff = . , and demethod = 'limma' for the lineagetraced dataset). initial clustering identified one macrophage (msr +) cluster consisting of cells; upon its removal and restarting from the normalization step a subsequent set of clusters was obtained. these clusters were used to filter out cells for which no stable clustering could be found (i.e., 'unassigned' cells), and four clusters respectively consisting of , and and cells. doublets were identified using doubletfinder and putative doublets were removed. inspection of the data in a three-dimensional umap embedding identified two groups of cells whose experimentally sampled timepoint did not match their position along the hbc differentiation trajectory, and these additional cells were also removed from subsequent analyses. analysis of wom scseq data were performed in python using the open-source scanpy software starting from the raw umi count matrix of the cells passing the initial filtering and qc criteria described above. umis were total-count normalized and scaled by , (tpt, tag per ten-thousands) and then log-normalized. for each gene, the residuals from linear regression models using the total number of umis per cell as predictors were then scaled via z-scoring. pca was then performed on a set of highlyvariable genes (excluding or genes) calculated using the "highly_variable_genes" function with parameters: min_mean= . , max_mean= , min_disp= . . a batch corrected neighborhood graph was constructed by the "bbknn" function with pcs with the parameters: local_connectivity= . , and embedding two-dimensions using the umap function with default parameters (min_dist = . ). cells were clustered using the neighborhood graph via the leiden algorithm (resolution = . ). identified clusters were manually merged and annotated based on known marker gene expression. we the filtered hbc lineage dataset containing cells was analyzing in python and processed for visualization using pipelines in spring and scanpy ( , ) . in brief, total counts were normalized to the median total counts for each cell and highly variable genes were selected using the spring gene filtering function ("filter_genes") using parameters ( , , ) . the dimensionality of the data was reduced to using principal components analysis (pca) and visualized in two-dimensions using the umap method with parameters (n_neighbors= , min_dist= . ). clustering was performed using the leiden algorithm (resolution= . ) and clusters were merged manually using known marker genes. expression of candidate cov- -related genes was defined if at least one transcript (umi) was detected in that cell, and the percent of cells expressing candidate genes was calculated for each cell type. in the wom dataset ace was only detected in out of , mature osns ( . %), and in the hbc lineage dataset, ace was not detected in any osns. furthermore, ace was not detected in immature sensory neurons (gbcs, inps, or iosns) in either dataset. single-cell rna-seq data from hbc-derived cells from fletcher et al. and gadye et al ( , ) , labeled via krt -creer driver mice, were downloaded from geo at accession gse using the file "gse _oehbcdiff_cufflinks_eset_counts_table.txt.gz". processing was performed as described above, including total counts normalization and filtering for highly variable genes using the spring gene filtering function "filter_genes" with parameters ( , , ) . the resulting data were visualized in spring and a subset of cells were removed for quality control, including a cluster of cells with low total counts and another with predominantly reads from ercc spike-in controls. putative doublets were also identified using scrublet and removed ( % of cells) ( ) . the resulting data were visualized in spring and partitioned using louvain clustering on the spring k-nearest-neighbor graph using the top principal components. cell type annotation was performed manually using the same set of markers genes listed above. three clusters were removed for quality control, including one with low total counts and one with predominantly reads from ercc spike-in controls (likely background), and one with high mitochondrial counts (likely stressed cells). for visualization, and clustering the remaining cells were projected to dimensions using pca and visualized with umap with parameters (n_neighbors= , min_dist= . , alpha= . , maxiter= ). clustering was performed using the leiden algorithm (resolution= . ) and cell types were manually annotated using known marker genes. the filtered dataset of mouse hbc-derived cells contained cells. the percent of cells expressing each marker gene was calculated as described above. of the osns identified, none of them expressed ace , and only out of inps and iosns expressed ace . in contrast, ace and tmprss were both detected in hbcs and sus cells. single-cell rnaseq data from whole mouse olfactory bulb ( ) were downloaded from mousebrain.org/loomfiles_level_l .html in loom format (l olfactory.loom) and converted to a seurat object. samples were obtained from juvenile mice (age postnatal day [ ] [ ] [ ] [ ] . this dataset comprises cells passing cell quality filters, excluding cells identified as potential doublets. a new dataset of whole olfactory bulb scseq was generated from adult male mice ( - weeks-old). all mouse husbandry and experiments were performed following institutional and federal guidelines and approved by harvard medical school's institutional animal care and use committee (iacuc). briefly, dissected olfactory bulbs (including the accessory olfactory bulb and fractions of the anterior olfactory nucleus) were dissociated in µl of dissociation media (dm: hbss containing mm hepes, mm mgcl , mm d-glucose) with u/ml papain and u/ml dnase i (worthington). minced tissue pieces were transferred to a ml round-bottom tube (bd). dm was added to a final volume of . ml and the tissue was mechanically triturated times with a p pipette tip. after -hour incubation with rocking at °c, the suspension was triturated with a ml pipette times and . ml was passed through µm cell strainer (bd). the suspension was then mechanically triturated with a p pipette tip times and µl were filtered on the same strainer. the cell suspension was further triturated with a p pipette tip times and filtered. ml of quench buffer ( ml of dm, . ml of protease inhibitor prepared by resuspending vial of protease inhibitor with ml of dm, and u of dnase i) was added to the suspension and centrifuged at g for min. cells were resuspended with ml of quench buffer and overlaid gently on top of ml of protease inhibitor, then spun down at g for min. the pellet was resuspended using dm supplemented with . % bsa and spun down at g for min. cells were suspended in µl of dm with . % bsa. drop-seq experiments were performed as previously described ( ) . microfluidics devices were obtained from flowjem and barcode beads were obtained from chemgenes. two min drop-seq runs were collected from a single dissociation preparation obtained from mice. two such dissociations were performed, giving total replicates. replicates of drop-seq samples were pooled and sequenced across runs on an illumina nextseq platform. paired end reads from the fastq files were trimmed, aligned, and tagged via the drop-seq tools ( - . ) pipeline, using star ( . . a) with genomic indices from ensembl release . the digital gene expression matrix was generated for , cells per replicate. cells with low numbers of genes ( ), low numbers of umis ( ) or high numbers of umis (> ) were removed ( % of cells). potential doublets were identified via scrublet and removed ( . % of cells). overall, this new dataset comprised cells. raw umi counts from juvenile and adult whole olfactory bulb samples were integrated in seurat ( ) . integrating the datasets ensured that clusters with rare cell types could be identified and that corresponding cell types could be accurately matched. as described below (see figure s ), although some cell types were observed with different frequencies, the integration procedure yielded stable clusters with cells from both datasets. briefly, raw counts were log-normalised separately and the most variable genes identified by variance stabilizing transformation for each dataset. the variable genes present in both datasets and the first principal components (pcs) were used as features for identifying the integration anchors. the integrated expression matrix was scaled and dimensionality reduced using pca. based on their percentage of explained variance, the first pcs were chosen for umap visualisation and clustering. graph-based clustering was performed using the louvain algorithm following the standard seurat workflow. cluster stability was analysed with clustree on a range of resolution values ( . to . ), with . yielding the most stable set of clusters ( ) . overall, clusters were identified, the smallest of which contained only cells with gene expression patterns consistent with blood cells, which were excluded from further visualisation plots. clustering the two datasets separately yielded similar results. moreover, the distribution of cells from each dataset across clusters was homogenous ( figure s ) and the clusters corresponded previous cell class and subtype annotations ( ) . as previously reported, a small cluster of excitatory neurons (cluster ) contained neurons from the anterior olfactory nucleus. umap visualisations of expression level for cell class and cell type markers, and for genes coding for coronavirus entry proteins, depict log-normalized umi counts. the heatmap in figure b shows the mean expression level for each cell class, normalised to the maximum mean value. the percentage of cells per cell class expressing ace was defined as the percentage of cells with at least one umi. in cells from both datasets, ace was enriched in pericytes but was not detected in neurons. acute olfactory bulb µm slices were obtained from dat-cre/flox-tdtomato (b .sjl-slc a tm . (cre) bkmn/j , jax stock / b .cg-gt(rosa) sor tm (cag-tdtomato)hze , jax stock ) p mice as previously described ( ) . as part of a wider study, at p these mice had undergone brief h unilateral naris occlusion via a plastic plug insert (n = mice) or were subjected to a sham control manipulation (n = mice); all observed effects here were independent of these treatment groups. single cell suspensions were generated using the neural tissue dissociation kit -postnatal neurons (miltenyi biotec. cat no. - - ), following manufacturer's instructions for manual dissociation, using fired-polished pasteur pipettes of progressively smaller diameter. after enzymatic and mechanical dissociations, cells were filtered through a µm cell strainer, centrifuged for minutes at ° c, resuspended in µl of acsf (in mm: nacl, . kcl, . nah po , hepes, glucose, mgcl , cacl ) with channel blockers ( . µm ttx, µm cnqx, µm d-apv) and kept on ice to minimise excitotoxicity and cell death. for manual sorting of fluorescently labelled dopaminergic neurons we adapted a previously described protocol ( ) . µl of single cell suspension was dispersed on . mm petri dishes (with a sylgard-covered base) containing ml of acsf + channel blockers. dishes were left undisturbed for minutes to allow the cells to sink and settle. throughout, dishes were kept on a metal plate on top of ice. tdtomato-positive cells were identified by their red fluorescence under a stereoscope. using a pulled glass capillary pipette attached to a mouthpiece, individual cells were aspirated and transferred to a clean, empty dish containing ml acsf + channel blockers. the same cell was then transferred to a third clean plate, changing pipettes for every plate change. finally, each individual cell was transferred to a . ml pcr tube containing µl of lysis buffer (rlt plus -qiagen). the tube was immediately placed on a metal plate sitting on top of dry ice for flash-freezing. collected cells were stored at - c until further processing. positive (more than cells) and negative (sample collection procedure without picking a cell) controls were collected for each sorting session. in total, we collected samples from . hours elapsed between mouse sacrifice and collection of the last cell in any session. samples were processing using a modified version of the smart-seq protocol ( ) . briefly, µl of a : , , dilution of ercc spike-ins (invitrogen. cat. no. ) was added to each sample and mrna was captured using modified oligo-dt biotinylated beads (dynabeads, invitrogen). pcr amplification was performed for cycles. amplified cdna was cleaned with a . : ratio of ampure-xp beads (beckman coulter). cdnas were quantified on qubit using hs dna reagents (invitrogen) and selected samples were run on a bioanalyzer hs dna chip (agilent) to evaluate size distribution. for generating the sequencing libraries, individual cdna samples were normalised to . ng/µl and µl was used for one-quarter standard-sized nextera xt (illumina) tagmentation reactions, with amplification cycles. sample indexing was performed using index sets a and d (illumina). at this point, individual samples were pooled according to their index set. pooled libraries were cleaned using a . : ratio of ampure beads and quantified on qubit using hs dna reagents and with the kapa library quantification kits for illumina (roche). samples were sequenced on two separate rapid-runs on hiseq (illumina), generating bp paired-end reads. an additional samples were sequenced on miseq (illumina). paired-end read fastq files were demultiplexed, quality controlled using fastqc (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/) and trimmed using trim galore (https://www.bioinformatics.babraham.ac.uk/projects/trim_galore/). reads were pseudoaligned and quantified using kallisto ( ) against a reference transcriptome from ensembl release (gencode release m grcm .p ) with sequences corresponding to the ercc spike-ins and the cre recombinase and tdt genes added to the index. transcripts were collapsed into genes using the sumacrossfeatures function in scater. cell level quality control and cell filtering was performed in scater ( ) . cells with < genes, < , reads, > % reads mapping to ercc spike-ins, > % reads mapping to mitochondrial genes or low library complexity were discarded ( % samples). the population of olfactory bulb cells labelled in dat-tdtomato mice is known to include a minor non-dopaminergic calretinin-positive subgroup ( ) , so calretininexpressing cells were excluded from all analyses. the sctransform function in seurat was used to remove technical batch effects. an analysis of single-cell gene expression data from studies was performed to investigate the expression of genes coding for coronavirus entry proteins in neurons from a range of brain regions and sensory systems. processed gene expression data tables were obtained from scseq studies that evaluated gene expression in retina (gse ) ( ) inner ear sensory epithelium (gse ) ( , ) and spiral ganglion (gse ) ( ) , ventral midbrain (gse ) ( ) , hippocampus (gse ) ( ), cortex (gse ) ( ), hypothalamus (gse ) ( ), visceral motor neurons (gse ) ( ) , dorsal root ganglia (gse ) ( ) and spinal cord dorsal horn (gse ) ( ) . smart-seq sequencing data from vsx -gfp positive cells was used from the retina dataset. a subset of the expression matrix that corresponds to day (i.e. control, undisturbed neurons) was used from the layer vi somatosensory cortex dataset. a subset of the data containing neurons from untreated (control) mice was used from the hypothalamic neuron dataset. from the ventral midbrain dopaminergic neuron dataset, a subset comprising dat-cre/tdtomato positive neurons from p mice was used. a subset comprising type i neurons from wild type mice was used from the spiral ganglion dataset. the "unclassified" neurons were excluded from the visceral motor neuron dataset. a subset containing neurons that were collected at room temperature was used from the dorsal root ganglia dataset. expression data from dorsal horn neurons obtained from c /bl wild type mice, vgat-cre-tdtomato and vglut -egfp mouse lines was used from the spinal cord dataset. inspection of all datasets for batch effects was performed using the scater package (version . . ) ( ) . publicly available raw count expression matrices were used for the retina, hippocampus, hypothalamus, midbrain, visceral motor neurons and spinal cord datasets, whereas the normalized expression data was used from the inner ear hair cell datasets. for datasets containing raw counts, normalization was performed for each dataset separately by computing pool-based size factors that are subsequently deconvolved to obtain cell-based size factors using the scran package (version . . ) ( ). violin plots were generated in scater. ( ) . normalized counts for each gene in the whole olfactory mucosa (wom) and olfactory sensory neurons (osns) are shown. each circle represents a biological replicate and each color indicates the category of the gene shown on the right (cov- and other covs: genes involved in the entry of these viruses, other categories: marker genes for specific cell types such fig. a for three bulk rna-sequencing datasets. mhv, mouse hepatitis virus. left plot is same as fig. a except for the addition of ceacam . (c) gene expression for cov-related genes including ace and tmprss as well as marker genes for olfactory and re subtypes are shown normalized by their maximum expression across cell types. ace and tmprss are expressed in wom respiratory and non-neuronal olfactory cell types, but not in osns. (d) umap representations of gene expression in the wom dataset for cov- related genes ace and tmprss , as well as marker genes for each cell type. each point represents an individual cell, and the color represents the normalized expression level for each gene (number of umis for a given gene divided by the total number of umis for each cell). (e) fluorescent in situ hybridization of an identified dorsal sustentacular cell marker, sult c (in yellow), combined with immunostaining for the known dorsal osn marker nqo (white). note that sult c rna fills the apical cytoplasm; given that sustentacular cells are ubiquitous in the epithelium, this is apparent as broad antisense signal for sult c in a pattern that is characteristic of the apical anatomy of sustentacular cells. sult c rna is detected in sustentacular cells in the nqo -positive dorsal olfactory epithelium. nuclei were stained with dapi (blue). bar = µm. granule cells ( ) granule cells ( ) immature neurons ( ) granule cells ( ) calretinin neurons ( ) astrocytes ( ) olfactory ensheathing cells ( ) immature neurons ( ) interneurons ( ) microglia ( ) oligodendrocytes ( ) dopaminergic neurons ( ) interneurons ( ) mitral/tufted cells -aon ( ) astrocytes ( ) vascular ( ) oligo precursor cells ( ) pericytes ( ) external tufted cells ( ) mitral/tufted cells ( ) perivascular 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the inner ear is shaped by activity sensory neuron diversity molecular diversity of midbrain development in resource molecular diversity of midbrain development in mouse, human and stem cells dissociable structural and functional hippocampal outputs via distinct subiculum cell classes variation in activity state, axonal projection, and position define the transcriptional identity of individual neocortical projection neurons molecular interrogation of hypothalamic organization reveals distinct dopamine neuronal subtypes visceral motor neuron diversity delineates a cellular basis for nipple-and pilo-erection muscle control unbiased classification of sensory neuron types by large-scale single-cell rna sequencing neuronal atlas of the dorsal horn defines its architecture and links sensory input to transcriptional cell types pooling across cells to normalize single-cell rna sequencing data with many zero counts we thank members of the datta lab, james schwob, bernardo sabatini, andreas schaefer, kevin franks, michael greenberg and vanessa ruta for helpful comments on the manuscript. we thank james lipscombe and andres crespo for technical support. data and materials availability: reanalyzed datasets are obtained from the urls listed in supplementary materials. all data is currently being deposited and will be made publicly accessible from the ncbi geo at accession gse . normalized expression * * * olfactory ensheathing cells (oec) and respiratory cells. (e) gene expression for cov-related genes including ace and tmprss as well as marker genes for olfactory and re subtypes are shown normalized by their maximum expression across cell types. ace and tmprss are expressed in wom respiratory and olfactory cell types, but not in osns. (f) cov- related genes ace and tmprss , as well as marker genes for cell types in fig. c ., in umap representation of wom dataset with normalized expression. gfap-positive oecs (olfactory ensheathing cells) and muc b-positive secretory cells are indicated by asterisks. key: cord- -xyo pe g authors: wang, ching-ying; lu, chien-yi; li, shih-wen; lai, chien-chen; hua, chun-hung; huang, su-hua; lin, ying-ju; hour, mann-jen; lin, cheng-wen title: sars coronavirus papain-like protease up-regulates the collagen expression through non-samd tgf-β signaling date: - - journal: virus res doi: . /j.virusres. . . sha: doc_id: cord_uid: xyo pe g sars coronavirus (cov) papain-like protease (plpro) reportedly induced the production of tgf-β through p mapk/stat -meidated egr- -dependent activation (sci. rep. , ). this study investigated the correlation of plpro-induced tgf-β with the expression of type i collagen in human lung epithelial cells and mouse pulmonary tissues. specific inhibitors for tgf-βri, p mapk, mek, and stat proved that sars-cov plpro induced tgf-β -dependent up-regulation of type i collagen in vitro and in vivo. subcellular localization analysis of smad and smad indicated that non-smad pathways in tgf-β signaling involved in the production of type i collagen in transfected cells with psars-plpro. comprehensive analysis of ubiquitin-conjugated proteins using immunoprecipitation and nanolc–ms/ms indicated that sars-cov plpro caused the change in the ubiquitination profile of rho gtpase family proteins, in which linked with the increase of rho-like gtpase family proteins. moreover, selective inhibitors tgf-βri and stat (as ) ascertained that stat activation was required for plpro-induced tgf-β -dependent up-regulation of type i collagen in human lung epithelial cells. the results showed that sars-cov plpro stimulated tgf-β -dependent expression of type i collagen via activating stat pathway. severe acute respiratory syndrome (sars)-associated coronavirus (cov), a member of betacoronoviruses in the coronaviridae family, is identified as the causative agent for the outbreak of sars in asia and other countries in [ ] [ ] . like other human coronaviruses (hcovs) hcov- e, hcov-oc , hcov-nl , hcov-hku , and mers-cov (li and lin, ; huang et al., ) , sars-cov genome is an approximately kb positive-strand rna consisting of a ′ cap, a ′ poly (a) tract, and open reading frames (orfs). the largest orfs orf a and orf ab encode for the polyprotein replicases a and ab mainly involving in the sars-cov replication, as cleaved in cis and in trans by orf a-encoded papain-like protease (plpro) and c-like protease ( clpro). plpro, a de-ubiquitinating/de-isgylating enzyme (barretto et al., ; ratia et al., ) , has the antagonistic activities of type i interferon (ifn) via blocking irf- and erk phosphorylation, preventing the iκbα degradation, and de-ubiquitinating the sting-traf -tbk complex (li et al., ; frieman et al., ; sun et al., ) . recently, plpro shows the inhibitory effect on tolllike receptor (tlr ) mediated cytokine production through removing lys -linked ubiquitin chains of traf and traf (li et al., a) . sars-cov up-regulates pro-inflammatory cytokines like ifn-γ, il- , tgf-β , tnf-α, il- , ip- , mcp- , mig, and il- (huang et al., ; he et al., ) , in which recruits immune responder cells into the lungs, triggers acute respiratory distress syndrome (ards), and even causes lung fibrosis in the late phase (huang et al., ; he et al., ) . among sars-cov proteins, the nucleocapsid induces a smad dependent induction of tgf-β expression (zhao et al., ) ; spike protein stimulates the il- up-regulation in lung cells (chang et al., ) ; nsp provokes the expression of ccl , cxvl , and ccl (law http://dx.doi.org/ . (law http://dx.doi.org/ . /j.virusres. received january ; received in revised form april ; accepted april et al., ) ; plpro elavates the production of tgf-β and pro-fibrotic markers via ubiquitin proteasome, p mapk, and erk / -mediated signaling (li et al., ) . recently, sars-cov plpro notably initiates ros/p mapk/stat pathway to activate egr- dependent expression of tsp- , tgf-β and vimentin in vitro and in vivo (li et al., b) . therefore, plpro becomes a virulent factor in sars pathogenesis. tgf-β plays a crucial mediator of tissue fibrosis in lung, skin, liver, heart, and kidney through modulating the expression of pro-fibrotic proteins including type i collagen, fibronectin, α-sma, and vimentin (kubiczkova et al., ) . in the canonical smad signalling pathway, tgf-β interacts with the tgf-β type i receptor, activates receptorregulated smads (smad and smad ) complexed with smad , and then translocate to the nucleus to synthesize tgf-β -induced transcriptional genes like type i collagen (leask and abraham, ; kubiczkova et al., ) . in non-canonical tgf-β signaling pathways, tgf-β activates mapks (erk, jnk and p mapk), rho-like gtpases (rhoa, rac and cdc ), pi k/akt, wnt/β-catenin, or ca + signaling cascades in tgf-β -induced transcriptional response (zhang, ). the activation of p mapk is responsible for tgf-β-induced epithelial-tomesenchymal transition of mouse mammary gland epithelial cells under a receptor independent of receptor-mediated smad activation (yu et al., ) . the activation of rhoa in tgf-β-induced emt response is also independent of smad and/or smad (bhowmick et al., ) . our prior study demonstrated sars-cov plpro triggering the tgf-β production in vitro and in vivo that linked with up-regulating the expression of pro-fibrotic proteins (vimentin and glial fibrillary acidic protein) (li et al., b) . this study assesses possible effects and mechanisms of sars-cov plpro-induced tgf-β upregulation on the expression of type i collagen. the induction ability of sars-cov plpro on in vitro and in vivo expression of type i collagen was characterized. in addition, subcellular localization of smad , smad , and stat was performed to elucidate the key factors involved in the induction of type i collagen by plpro. the relationship between tgf-β upregulation and the mechanism of type i collagen induction by plpro was validated by the specific inhibitors of tgf-β receptor kinase, p mapk and stat . human alveolar basal epithelial a cells grew in dulbecco's modified eagle's medium (hyclone laboratories) and were transfected with control vector pcdna . /his c (invitrogen), or psars-plpro containing sars-cov plpro gene, as described in our prior reports (li et al., (li et al., , (li et al., , a (li et al., , b . in addition, psars-plpro (h a) that had the alanine substitution for histidine at position by ala within plpro gene was constructed using pcr-based sitedirected mutagenesis with a mutated primer pair ( ′-ggtaact atc-agtgtggtgcttacactcatataactgctaag- ′ and ′-cttagcagt tatatgagtgtaagcaccacactgatagttacc- ′). a cells transiently expressing recombinant plpro days post transfection was analyzed using western blotting, real-time rt-pcr, sirius staining, and immunofluorescent staining assays. to detect the mrna expression of type i collagen, total rnas extracted from transfected cells treated with or without a tgf-β inhibitor (sb- ) were further examined the mrna levels of type i collagen using quantitative real-time pcr. the relative fold mrna levels were normalized by gapdh mrna, presented as the relative ratio (b). for verifying the protein levels of type i collagen, transfected cells with serial doses of indicated plasmids were stained using sirius red staining kit (c). ** p value < . by student's t-test. the lysate of transfected cells was performed by western blotting with primary antibodies including rabbit anti-tgf-β (cell signaling), anti-e. coli synthesized plpro mouse serum, anti-phospho stat (tyr ) (cell signaling), and anti-β-actin mab (abcam), and hrpconjugated secondary antibodies like goat anti-mouse or anti-rabbit igg. immune complexes were detected using enhanced chemiluminescent hrp substrate (millipore). to measure the expression of type i collagen, tgf-β , and vimentin in transfected cells, total rnas extracted from transfected cells days post transfection and mouse lung tissues were analyzed using two-step real time rt-pcr with sybr green i, as described in our prior reports (li et al., b) . primer pairs included ( ) ′-gttcgtgaccgtgac-ctcg- ′ and ′-tcttgtccttggggttcttgc- ′ for human type i collagen, ( ) ′-gagcggagagtactggatcg- ′ and ′-tactcgaac-gggaatccatc- ′ for mouse type i collagen, ( ) ′-ggcctttcctgc-ttctcatgg- ′ and ′-ccttgctgtactgcgtgtcc- ′ for human tgf-β , ( ) ′-tctctgaggctgccaaccg- ′ and ′-cgaaggtgacgagc-catttcc- ′ for human vimentin, ( ) ′-cagaacagcctcccgaatg- ′ and ′-tgctacgctcactccattac- ′ for human rac , ( ) ′-agcc-acatcgctcagacac- ′ and ′-gcccaatacgaccaa atcc- ′ for human gapdh, and ( ) ′-tgaggccggtgctgagtatgtcg- ′ and ′-ccacagtcttctgggtggcagtg- ′ for mouse gapdh. specific pcr product was quantified using the abi prism ht sequence detection system (pe applied biosystems). relative mrna levels of indicated genes were normalized relative to gapdh mrna. for the detection of collagen expression, the tissue sections were stained with sirius red solution for h, and then rinsed times with . % glacial acetic acid in pbs. after dehydrating with ethanol, stained sections were mounted on the glass slides, and then examined using light microscopy (olympus, bx ). the mouse mode with a direct chest injection was performed as described in our prior report (li et al., b) . empty vector pcdna . or recombinant plasmin psars-plpro ( μg/ μl) in % sucrose/ pbs was injected into the right chest of eight-weeks-old balb/c male mice using a -ml syringe with a -gage needle every days. after injections, the mice were sacrificed; the lung tissues were fixed, dehydrated, embedded in paraffin, and cut at - μm thickness using a rotary microtome. for immunohistochemistry (ihc) staining, mouse lung tissues were performed with anti-e. coli synthesized plpro serum, as descried in our previous report (li et al., b) . for h & e staining, sections were stained with hematoxylin for min, eosin for min, dehydrated in ethanol, and then mounted as slides that were examined and photographed using light microscopy (olympus, bx ). sirius staining and sybr green real time rt-pcr assays were mentioned above. for determining the effects of sars-cov plpro on the nuclear translocalization of smad , smad , and stat , a cells grew on the glass coverslip in -wellt were transfected with psars-cov plpro or pcdna . , and treated with or without μm kartogenin (sigma). for testing the role of rac in stat signal, the rac mutant plasmid, pmx-ig-rac t n provided by dr. takehito uruno (kyushu university, japan), was co-transfected into cells. after -day incubation, cells were fixed with . % formaldehyde in pbs for h, blocked with % bovine serum albumin in pbs for h, and then incubated with specific primary antibodies against smad , smad , and stat at °c to detect the mrna expression of type i collagen, total rnas extracted from lung tissues were examined the mrna levels of type i collagen using quantitative real-time pcr. the relative fold mrna levels were normalized by gapdh mrna, presented as the relative ratio (b). ** p value < . by student's t-test. overnight. subsequently, cells were reacted with fitc-or af conjugated secondary antibodies in a dark box for h, finally, cells were stained with ′, -diamidino- -phenylindole (dapi) for min. after washing with pbs, stained cells were photographed using the immunofluorescence microscopy (olympus, bx ). the lysates from plpro-expressing and empty vector cells were reacted with anti-ubiquitin antibodies for h at °c, and then incubated with protein a-sepharose beads. the ubiquitin-conjugated proteins were collected after centrifugation, washed four times with net buffer, embedded in sds-page gel, and then digested in gel. the peptides of ubiquitin-conjugated proteins were recovered for nanolc-ms/ms spectra. proteins were identified according to mass spectra obtained were compared to swissport database (release . ) via mascot algorithm (version . . ), as described in our prior reports (li et al., ) . peptides were identified if mascot individual ion scores exceeded . all data were collected from independent experiments and analyzed using student's t-test or χ test. statistical significance was considered at p < . . to examine the association of sars-cov plpro-induced tgf-β production with the collagen up-regulation, a lung epithelial cells transiently transfected with pcdna . and psars-plpro were analyzed the production of tgf-β and type i collagen using western blot, realtime rt-pcr and sirius red staining assays (fig. ) . transfected cells with psars-plpro, but not pcdna . , secreted the active form of tgf-β , and significantly increased the mrna and protein expression of type i collagen. importantly, sb- (a selective tgf-βri inhibitor) treatment at nm caused the -fold reduction of type i collagen mrna in transfected cells with psars-plpro (fig. b) . in a mouse model, the expression of type i collagen in lung tissues from chest injection with pcdna . or psars-plpro was examined using sirius staining and quantitative rt-pcr (fig. ) . the expression of sars-plpro in lung tissues of mice was determined using ihc staining with anti-e. coli synthesized plpro serum, and ihc positivity for plpro expression within lung tissues was observed in the group infected with psars-plpro ( fig. a) . h & e and sirius staining assays indicated that pulmonary inflammation with the infiltration of immune cells and the increase of type i collagen was identified in the psars-plpro group, but not vector control and solvent groups ( fig. a) . real-time pcr confirmed that plpro triggered the mrna expression of type i collagen fig. . analysis of tgf-β and type i collagen levels in transiently transfected cells in response to the inhibitors for p mapk and stat . to detect the mrna expression of tgf-β or type i collagen, total rnas extracted from transfected cells treated with or without the rac mutant plasmid (a), an inhibitor for p mapk (sb ) (c) or stat (stattic) (d) were examined the mrna levels of type i collagen using quantitative real-time pcr. the relative fold mrna levels were normalized by gapdh mrna, presented as the relative ratio (a, b). for verifying the protein levels of type i collagen, transfected cells treated with or without an inhibitor for p mapk (sb ) or mek (u ) were stained using sirius red staining kit (c). ** p value < . by student's t-test. c.-y. wang et al. virus research ( ) [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] in mouse lung tissues in comparison with vector control and solvent groups (fig. b) . to examine the proteolytic enzymatic activity of plpro on the production of tgf-β and type i collagen, the catalytic mutant of plpro (h a) was constructed, and then used to investigate whether plpro (h a) up-regulated tgf-β and type i collagen in vitro (fig. a) . importantly, plpro(h a) with the catalytic mutation lose the ability to induce the expression of tgf-β and type i collagen in vitro. since sars-plpro had been demonstrated to stimulate p mapk/stat mediated activation of tgf-β production (li et al., b) , sb (a specific p -mapks inhibitor), u (a mek / inhibitor), and stattic (a small-molecule inhibitor of stat activation) were used to further confirm the correlation between the tgf-β production and type i collagen up-regulation in transfected cells with psars-plpro and pcdna . (fig. b-d) . sb significantly reduced the mrna expression of tgf-β , in which was linked with down-regulation of type i collagen in transfected cells with psars-plpro in presence of sb or u ( fig. b and c) . stattic also suppressed the mrna expression of type i collagen in transfected cells with psars-plpro ( fig. d) . overall, results of the in vitro and in vivo data demonstrated that the proteolytic enzymatic activity was required for sars-cov plpro-dependent tgf-β -mediated up-regulation of pro-fibrotic gene type i collagen. to examine whether smad-dependent pathways involve in tgf-β mediated up-regulation of type i collagen in response sars-cov plpro, subcellular localization of receptor-regulated smad and inhibitory smad in transfected cells were detected using the immunofluorescent and dapi staining (fig. ) . imaging analysis of transfected cells indicated that smad localized in the nucleus of pcdna . -transfected cells, but not psars-plpro-transfected cells (fig. a) . moreover, smad was detected in the nucleus of both transfected cells (fig. b) . interestingly, kartogenin, a stimulator for tgf-β /smad signal pathway , was used to verify the inhibitory effect of plpro on tgf-β /smad signal in transfected cells (fig. c) . after h treatment with kartogenin, samd nuclear translocation was spotted within the nucleus in vector control cells, but not in plproexpressing cells. most samd was in the cytoplasm of psars-plpro transfected cells treated with kartogenin. the result indicated that sars-cov plpro inactivated smad-dependent pathways, implying that non-smad pathways in tgf-β signaling for the production of type i collagen would be initiated by sars-cov plpro. non-smad pathways in tgf-β signaling include map kinase, rholike gtpase, and phosphatidylinositol- -kinase/akt pathways (zhang, ) . to examine the possible pathways involved in tgf-β -dependent up-regulation of type i collagen by sars-cov plpro, the profiles of ubiquitin-conjugated proteins in transfected cells with vector control and psars-plpro were determined using immune-precipitation and nanolc-ms/ms. interestingly, several proteins of the ras gtpase family were identified (table ), in which indicated the ubiquitination of these ras family gtpase proteins was influenced by sars-cov plpro. fig. a represented the mass spectrum of rac identified by lc-ms/ms. rac , a rho gtpase, had an increase of the ubiquitination in plpro-expressing cells compared to vector control cells. real-time rt-pcr and western blotting assays revealed the elevation of rac mrna and protein expression in transfected cells with psars-plpro compared to vector control ( fig. b and c) . rho gtpases were identified in the regulation of g protein-coupled receptor signaling through activation of jak/stat and modulated stat-dependent gene expression (pelletier et al., ) . in addition, stat- -dependent collagen production has been demonstrated in human skin fibroblasts and mouse airway fibroblasts in response to bovine milk and platelet-derived growth factor, respectively (kippenberger et al., ; lu et al., ) . for analyzing the correlation between plpro-induced rac upregulation and stat activation, co-transfection of pcdna . or psars-plpro plus the rac mutant plasmid pmx-ig-rac t n was performed, and the stat activation in co-transfected cells was measured using immunofluorescence staining with anti-stat and fitc-conjugated secondary antibodies. plpro expression caused the stat expression and induced the translation of stat in the nucleus. remarkably, the rac mutant slightly affected the stat up-regulation by plpro, but significantly reduced plpro-induced stat nuclear translocation (fig. d) . later, the functional activity of stat in tgf-β -dependent collagen up-regulation was further characterized in vector control and plpro-expressing cells (figs. and ) . nuclear localization and phosphorylation of stat was observed in transfected cells with psars-plpro, but not vector control cells (figs. and a) . importantly, sb- , a selective tgf-βr inhibitor, significantly reduced the entry of stat in the nucleus of plpro-expressing cells (fig. ) . the result indicated that rho gtpases/stat was responsible for one of non-smad pathways in sars plpro-induced tgf-β signals. furthermore, a stat inhibitor as markedly suppressed tgf-β -dependent collagen expression in plpro-expressing cells (fig. ) . the results demonstrated that stat activation was required for sars plpro-induced tgf-β -dependent production of type i collagen. plpro has been demonstrated to trigger the tgf-β production in human promonocytes, human lung epithelial cells, and pulmonary tissues in mouse models (li et al., (li et al., , b . sars-cov plpro induced the ros-mediated p mapk and stat activation of egr- expression, in which egr- specifically bound to the tgf-β promoter region between − to − , resulting in the increase of tgf-β production. this study indicated that sars-cov plpro stimulated the production of type i collagen in vitro and in vivo ( fig. - ) . sars-cov plpro-induced collagen deposition in pulmonary tissues was associated with lung inflammation and pulmonary fibrosis in mice injected with psars-plpro (fig. ) . the specific inhibitor for tgf-β receptor, sb- , blocked the up-regulation of type i collagen in transfected cells with psars-plpro. moreover, the inhibitors for p mapk and stat that involved in egr- -dependent tgf-β production significantly reduced the expression of tgf-β and type i collagen. overall, the results revealed that sars-cov plpro elicited tgf-β dependent up-regulation of type i collagen. tgf-β -dependent profibrotic response was considered as the crucial character in sars-cov plpro-induced pathogenesis. an elevated level of pro-inflammatory cytokines including tgf-β was detected in sars-cov-infected cells in autopsy tissues from died sars patients (he et al., ) , in which implied the association of pro-inflammatory cytokines with the severity and mortality of sars. the increase of serum tgf-β concentration during the early phase of sars could be associated with lung infiltration (beijing group of national research project for sars, ) . meanwhile, the decrease of serum tgf-β concentration markedly lowered in the severity of sars patients (zhang et al., ) . in canonical pathway, tgf-β interacts with type i and ii tgf-β receptors and causes the activation of a serine/threonine kinase domain in tgf-β receptors and the recruitment and phosphorylation of smad and smad . the phosphorylated smad / complexed with smad was trans-localized into nucleus to regulate the target gene expression (kubiczkova et al., ) . subcellular localization analysis demonstrated that smad was predominant in cytoplasmic, but not in the nucleus in transfected cells with psars-plpro compared to vector control (fig. ) , revealing that canonical smad-dependent signaling pathway was not involved in plpro-induced tgf-β -dependent upregulation of type i collagen. tgf-β also activates non-canonical non-smad pathways, including mapks (erk /erk , jnk and p ), pi k kinases, akt/pkb, mtor, and rho-like gtpase family proteins (ras, rhoa, rac , and cdc ) (zhang, ; kubiczkova et al., ) . particularly, activation of rhoa pathway was required for tgf-βmediated process of epithelial-to-mesenchymal trans-differentiaion in a dominant-negative smad cells (bhowmick et al., ) . in addition, rac activation promoted tgf-β-dependent collagen expression in mesangial cells (hubchak et al., ) . lc-ms/ms analysis demonstrated that sars-cov plpro influenced the ubiquitination status of ras-related c botulinum toxin substrate (rac ), ras gtpaseactivating-like protein iqgap , ras-related protein rab (rab c), putative ras-related protein rab (rab c), and ras gtpase-activating protein-binding protein (g bp ) ( table ) . real-time rt-pcr and western blotting assays indicated the increased expression of rac in transfected cells with psars-plpro compared to vector control (fig. ) . since activation of jak/stat pathway in a rac-dependent manner was identified in response to the agonist of protein-coupled receptors (pelletier et al., ) , stat-dependent signals were investigated in this study. the tgf-βri inhibitor sb- significantly attenuated the phosphorylation and nuclear localization of stat in transfected cells with psars-plpro (fig. ) . meanwhile, the stat inhibitor as meaningfully reduced tgf-β -dependent up-regulation of type i collagen in plpro-expressing cells (fig. ) . the results indicated fig. . nuclear localization of stat in transfected lung epithelial cells in response to sb- . for analyzing the effect of sb- on nuclear localization of stat , transfected cells were treated with or without sb- for days, washed, fixed, and reacted with primary antibodies against phospho-stat , followed by fitc-conjugated secondary antibodies. after staining with dapi for min, imaging was analyzed by immunofluorescent microscopy. that stat activation was required for plpro-induced tgf-β -dependent expression of type i collagen in human lung epithelial cells and mouse lung tissues. the result was accordant with the previous reports in that stat -dependent collagen production has been demonstrated in human skin fibroblasts and mouse airway fibroblasts (kippenberger et al., ; lu et al., ) . sars-cov plpro affected on the ubiquitination and expression profile of rho-like gtpase family proteins that could link with the activation of stat signaling in tgf-β dependent collagen expression, in which suggested that a new gtpase proteins/stat pathway might play a critical role in tgf-β -dependent collagen production in plpro-expressing cells. in summary, sars-cov plpro induced p mapk/stat -mediated tgf-β -dependent up-regulation of type i collagen, causing pulmonary pro-fibrotic responses. plpro diminished the nuclear localization of smad , changed the expression profiling of rho-like gtpase family proteins, and activated stat -mediated tgf-β -dependent production of type i collagen. the results let us conclude that sars-cov plpro induced non-smad signals including stat activation in tgf-β dependent pulmonary pro-fibrotic responses. fig. . analysis of stat -mediated plpro-induced production of type i collagen. to determine the phosphorylation of stat , transfected cells with . , , , or μg of pcdna . or pplpro were harvested days post transfection; the cell lysates were examined using western blotting with anti-phospho-stat (tyr ) or anti-β actin antibodies (a). for analyzing mrna levels of type i collagen (b) and vimentin (c), transfected cells were treated with or without as for h, and then their mrna levels of type i collagen and vimentin were measured by quantitative pcr. relative mrna levels were normalized by gapdh mrna, presented as relative ratio. ** p value < . by student's t-test. the papain-like protease of severe acute respiratory syndrome coronavirus has deubiquitinating activity dynamic changes in blood cytokine levels as clinical indicators in severe acute respiratory syndrome transforming growth factor-beta mediates epithelial to mesenchymal transdifferentiation through a rhoa-dependent mechanism induction of il- release in lung cells via activator protein- by recombinant baculovirus displaying severe acute respiratory syndrome-coronavirus spike proteins: identification of two functional regions severe acute respiratory syndrome coronavirus papain-like protease ubiquitin-like domain and catalytic domain regulate antagonism of irf and nf-kappab signaling expression of elevated levels of pro-inflammatory cytokines in sars-cov-infected ace + cells in sars patients: relation to the acute lung injury and pathogenesis of sars an interferon-gamma-related cytokine storm in sars patients epidemiology of human coronavirus nl infection among hospitalized patients with pneumonia in taiwan rac promotes tgfbeta-stimulated mesangial cell type i collagen expression through a pi k/aktdependent mechanism stat -dependent collagen synthesis in human fibroblasts is induced by bovine milk tgf-β-an excellent servant but a bad master role for nonstructural protein of severe acute respiratory syndrome coronavirus in chemokine dysregulation tgf-beta signaling and the fibrotic response human coronaviruses: clinical features and phylogenetic analysis severe acute respiratory syndrome coronavirus papain-like protease suppressed alpha interferon-induced responses through downregulation of extracellular signalregulated kinase -mediated signalling pathways correlation between tgf-β expression and proteomic profiling induced by severe acute respiratory syndrome coronavirus papain-like protease sars coronavirus papain-like protease inhibits the tlr signaling pathway through removing lys -linked polyubiquitination of traf and traf sars coronavirus papain-like protease induces egr- -dependent upregulation of tgf-β via ros/p mapk/stat pathway platelet-derived growth factor mediates interleukin- -induced collagen i production in mouse airway fibroblasts rho family gtpases are required for activation of jak/stat signaling by g protein-coupled receptors severe acute respiratory syndrome coronavirus papain-like protease: structure of a viral deubiquitinating enzyme coronavirus papain-like proteases negatively regulate antiviral innate immune response through disruption of sting-mediated signaling a heterocyclic molecule kartogenin induces collagen synthesis of human dermal fibroblasts by activating the smad / smad pathway tgf-β receptor-activated p map kinase mediates smad-independent tgf-β responses analysis of serum cytokines in patients with severe acute respiratory syndrome non-smad pathways in tgf-beta signaling severe acute respiratory syndrome-associated coronavirus nucleocapsid protein interacts with smad and modulates transforming growth factor-beta signaling this study was supported by china medical university under the aim for top university plan of the ministry of education, taiwan (chm - - ). this project was also funded by grants from the ministry of science and technology, taiwan (most - -b- - -my , most - -b- - -my ) and china medical university (cmu -s- , cmu -asia- , and cmu -asia- ). key: cord- -lrsqrc u authors: yañez-guerra, luis alfonso; zhong, xingxing; moghul, ismail; butts, thomas; zampronio, cleidiane g; jones, alexandra m; mirabeau, olivier; elphick, maurice r title: echinoderms provide missing link in the evolution of prrp/snpf-type neuropeptide signalling date: - - journal: elife doi: . /elife. sha: doc_id: cord_uid: lrsqrc u neuropeptide signalling systems comprising peptide ligands and cognate receptors are evolutionarily ancient regulators of physiology and behaviour. however, there are challenges associated with determination of orthology between neuropeptides in different taxa. orthologs of vertebrate neuropeptide-y (npy) known as neuropeptide-f (npf) have been identified in protostome invertebrates, whilst prolactin-releasing peptide (prrp) and short neuropeptide-f (snpf) have been identified as paralogs of npy/npf in vertebrates and protostomes, respectively. here we investigated the occurrence of npy/npf/prrp/snpf-related signalling systems in a deuterostome invertebrate phylum – the echinodermata. analysis of transcriptome/genome sequence data revealed loss of npy/npf-type signalling, but orthologs of prrp-type neuropeptides and snpf/prrp-type receptors were identified in echinoderms. furthermore, experimental studies revealed that the prrp-type neuropeptide pqdrskamqaertgqlrrlnprf-nh( ) is a potent ligand for a snpf/prrp-type receptor in the starfish asterias rubens. our findings indicate that prrp-type and snpf-type signalling systems are orthologous and originated as a paralog of npy/npf-type signalling in urbilateria. neuropeptides are neuronally secreted signalling molecules that regulate many physiological processes and behaviours in animals, including feeding, digestion, reproduction and social behaviour. they typically exert effects by binding to cognate g-protein coupled receptors (gpcrs) on target cells, which leads to changes in the activity of downstream effectors (e.g. ion channels, enzymes) (jékely et al., ) . investigation of the evolution of neuropeptide signalling has revealed that many of the neuropeptide systems found in vertebrates have orthologs in invertebrate deuterostomes (urochordates, cephalochordates, hemichordates, echinoderms) and protostomes (e.g. arthropods, nematodes, molluscs, annelids, platyhelminthes). thus, the evolutionary origin of over thirty neuropeptide signalling systems has been traced back to the common ancestor of the bilateria (urbilateria) (jékely, ; mirabeau and joly, ; elphick et al., ) . one of the neuropeptide systems that originated in urbilateria is neuropeptide y (npy)-type signalling. npy is a -residue peptide that was first isolated from the porcine hypothalamus tatemoto, ) but which is also expressed by neurons in many other regions of the nervous system (adrian et al., ; morris, ) and in peripheral organs such as the gut and cardiovascular system (holzer et al., ; farzi et al., ) . accordingly, npy is pleiotropic (pedrazzini et al., ) , although it is most widely known as a potent stimulant of food snpf-type signalling may be restricted to protostomes (mirabeau and joly, ) . subsequently, snpf-type peptides and a cognate receptor have been characterised in the bivalve mollusc crassostrea gigas, confirming the occurrence of this signalling system in the lophotrochozoan branch of the protostomes (bigot et al., ) . furthermore, the physiological roles of snpf-type neuropeptides have been characterised in c. gigas and in other molluscs (hoek et al., ; zatylny-gaudin et al., ; bigot et al., ) . important insights into neuropeptide evolution have been obtained recently by pharmacological characterisation of g-protein coupled neuropeptide receptors in invertebrate deuterostomes (kawada et al., ; roch et al., ; bauknecht and jékely, ; semmens et al., ; tian et al., ; yañez-guerra et al., ) . however, currently little is known about the occurrence and characteristics of npy/npf/prrp/snpf-related signalling systems in invertebrate deuterostomes. phylogenetic analysis of bilaterian g-protein coupled neuropeptide receptors has demonstrated the occurrence of npy/npf receptor-related proteins in ambulacrarians -the echinoderm strongylocentrotus purpuratus and the hemichordate saccoglossus kowalevskii (mirabeau and joly, ) . furthermore, the precursor of a putative npy/npf-type peptide was identified in s. kowalevskii (mirabeau and joly, ; elphick and mirabeau, ) . a candidate npy/npf-type precursor has also been identified in the cephalochordate branchiostoma floridae, but an npy/npf-type receptor has yet to be identified in this species (mirabeau and joly, ; elphick and mirabeau, ) . a more recent finding was the discovery of a family neuropeptide precursor-type proteins in echinoderms that contain a peptide that shares sequence similarity with npy/npf-type peptides (zandawala et al., ) . however, it is not known if these proteins are orthologs of vertebrate npy-type precursors and protostome npftype precursors. to address this issue, detailed analysis of the sequences of the echinoderm npy/npf-like peptides and precursors and the genes encoding these peptides/proteins is needed. furthermore, the receptors for echinoderm npy/npf-like peptides need to be identified. accordingly, here we show that npy/npf-type signalling has in fact been lost in echinoderms and report the discovery and pharmacological characterisation of a prrp/snpf-type signalling system in an echinoderm -the starfish asterias rubens. these findings provide important new insights into the evolution of neuropeptide signalling in the bilateria. the sequence of a transcript (contig ; genbank accession number mk . ) encoding the precursor of an npy-like neuropeptide has been reported previously based on analysis of neural transcriptome sequence data from the starfish a. rubens (zandawala et al., ) . here, a cdna encoding this precursor was cloned and sequenced, revealing that the open reading frame encodes a -residue protein comprising a predicted -residue signal peptide, a -residue npy-like peptide sequence with an n-terminal glutamine residue and a c-terminal glycine residue, followed by a putative monobasic cleavage site (figure -figure supplement a) . analysis of radial nerve cord extracts using mass spectrometry (lc-ms-ms) revealed the presence of a peptide with the structure pqdrskamqaertgqlrrlnprf-nh , showing that the n-terminal glutamine and c-terminal glycine in the precursor peptide are post-translationally converted to a pyroglutamate residue and an amide group, respectively ( figure -figure supplement b) . alignment of the sequences of the a. rubens neuropeptide and orthologs from other echinoderms with related peptides in other taxa revealed that they share sequence similarity with both prrp-type neuropeptides ( figure a ) and with npy/npf-type neuropeptides ( figure b) . however, the echinoderm peptides comprise - residues and are similar in length to vertebrate prrps, which are - residues as full-length peptides and in some species can occur as n-terminally truncated peptides due the presence of a monobasic cleavage site (hinuma et al., ; tachibana and sakamoto, ) . this contrasts with npy/npf-type neuropeptides, which are longer peptides ranging in length from to residues (fadda et al., ) . furthermore, by analysing sequence data from the hemichordate s. kowalevskii and the cephalochordate b. floridae, here we identified novel neuropeptides that share sequence similarity with the echinoderm figure . comparison of the sequences of echinoderm npy/npf/prrp-like peptides with related peptides in other taxa. (a) comparison with prrp-type neuropeptides. conserved residues are highlighted in black (identical) or grey (conservative substitutions) (b) comparison with npy/npf-type neuropeptides. conserved residues are highlighted in black (identical) or grey (conservative substitutions). the arrowheads indicate residues that have been shown to be important for the three-dimensional structure of the npy/npf-type peptides but which are not present in the echinoderm peptides. the colour coding of phyla is as follows: dark blue (echinodermata), light blue (hemichordata), purple (chordata), orange (platyhelminthes), red (lophotrochozoa), yellow (priapulida), green (arthropoda), grey (nematoda). the full names of the species and the accession numbers of the sequences are listed in figure -source data . figure continued on next page neuropeptides and with vertebrate prrps ( figure a) . thus, sequence alignment reveals that, in addition to a shared characteristic of a c-terminal rfamide or a ryamide (y and f being conservative substitutions), there are other residues in the echinoderm peptides that are identical or structurally similar to equivalently positioned residues in chordate prrps ( figure a) . contrastingly, the echinoderm peptides lack two proline (p) residues that are a conserved feature of the n-terminal region of many npy/npf-type peptides, with the exception of some peptides that have only one of these proline residues and a peptide in the cephalochordate branchiostoma floridae that has neither ( figure b) . furthermore, there are four other residues that are highly conserved in bilaterian npy/npf-type peptides -tyrosine (y), leucine (l), tyrosine (y), and isoleucine (i) residues, which are marked with arrowheads in figure b . these residues have been shown to be important for the formation of the three-dimensional structure in vertebrate npytype peptides (blundell et al., ; glover et al., ; glover et al., ; allen et al., ) , so these residues may likewise be important for npf receptor activation and bioactivity. importantly, none of these residues are present in the echinoderm peptides. it is noteworthy, however, that all but one of the aforementioned six conserved residues in npy/ npf-type peptides are present in a peptide from a species belonging to a sister phylum of the echinoderms -the hemichordate saccoglossus kowalevskii ( figure b ; mirabeau and joly, ; elphick and mirabeau, ) . collectively these findings indicate that the echinoderm neuropeptides originally described as npy-type peptides (zandawala et al., ) are not orthologs of npy/ npf-type peptides but are orthologs of chordate prrp-type peptides. therefore, henceforth we will refer to the a. rubens neuropeptide pqdrskamqaertgqlrrlnprf-nh as arprrp and we will refer to orthologs in other echinoderms equivalently. echinoderm prrp-like peptide genes have the same exon-intron structure as chordate prrp genes to investigate further the proposition that arprrp and other echinoderm prrp-like neuropeptides are orthologs of chordate prrps, we compared the exon-intron structure of genes encoding these peptides ( figure ). this revealed that a common characteristic is the presence of an intron that interrupts the coding sequence at a position corresponding to the n-terminal or central region of the echinoderm prrp-like peptides and vertebrate prrps. furthermore, in echinoderm prrp-like peptide genes and vertebrate prrp genes the intron interrupts the coding sequence in the same frame, at a position between the first and second nucleotide of the interrupted codon (a phase one intron), which is denoted by + in figure . genes encoding novel precursors of prrp-like peptides in s. kowalevskii and b. floridae also have a phase one intron. furthermore, in the b. floridae gene and in one of the s. kowalevskii genes (skow ) the intron is located in the region of the gene encoding the n-terminal part of the neuropeptide, whereas in the other s. kowalevskii gene (skow ) the intron is located in a region encoding the c-terminal part of the neuropeptide. the presence of a conserved intron in the same frame in echinoderm prrp-like peptide genes, the two s. kowalevskii prrp-like peptide genes and chordate prrp-type genes supports our hypothesis that the echinoderm and hemichordate prrp-like peptides are orthologs of chordate prrp-type neuropeptides. by way of comparison, echinoderm prrp-like peptide genes have a different exon-intron structure to npy/npf genes. previous studies have reported that a conserved feature of npy/npf genes is an intron that interrupts the coding sequence for npy/npf-type peptides, with the intron located between the second and third nucleotide of the codon for the arginine residue of the c-terminal rf or ry dipeptide (mair et al., ) . here we show this conserved feature in npy/npf genes in source data . accession numbers of the precursor sequences used for the peptide alignments in figure . species from several animal phyla, including a hemichordate (sister phylum to the echinoderms), chordates, molluscs, an annelid, a priapulid, an arthropod and a nematode (figure -figure supplement ). in echinoderm prrp-like peptide genes, the exon encoding the neuropeptide is likewise interrupted by an intron but it is located in a different position to the intron that interrupts the coding sequence for npy/npf-type peptides. thus, it does not interrupt the codon for the arginine of the c-terminal rf or ry motif, but instead it is located between the first and second nucleotide of the codon for a residue located in the n-terminal or central regions of echinoderm prrp-like peptides ( figure -figure supplement ) . another difference is that typically in npy/npf genes there is another intron that interrupts the coding sequence in the c-terminal region of the precursor protein, whereas in the echinoderm prrp-like peptide precursor genes the coding sequence for the c-terminal region of the precursor protein is not interrupted by an intron (figure -figure supplement ) . collectively, these findings provide further evidence that echinoderm prrp-like peptides are not orthologs of npy/npf-type neuropeptides. . the protein-coding exons are colour-coded to show regions that encode the n-terminal signal peptide (blue), the neuropeptide (red), monobasic or dibasic cleavage sites (green) and other regions of the precursor protein (grey). note that a common characteristic is that an intron interrupts the coding sequence in the n-terminal or central region of the neuropeptide, with the intron consistently located between the first and second nucleotides (phase one intron represented by + ) of the codon for the amino acid shown after intron. taxa are highlighted in phylum-specific colours: dark blue (echinodermata), light blue (hemichordata), purple (chordata). the full names of the species and the accession numbers of the sequences are listed in figure -source data . the online version of this article includes the following source data and figure supplement(s) for figure : source data . accession numbers of the sequences used for the gene structure analysis in figure and discovery of orthologs of snpf/prrp-type receptors in a. rubens and other echinoderms having obtained evidence that echinoderm npy/prrp-like peptides are not orthologs of npy/npftype neuropeptides but are orthologs of prrp-type peptides, we then investigated the occurrence in a. rubens and other echinoderms of proteins related to gpcrs that mediate effects of npy/npftype peptides, prrp-type peptides and snpf-type peptides in other bilaterians. using receptor sequences of h. sapiens npy-type, d. melanogaster npf-type, h. sapiens prrp-type and d. melanogaster snpf-type receptors as queries for similarity-based analysis of a. rubens neural figure . blosum cluster map of npy/npf/prpr/snpf-type receptors and closely related tachykinin-type receptors (tkr) and luqin/ryamide-type receptors (lq/ryar). nodes are labelled with phylum-specific colours, as shown in the key, and connections represent blast relationships with a p value > e- . note that the echinoderm receptors (boxed) have more connections with prrp/snpf-type receptors than with npy/npf-type receptors. the sequences of the receptors included in this figure are listed in figure -source data . the online version of this article includes the following source data and figure supplement(s) for figure : source data . accession numbers of the receptor sequences used for the clans analysis in figure . transcriptome sequence data, a transcript (contig ) encoding a -residue protein was identified as the best hit (figure -figure supplement ). furthermore, homologs of the a. rubens protein encoded by contig were also identified in other echinoderms for which genome sequences have been obtained, including the starfish a. planci, the sea urchin s. purpuratus and the sea cucumber a. japonicus, and importantly no other npy/npf/prrp/snpf-type receptors were identified in these species. to investigate relationships of the novel echinoderm receptors with other bilaterian neuropeptide receptors, we generated a sequence database including bilaterian npy/ npf/prrp/snpf-type receptors and other closely related receptors (tachykinin-type, luqin-type receptors) as outgroups. these receptor sequences were then analysed using two different methodologies. firstly, we performed a cluster-based analysis of the receptor sequences using clans ( figure ). this analysis revealed three main clusters: . a cluster comprising the outgroup receptors (tachykinin/luqin), . a cluster comprising npy/npf-type receptors and . a cluster comprising snpf-type receptors and prrp-type receptors. interestingly, the echinoderm receptors showed stronger connections with the snpf/prrp receptor cluster ( figure , black square) than with the npy/npf receptor cluster. these findings indicate that snpf-type receptors and prrp-type receptors are orthologous, as has been proposed previously based on cluster-based analysis of receptor sequences (jékely, ). furthermore, these findings indicate that npy/npf/prrp/snpf-type receptors in echinoderms are not orthologs of npy/npf-type receptors but are orthologs of snpf/prrp-type receptors. however, it is noteworthy that the lines linking the echinoderm receptors and nematode snpf-type receptors with other snpf/prrp-type receptors in clans are quite long (figure ) , which is indicative of sequence divergence. secondly, we performed a phylogenetic analysis of the receptor sequences using the maximum likelihood method. for this analysis, in addition to bilaterian npy/npf-type receptors, deuterostome prrp-type receptors and protostome snpf-type receptors, we included tachykinintype, luqin-type and gpr -type receptors as outgroups. this revealed that the echinoderm receptors are positioned within a branch of the phylogenetic tree that comprises npy/npf-type, prrp-type and snpf-type receptors, with the other receptor types included in the analysis occupying an outgroup position ( figure ). more specifically, the echinoderm receptors are positioned in a clade comprising snpf-type receptors, with bootstrap support of > %, indicating that the echinoderm receptors are orthologs of protostome snpf-type receptors. however, it is noteworthy that snpf-type receptors and prrp-type receptors do not form a monophyletic clade as would be expected for orthologous receptors. this may be a consequence of sequence divergence in the echinoderm and nematode snpf/prrp-type receptors that is reflected in the long branches leading to these receptors. because the phylogenetic analysis revealed that the echinoderm receptors are positioned in a clade comprising protostome snpf-type receptors (figure ), we also compared the sequences of echinoderm prrp-type peptides and protostome snpf-type peptides (figure -figure supplement ) and the structures of the genes encoding these neuropeptides (figure -figure supplement ). this revealed that sequence identity is restricted to a few residues in the c-terminal regions of the peptides and, furthermore, the echinoderm prrp-type peptides are much longer than protostome snpf-type peptides (figure -figure supplement ). this contrasts with the much higher levels of sequence similarity shared between echinoderm prrp-type neuropeptides and chordate prrp-type neuropeptides, as shown in figure a . another difference is that protostome snpf-type neuropeptide precursors typically give rise to multiple snpf-type peptides, whereas echinoderm prrp-type precursors are similar to chordate prrp-type precursors in containing a single prrp-type neuropeptide that is located adjacent to the signal peptide (figure -figure supplement ). accordingly, comparison of the exon/intron structure of the genes encoding prrp-type precursors in echinoderms and snpf-type precursors in protostomes also revealed limited similarity (figure -figure supplement ). collectively, our analysis of sequence data indicates that npy/npf/prrp/snpf-type receptors in echinoderms are not orthologs of npy/npf-type receptors but are orthologs of snpf/prrp-type receptors. therefore, henceforth we refer to these echinoderm receptors as snpf/prrp-type receptors and specifically refer to the snpf/prrp-type receptor in the starfish a. rubens as ar-snpf/ prrpr. furthermore, having identified ar-snpf/prrpr we proceeded to investigate if arprrp acts as a ligand for this receptor. . phylogenetic tree showing that a candidate receptor for the a. rubens neuropeptide arprrp is an ortholog of protostome snpf-type receptors. the tree includes npy/npf-type receptors, chordate prrp-type receptors and protostome snpf-type receptors, with gpr -type, luqin-type, and tachykinin-type receptors as outgroups to root the tree. interestingly, the candidate receptor for the a. rubens neuropeptide arprrp (red arrow) and orthologs from other echinoderms are positioned in a clade comprising protostome snpf-type receptors, whereas candidate receptors for prrptype peptides in the hemichordate s. kowalevskii are positioned in a clade containing chordate prrp-type receptors. note that npy/npf-type receptors form a distinct clade that includes an npy/npf-type receptor from the hemichordate s. kowalevskii, but no echinoderm receptors are present in this clade. the tree was generated in w-iq-tree . using the maximum likelihood method. the stars represent bootstrap support ( replicates, see legend) and the coloured backgrounds represent different taxonomic groups, as shown in the key. the names with text in blue represent the receptors for which ligands have been experimentally confirmed. the asterisks highlight receptors where the reported ligand is atypical when compared with ligands for receptors in the same clade. species names are as follows: aaeg (aedes aegypti), acal (aplysia californica), ajap (apostichopus japonicus), amis (alligator mississippiensis), apla (acanthaster planci), arub (asterias rubens), bbel (branchiostoma belcheri), bdor (bactrocera dorsalis), bflo a cdna encoding ar-snpf/prrpr was cloned and sequenced ( figure -figure supplement ) and its sequence has been deposited in genbank under accession number mh . . analysis of the sequence of ar-snpf/prrpr using protter revealed seven predicted transmembrane domains, as expected for a gpcr ( figure -figure supplement ) . the cloned receptor was then co-expressed with ga in cho-k cells expressing apoaequorin to produce the cell system cho-ar-snpf/ prrpr. synthetic arprrp (pqdrskamqaertgqlrrlnprf-nh ) was tested as a candidate ligand for ar-snpf/prrpr at concentrations ranging from À m to À m, comparing with cells incubated in assay media without the addition of the peptide. this revealed that arprrp at a concentration of À m triggers luminescence responses (defined as %) in cho-ar-snpf/prrpr cells that were approximately five times the background luminescence detected with the assay media used to dissolve the peptide ( figure a) , demonstrating that arprrp acts as a ligand for the receptor. furthermore, arprrp induced dose-dependent luminescence in cho-ar-snpf/prrpr cells with a halfmaximal response concentration (ec ) of . Â À m ( figure b ). importantly, no response to arprrp was observed in cho-k cells transfected with the vector alone, demonstrating that the signal observed in cho-ar-snpf/prrpr cells exposed to arprrp can be attributed to activation of the transfected receptor ( figure -figure supplement ) . because arprrp contains a potential dibasic cleavage site (see underlined arginine residues in its sequence: pqdrskamqaertgqlrrlnprf-nh ), we hypothesised that the c-terminal pentapeptide of arprrp (lnprfamide) may also be generated from arprrpp in vivo. therefore, we also tested synthetic lnprfamide as a candidate ligand for ar-snpf/prrpr. however, this peptide did not induce luminescence responses in cho-ar-snpf/ prrpr cells ( figure b) . therefore, we conclude that the -residue amidated peptide arprrp is the natural ligand for ar-snpf/prrpr in a. rubens. the a. rubens luqin-type neuropeptide arlq also did not induce luminescence responses in cho-ar-snpf/prrpr cells, demonstrating the selectivity of ar-snpf/prrpr for arprrp as a ligand ( figure b ). the discovery of an npy-like neuropeptide, named npf, in a platyhelminth provided the first definitive molecular evidence that npy-type neuropeptides originated in a common ancestor of the bilateria (maule et al., ) . subsequently, analysis of transcriptomic/genomic sequence data has enabled identification of npy/npf-type neuropeptides and their cognate receptors in a variety of invertebrate taxa, revealing a high level of conservation of this signalling system in bilaterian phyla (zatylny-gaudin and favrel, ; fadda et al., ) . here we report the first detailed analysis npy/npf-related signalling systems in echinoderms -invertebrate deuterostomes that have provided key insights into the evolution of other neuropeptide signalling systems (semmens et al., ; tian et al., ; elphick et al., ; yañez-guerra et al., ) . recently, we reported the discovery of echinoderm proteins comprising putative neuropeptides that share sequence similarity with npy/npf-type peptides (zandawala et al., ) . however, here our detailed analysis of the sequences of these peptides and the genes encoding them has revealed that they are not orthologs of the npy/npf-type neuropeptides. consistent with this finding, orthologs of npy/npf-type receptors were also not found in echinoderms. therefore, we conclude that npy/npf-type neuropeptide signalling has been lost in the phylum echinodermata ( figure ). this is a noteworthy because, to the best of our knowledge, the only other taxon in which loss of npy/npf-type signalling has been reported are the urochordates, a sub-phylum of the phylum chordata (mirabeau and joly, ; figure ). the evolutionary and functional significance of loss of npy/npf-type signalling in echinoderms and urochordates is rubens prrp/snpf-type receptor ar-snpf/prrpr, the promiscuous g-protein g a and the calcium-sensitive luminescent gfp-apoaequorin fusion protein g a. for comparison, the background luminescence of cells that were not exposed to arprrp is shown (basal media; grey bar). mean values (± s.e.m) were determined from three independent experiments performed in triplicate (b). graph showing the selectivity of arprrp as a ligand for ar-snpf/prrpr. arprrp causes dose-dependent luminescence in cho-k cells expressing ar-snpf/prrpr, with an ec of . nm. ar-snpf/prrpr is not activated by a c-terminal pentapeptide fragment of arprrp (lnprfamide) or by the a. rubens luqin-type peptide arlq. each point represents mean values (± s.e. figure continued on next page unknown. however, insights into this issue may emerge from functional characterisation of npy/ npf-type signalling in other invertebrates. the nematode c. elegans is a powerful model system for functional characterisation of neuropeptide signalling systems (frooninckx et al., ) . however, npy/npf-type signalling has thus far only been partially characterised in this species. here, our phylogenetic analysis (figure ) indicates that there are two c. elegans receptors that are orthologs of npy/npf-type receptors: npr- , which is an orphan receptor, and npr- , which has been shown to be activated by the peptide mdanafrmsfamide (chalasani et al., ) . however, this peptide shares little sequence similarity with npy/npf-type peptides from other bilaterians. furthermore, receptor assays only showed activation at peptide concentrations of and mm (chalasani et al., ) , which are high when compared to other npy/npf-type receptors that are typically activated by ligands in the nanomolar range (bard et al., ; lundell et al., ; garczynski et al., ; saberi et al., ) . recently, based on similarity-based sequence alignments, it has been suggested that the mature peptide derived from the c. elegans protein flp- may be an ortholog of npy/npf-type peptides (fadda et al., ) . here, our analysis of the structure of the gene encoding the flp- precursor has revealed that it has the characteristic structure of npy/npf-type genes, with an intron interrupting the codon for the c-terminal arginine of the npf-type peptide sequence (figure -figure supplement ). thus, based on our analysis of c. elegans sequence data, we conclude that the npy/ npf-type peptide derived from the flp- precursor protein is likely to act as a ligand for the npr- and/or npr- receptors. this finding provides a basis for functional characterisation of npy/ npf-type signalling in c. elegans. if the echinoderm npy-like peptides are not orthologs of npy/npf-type neuropeptides, then what are they? here we show that these peptides share sequence similarity with vertebrate prrp-type neuropeptides ( figure a) . furthermore, analysis of the structure of the genes encoding the echinoderm neuropeptides revealed that the coding sequence for the neuropeptides is interrupted by an intron in the phase one frame, a feature that is also a characteristic of genes encoding vertebrate prrp-type neuropeptides (figure ) . these findings indicate that the echinoderm neuropeptides are orthologs of vertebrate prrp-type neuropeptides. to further address this issue we analysed echinoderm genome/transcriptome sequence data to identify candidate cognate receptors for the echinoderm prrp-like peptides. a cluster-based analysis of receptor sequence data using clans revealed the presence in echinoderms of receptor proteins that show strong connections with a receptor cluster comprising vertebrate prrp-type receptors and protostome snpf-type receptors (figure ) . accordingly, a previous cluster-based analysis of receptor sequence data has reported that vertebrate prrp-type receptors cluster with protostome snpf-type receptors, indicating that these receptors may be orthologous (jékely, ). a novelty of our analysis is the inclusion of several echinoderm receptor sequences. it is noteworthy, however, that whilst strong connections between the echinoderm receptors and prrp/snpf-type receptors in other taxa can be seen using clans, the lines linking to the echinoderm receptors are quite long (figure ). this suggests that the echinoderm receptors are orthologs of prrp/snpf-type receptors but have undergone sequence divergence. interestingly, a group of snpf-type receptors in the nematode c. elegans appears to be similarly divergent with respect to other snpf/prrp-type receptors (figure ) . . phylogenetic diagram showing the occurrence of npy/npf-type, snpf-type and prrp-type neuropeptide signalling in the bilateria. the tree shows the phylogenetic relationships of selected bilaterian phyla. a gene duplication event giving rise to the paralogous npy/npf-type (green) and prrp/snpf (purple) signalling systems is shown at a position in the tree corresponding to the common ancestor of the bilateria. phyla in which npy/ npf-type peptides/precursors and npy/npf-type receptors have been identified are labelled with green-filled squares. phyla in which prrp-type peptides/precursors and prrp-type receptors have been identified are labelled with blue-filled squares. phyla in which snpf-type peptides/precursors and snpf-type receptors have been identified are labelled with red-filled squares. the inclusion of an asterisk in filled squares indicates that activation of a receptor by a peptide ligand has been demonstrated experimentally. note that in the starfish asterias rubens (this study) a prrp-type peptide (blue triangle) is the ligand for receptor that has been found to be an ortholog snpf/prrp-type receptors (figure ) or an ortholog of snpf-type receptors ( figure ) ; hence this receptor is represented here as a red triangle. note also the mutually exclusive patterns in the phylogenetic distribution of snpftype signalling and prrp-type signalling, with the former found in protostomes and the latter found in vertebrates, cephalochordates and hemichordates, which is supportive of the hypothesis that these signalling systems are orthologous. our discovery of a prrp/snpf-type signalling system in echinoderms provides a missing link in the evolution of this neuropeptide signalling system. npy/npf-type signalling occurs in most phyla, but it has been lost in echinoderms and urochordates. the inclusion of a question mark for the putative npy/npf-type peptide identified in the cephalochordate b. floridae (mirabeau and joly, ; elphick and mirabeau, ) signifies that it is atypical of npy/npf-type peptides, which may explain why npy/npf-type receptors have yet to be identified in cephalochordates. the inclusion of a question mark in the c. elegans green square figure continued on next page to further investigate the relationship of the echinoderm receptors with snpf/prrp-type receptors, we performed a phylogenetic analysis of sequence data using the maximum likelihood method (figure ) . in this analysis, the echinoderm receptors are positioned in a clade comprising protostome snpf-type receptors. however, snpf-type receptors and prrp-type receptors do not form a monophyletic clade in the tree. interestingly, this finding has been reported previously as part of a wider analysis of neuropeptide receptor relationships in the bilateria (mirabeau and joly, ) . thus, there is inconsistency in the findings from cluster-based analysis (clans) (jékely, ; figure ) and phylogenetic tree-based analysis (mirabeau and joly, ; figure ) of receptor relationships. one possible explanation for this inconsistency would be that gene duplication in a common ancestor of the bilateria gave rise to two snpf/prrp-type signalling systems, which were then differentially lost/retained in bilaterian lineages, but in such a scenario gene loss in several lineages would have to be invoked. alternatively, the inconsistency may, at least in part, be a consequence of sequence divergence in echinoderm and nematode snpf/prrp-type receptors with respect snpf/prrp-type receptors in other taxa, which is reflected in their position peripheral to the main cluster of snpf/prrp-type receptors in the clans. accordingly, it is noteworthy that in the phylogenetic tree (figure ) there is a long branch leading to the echinoderm receptor clade and likewise nematode snpf-type receptors also have long branches (figure ) . nevertheless, collectively our sequence analysis indicates that the echinoderm receptors are orthologs of snpf/prrptype receptors. therefore, it was of interest to determine if echinoderm prrp-type neuropeptides act as ligands for snpf/prrp-type receptors in this phylum. here we show that the a. rubens prrp-type neuropeptide arprrp (pqdrskamqaertg qlrrlnprf-nh ) is a potent ligand for the a. rubens snpf/prrp-type receptor ar-snpf/prrpr ( figure ). these findings demonstrate for the first time the existence and molecular identity of a prrp-type signalling system in an echinoderm. furthermore, our identification of orthologs of arprrp and ar-snpf/prrpr in other echinoderms, including for example the sea urchin s. purpuratus, demonstrates the conservation of this signalling system in this phylum. in addition, our comparative analysis of sequence data has also enabled identification of genes/transcripts encoding prrp-type neuropeptides in the hemichordate s. kowalevskii and the cephalochordate b. floridae (figure ). previous studies have concluded that snpf-type signalling is paralogous to npy/npf-type signalling in protostomes (nässel and wegener, ) and that prrp-type signalling is paralogous to npy/ npf-type signalling in vertebrates (lagerströ m et al., ) . evidence that the prrp-type and snpf-type signalling systems may be orthologous has also been reported previously (jékely, ), but this hypothesis has not been tested experimentally. our discovery of a starfish prrp-type neuropeptide that acts as a ligand for a starfish ortholog of snpf-type receptors is important because it provides a missing link for reconstruction of the evolutionary history of prrp/snpf-type neuropeptide signalling (figure ) . comparison of the sequences of vertebrate prrp-type neuropeptides and protostome snpf-type neuropeptides reveals low levels of sequence similarity, which no doubt in part explains why prrptype and snpf-type neuropeptides have not been recognised as orthologs. in figure -figure indicates that the peptide identified as a ligand for the c. elegans npy/npf-type receptor (chalasani et al., ) does not have the typical features of an npy/npf-type peptide. the grey square for snpf in m. expansa, for which only transcriptome sequence data are available, indicates that snpftype peptides and snpf-type receptor(s) are likely to be present in this species because snpf-type peptides and snpf-type receptors have been identified in another platyhelminth species, s. mediterranea, for which a genome sequence is available. species names are as follows: h. sapiens (homo sapiens), c. intestinalis (ciona intestinalis), b. floridae (branchiostoma floridae), s. kowalevskii (saccoglossus kowalevskii), a. rubens (asterias rubens), p. dumerilii (platynereis dumerilii), l. stagnalis (lymnaea stagnalis), m. expansa (moniezia expansa), s. mediterranea (schmidtea mediterranea), c. gigas (crassostrea gigas), d. melanogaster (drosophila melanogaster), c. elegans (caenorhabditis elegans). silhouettes of representative animals from each phylum are from www.openclipart.com and they are free from copyright. supplement we illustrate this in an alignment of the echinoderm prrp-type neuropeptides and protostome snpf-type neuropeptides, with sequence identity restricted to a few residues in the c-terminal regions of these peptides. this contrasts with the higher levels of sequence similarity shared between echinoderm prrp-type neuropeptides and vertebrate prrp-type neuropeptides, as shown in figure a . furthermore, echinoderm prrp-type precursors are similar to chordate prrptype precursors in containing a single long neuropeptide, whereas protostome snpf-type precursors typically contain multiple smaller neuropeptides. thus, there is little evidence of orthology from comparison of echinoderm prrp-type and protostome snpf-type neuropeptide, precursor and gene sequences. consequently, our conclusion that the echinoderm prrp-type peptides are orthologs of protostome snpf-type peptides is principally based on the orthology of their receptors (figure ) and our experimental demonstration that a prrp-like peptide (arprrp) acts as a ligand for a snpf/ prrp-type receptor (ar-snpf/prrpr) in the starfish a. rubens ( figure ) . it is important to note, however, that this is not unprecedented in investigations of the evolution of neuropeptide signalling. thus, whilst the sequences of some neuropeptides and neuropeptide precursors are highly conserved throughout the bilateria, others are so divergent that they can be unrecognisable as orthologs. an example of the former are vasopressin/oxytocin (vp/ot)-type neuropeptides and precursors. an example of the latter are neuropeptide-s (nps)/crustacean cardioactive peptide (ccap)-type neuropeptides and precursors, which are paralogs of vp/ot-type neuropeptides and precursors (semmens et al., ) . thus, by way of comparison, npy/npf-type neuropeptides are similar to vp/ot-type neuropeptides in exhibiting a high level of sequence conservation throughout the bilateria. conversely, prrp/snpf-type neuropeptides are similar to nps/ccap-type neuropeptides in being highly divergent, with neuropeptides in protostomes and deuterostomes exhibiting modest sequence similarity. the discovery of prrp/snpf-type signalling in echinoderms has provided a unique opportunity to speculate on the ancestral characteristics of this signalling system in urbilateria. it is noteworthy that, by comparison with the protostome snpf-type peptides, the echinoderm prrp-type peptides have more features in common with the paralogous npy/npf-type peptides. prrp-type peptides are not as long as npy/npf-type peptides but they are nevertheless much longer than protostome snpf-type peptides. furthermore, it was the sequence similarity that echinoderm prrp-type peptides share with npy/npf-type peptides that originally facilitated their discovery (zandawala et al., ) . additionally, the structure of the prrp-type precursors is similar to npy/npf-type precursors because the neuropeptide is located immediately after the signal peptide, whereas this is not a feature of protostome snpf-type precursors. based on these observations, we propose that prrp-type peptides and precursors may more closely resemble the ancestral characteristics of the prrp/snpf type signalling system in urbilateria. furthermore, we speculate that the common ancestor of the paralogous npy/npf-type and prrp/snpf-type neuropeptide precursors may have been similar to npy/npf-type precursors with respect peptide, precursor and gene structure. then, following gene duplication, these ancestral characteristics were retained in the paralog that gave rise to the bilaterian npy/npf-type peptides/precursors. in contrast, the paralog that gave rise to prrp/snpf-type signalling diverged from the ancestral condition. however, the extent of divergence varies in the deuterostome and protostome lineages. in deuterostomes, the prrp-type peptides/precursors have many npy/npf-type characteristics and we conclude that this reflects less divergence from the proposed ancestral condition. conversely, in the protostomes, the snpf-type peptides/precursors exhibit little similarity with npy/npf-type peptides/precursors and we conclude that this reflects more divergence from the proposed ancestral condition. in conclusion, our discovery of a prrp/snpf-type signalling system in echinoderms has provided a missing link that unites prrp-type peptides in vertebrates and snpf-type peptides in protostomes as members of a bilaterian family of neuropeptides, as illustrated in figure . this represents an important advance in our knowledge of neuropeptide signalling systems in the bilateria and illustrates the value of insights from echinoderms in enabling reconstruction of the evolutionary history of neuropeptides. animals starfish (asterias rubens) were obtained from a fisherman based at whitstable (kent, uk). they were then maintained in a circulating seawater aquarium at~ ˚c in the school of biological and chemical sciences at queen mary university of london and were fed on mussels (mytilus edulis) collected near margate (kent, uk). cloning and sequencing of a cdna encoding the precursor of an a. rubens npy/npf/prrp-like peptide a transcript encoding the a. rubens precursor of an npy/npf-like peptide was reported previously (genbank: mk ) (zandawala et al., ) . however, in this paper we show that the npy/npflike peptide derived from this precursor shares more sequence similarity with prrp-type peptides. a cdna containing the complete open reading frame of the precursor was amplified by pcr using a. rubens radial nerve cord cdna, the forward primer aagtcaaaaggcgagcaaga, the reverse primer aaagggatgtggtgttggtg and q polymerase (neb; cat. no. m s). the pcr products were ligated into the pbluescript ii ks (+) vector (invitrogen; cat. no. k ) that had been cut previously with the restriction enzyme ecorv by performing blunt-end ligation with t dna ligase (neb; cat. no. m s). the cloning was confirmed by restriction enzyme digestion and sequencing (tubeseq service; eurofins genomics). structural characterisation of the a. rubens npy/npf/prrp-like peptide using mass spectrometry after confirming the nucleotide sequence of the a. rubens precursor of a npy/npf/prrp-like peptide by cloning and sequencing, mass spectrometry was used to determine the mature structure of the peptide. the methods employed, including extraction of peptides from a. rubens radial nerve cords, treatment of samples, equilibration of columns, reverse phase chromatography for the initial separation and injection into a orbitrap-fusion (thermoscientific) for tandem mass spectrometry (ms/ms), were performed using a previously reported protocol for the identification of the starfish neuropeptides (lin et al., ) . the methods employed for data analysis are described below. mass spectra were searched using sequest proteome discoverer (thermo fisher scientific, v. . ) against a database comprising forty-three different precursor proteins identified by analysis of a. rubens neural transcriptome data, including the a. rubens arprrp precursor and all proteins in gen-bank from species belonging to the asteriidae family and the common repository of adventitious proteins database (http://www.thegpm.org/crap/index.html). theoretical peptides were generated allowing up to two missed cleavages and variable modifications, including amidation (À . ) of c-terminal glycines and pyroglutamate (À . ) of n-terminal glutamines, and oxidation of methionine (+ . ). precursor mass tolerance was ppm and fragment ions were searched at . da tolerances. results from discoverer were collated and annotated in scaffold version . . (proteome software). sequence alignment of echinoderm npy/npf/prrp-like peptides with npy/npf-type peptides, prrp-type peptides, and snpf-type peptides from other taxa the amino acid sequences of echinoderm npy/npf/prrp-like peptides were aligned with the sequences of npy/npf-type peptides, prrp-type peptides and snpf-type peptides from a variety of bilaterian species (see figure -source data and figure -figure supplement -source data for lists of the sequences). to identify candidate ligands for prrp-type receptors in the cephalochordate b. floridae and the hemichordate s. kowalevskii, we analysed transcriptomic and genomic sequence data for these species (putnam et al., ; simakov et al., ) . the data analysed also included a list of predicted s. kowalevskii proteins kindly provided to o. mirabeau by dr. r.m. freeman (harvard medical school, usa). the methods employed to identify candidate neuropeptide precursors have been reported previously (mirabeau and joly, ) but here we had the more specific objective of identifying proteins with an n-terminal signal peptide followed by a neuropeptide with a predicted c-terminal rfamide or ryamide motif. this resulted in discovery of one candidate prrp-type precursor in the cephalochordate b. floridae and two candidate prrp-type precursors in the hemichordate s. kowalevskii. alignments were performed using mafft version ( iterations, substitution matrix; blosum ) and then manually curated. highlighting of the conserved residues was done using boxshade (www.ch.embnet.org/software/box_form.html) with % conservation as the minimum for highlighting. finally, the sequences were highlighted in phylum-specific or superphylum-specific colours: dark blue (echinodermata), light blue (hemichordata), purple (chordata), orange (platyhelminthes), red (lophotrochozoa), yellow (priapulida), green (arthropoda), grey (nematoda). comparison of the exon/intron structure of genes encoding npy/npf/ prrp-like peptides in echinoderms and genes encoding npy/npf-type peptides, prrp-type peptides and snpf-type peptides in other taxa the sequences of transcripts and genes encoding precursors of echinoderm precursors of npy/npf/ prrp-like peptides and precursors of npy/npf-type, prrp-type and snpf-type peptides from other taxa were obtained from genbank. the sequence of a predicted transcript encoding a second s. kowalevskii precursor (skow ) of a prrp-like peptide was determined based on a genscan prediction (burge and karlin, ; burge and karlin, ) from scaffold (genbank accession number nw_ . ). see figure -source data and figure -figure supplement source data for a list of the transcript and gene sequences analysed. the online tool splign (kapustin et al., ) (https://www.ncbi.nlm.nih.gov/sutils/splign/splign.cgi) was employed to determine the exon/intron structure of genes and schematic figures showing gene structure were generated using ibs . (liu et al., ) . identification of a candidate receptor for the npy/npf/prrp-like peptide in a. rubens and analysis of its relationship with npy/npf/ prrp/snpf-type receptors in other taxa to identify a candidate receptor for the a. rubens npy/npf/prrp-like peptide, a. rubens neural transcriptome sequence data were analysed using the blast server sequenceserver (priyam et al., ) , submitting npy-type receptors from h. sapiens (genbank np_ . , np_ . , np_ . ), an npf-type receptor from d. melanogaster (genbank aaf . ), a prrp-type receptor from h. sapiens (np_ . ) and snpf-type receptors from d. melanogaster (genbank; np_ . ) and c.gigas (genbank xp_ . ) as query sequences. a transcript (contig ) encoding a -residue protein (http://web.expasy.org/translate/) was identified as the top hit in all blast searches and this has been deposited in genbank under the accession number mh . the protein sequence was also analysed using protter v . (omasits et al., ) . using blast, homologs of the a. rubens protein were identified in other echinoderms for which genome sequences are available, including the starfish acanthaster planci (xp_ . ), the sea urchin strongylocentrotus purpuratus (xp_ . ) and the sea cucumber apostichopus japonicus (pik . ). furthermore, no other npy/npf/prrp/snpf-type receptors were identified in these species. to investigate the relationship of the echinoderm receptors with neuropeptide receptors from other bilaterians, a database of receptor sequences was generated that included npy/npf-type, prrp-type, snpf-type, tachykinin-type, luqin-type and gpcr -type receptors (the latter three receptor types being included as outgroups), including representative species from the phyla chordata, hemichordata, echinodermata, mollusca, annelida, platyhelminthes, nematoda, priapulida, and arthropoda (see figure -source data for a list of the sequences used). a cluster-based analysis of the receptor sequences was performed using clans (frickey and lupas, ). an allagainst-all blast was performed using the scoring matrix blosum and linkage clustering was performed with an e-value of e- to identify coherent clusters. the clustering was first performed in d and then the map was collapsed to d to enable generation of the diagram shown in figure (see figure -source data for a list of sequences used). using the same receptor sequences, a phylogenetic tree was generated using the maximum-likelihood method. receptor sequences were aligned using muscle in the online tool ngphylogeny (iterative, iterations, upgmb as clustering method) (edgar, ; lemoine et al., ) and the alignment was automatically trimmed using trimal with automatic selection of trimming method using the online tool ngphylogeny (capella-gutierrez et al., ) . the trimming contained a total of residues that were used to generate the maximum-likelihood tree using w-iq-tree online version . (the model was automatically selected, being lg+g+i+f the chosen substitution model, branch tests used were ultrafastbootstrap replicates and sh-alrt replicates) (trifinopoulos et al., ) . the sequence database used for this tree, together with the trimmed alignment, and the raw tree are available at zenodo (https://zenodo.org/record/ ). to enable the pharmacological characterisation of a candidate receptor for the a. rubens npy/npf/ prrp-like peptide, a cdna encoding this receptor was cloned into the eukaryotic expression vector pcdna . (+) (invitrogen; . to facilitate expression of the cloned receptor, the forward primer included a partial kozak consensus sequence (acc) and a sequence corresponding to the first bases of the open reading frame of contig (accatgcagatgacaacc) and the reverse primer consisted of a stop codon and a sequence reverse complementary to the ' region of the open reading frame of contig (gcgtcacatagtggtatcatg). pcr was performed using the forward primer and reverse primers, a. rubens radial nerve cord cdna and q polymerase (neb; cat. no. m s). pcr products were ligated into the pcdna . (+) vector that had been cut previously with the restriction enzyme ecorv by performing blunt-end ligation with t dna ligase (neb; cat. no. m s). successful ligation and the direction of the insert was determined by restriction enzyme digestion and sequencing (tubeseq service; eurofins genomics). cell lines and pharmacological characterisation of a candidate receptor for the npy/npf/prrp-like peptide in a. rubens chinese hamster ovary (cho)-k cells stably expressing the calcium sensitive apoaequorin-gfp fusion protein (g a) (baubet et al., ) were used here for receptor assays. these cells have been used previously for neuropeptide receptor deorphanisation (bauknecht and jékely, ) and were generously supplied to us by dr gá spá r jé kely (university of exeter). the cell line was generated using the cho-k cell line from sigma-aldrich ( ), which is certified by the european collection of authenticated cell cultures (ecacc). following transfection with a plasmid encoding g a, cells were selected for stable transfection using geneticin g sulfate (thermo fisher scientific, cat. no. ) . the methods we used for cell culture and receptor assays have been described previously (yañez-guerra et al., ) . upon reaching a confluency of approximately %, cells were transfected with a plasmid containing the ar-snpf/prrp receptor cdna and a plasmid containing the promiscuous gaÀ protein that can couple a wide range of gpcrs to the phospholipase c signalling pathway. the transfection was achieved using mg of each plasmid and ml of the transfection reagents p and lipofectamine (thermo fisher scientific; cat. no. l ), as recommended by the manufacturer. it was not possible to authenticate the cho-k (g a) cells or test the cells for mycoplasma contamination at the time of manuscript submission due to laboratory closure during the covid- pandemic. after transfection with the a. rubens receptor, cells were exposed to the a. rubens npy/npf/ prrp-like peptide pqdrskamqaertgqlrrlnprf-nh (custom synthesised by peptide protein research ltd., fareham, uk), which was diluted in dmem/f nutrient mixture medium at concentrations ranging from À m to À m in clear bottom -well plates (sigma-aldrich; cat. no. cls - ea). luminescence was measured over a s period using a fluostar omega plate reader (bmg labtech; fluostar omega series multi-mode microplate reader) and data were integrated over the s measurement period. for each concentration, measurements were performed in triplicate, and the average of each was used to normalise the responses. the responses were normalised to the maximum luminescence measured in each experiment ( % activation) and to the background luminescence with the vehicle media ( % activation). dose-response curves were fitted with a four-parameter curve and ec values were calculated from dose-response curves based on at least three independent transfections using prism (graphpad, la jolla, usa). editing; ismail moghul, software, formal analysis, writing 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(bb/ m / ) and amj (bb/m / ). layg was supported by a phd studentship awarded by the mexican council of science and technology (conacyt studentship no. ) and queen mary university of london and by a leverhulme trust grant (rpg- - ) awarded to mre. xz was supported by a phd studentship awarded by the china scholarship council and queen mary university of london. we are grateful to gá spá r jé kely (university of exeter) for providing the cho-k (g a) cell line used here for receptor deorphanisation assays. the funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. luis alfonso yañ ez-guerra, xingxing zhong, conceptualization, data curation, formal analysis, validation, investigation, visualization, methodology, writing -original draft, writing -review and key: cord- -wmbgpnr authors: katsarou, maria; grassi, viviana; lomazzi, chiara; domanin, maurizio; trimarchi, santi title: acute retrograde type a intramural hematoma during sars-cov- time date: - - journal: j vasc surg doi: . /j.jvs. . . sha: doc_id: cord_uid: wmbgpnr nan mostly affecting the descending aorta. type a imhs involve, type b imhs do not involve the ascending aorta. retrograde type a imh (retro-taimh) origins in the descending aorta and extend into the arch or ascending aorta. taimhs with distal ad carry an in-hospital mortality risk of - %. - we report the case of an -year-old woman with acute retro-taimh and distal ad. the patient's consent for publication was obtained. she was admitted to the emergency room with acute onset dyspnea, chest pain but no evidence of malperfusion. emergency computed tomography angiography (cta) identified a retro-taimh with ad with proximal entry tear above the celiac axis (a/cover). the patient was hemodynamically stable. she was treated with hypotensive and analgesic therapy and hospitalized for intensive monitoring. follow-up cta was performed at hours (b) and days (c) showing progressive to complete thrombosis of the entry tear, with reduction in aortic diameter which is the most important predictor of imh regression and positive outcome. complete symptom regression occurred. the event was observed during the sars- cov- pandemic peak in lombardy and the patient was found to be positive to the virus five days after symptom onset, with progressive dyspnea and worsening findings on chest x rays (d). she died due to pulmonary complications at days. hybrid treatment with ascending aortic replacement and distal thoracic aortic endovascular repair (tevar), or with frozen elephant trunk is the most appropriate treatment for acute retro-taimh. tevar is a valid alternative only in patients with prohibitive surgical risk, although landing zones may be unsuitable and the risk of neurological and cardiac complications may be high. medical treatment appears to be appropriate in asymptomatic patients, in those with non-complicated retro-taimh and in patients with high open surgical / tevar risks. considering both the absence of end-organ malperfusion and the advanced age of the patient, we chose medical treatment, that allows to reduce mortality by - %. this choice was proven effective with symptom recovery and clinical stability, until the deadly overlap of the sars- cov- . j o u r n a l p r e -p r o o f the differences and similarities between intramural hematoma of the descending aorta and acute type b dissection prognostic value of clinical and morphologic findings in short- term evolution of aortic intramural haematoma. therapeutic implications management of retrograde type a imh with acute arch tear/type b dissection diagnosis and management of patients with aortic dissection key: cord- -bs p ig authors: luk, andrea o. y.; ke, calvin; lau, eric s. h.; wu, hongjiang; goggins, william; ma, ronald c. w.; chow, elaine; kong, alice p. s.; so, wing-yee; chan, juliana c. n. title: secular trends in incidence of type and type diabetes in hong kong: a retrospective cohort study date: - - journal: plos med doi: . /journal.pmed. sha: doc_id: cord_uid: bs p ig background: there is very limited data on the time trend of diabetes incidence in asia. using population-level data, we report the secular trend of the incidence of type and type diabetes in hong kong between and . methods and findings: the hong kong diabetes surveillance database hosts clinical information on people with diabetes receiving care under the hong kong hospital authority, a statutory body that governs all public hospitals and clinics. sex-specific incidence rates were standardised to the age structure of the world health organization population. joinpoint regression analysis was used to describe incidence trends. a total of , cases of incident diabetes (type diabetes [n = , ]: mean age at diagnosis is . years, . % men; type diabetes [n = , ]: mean age at diagnosis is . years, . % men) were included. among people aged < years, incidence of both type and type diabetes increased. for type diabetes, the incidence increased from . ( % ci . – . ) to . ( % ci . – . ) per , person-years (average annual percentage change [aapc] . % [ % ci . – . ], p < . ) in boys and from . ( % ci . – . ) to . ( % ci . – . ) per , person-years (aapc . % [ % ci . – . ], p < . ] in girls; for type diabetes, the incidence increased from . ( % ci . – . ) to . ( % ci . – . ) per , person-years (aapc . % [ % ci . – . ], p < . ) in boys and from . ( % ci . – . ) to . ( % ci . – . ) per , person-years (aapc . % [ % ci . – . ], p < . ) in girls. in people aged to < years, incidence of type diabetes remained stable, but incidence of type diabetes increased over time from . ( % ci . – . ) to . ( % ci . – . ) per , person-years (aapc . % [ % ci . – . ], p < . ) in men and from . ( % ci . – . ) to . ( % ci . – . ) per , person-years (aapc . % [ % ci . – . ], p < . ) in women. in people aged to < years, incidence of type diabetes increased until / and then flattened. in people aged ≥ years, incidence was stable in men and declined in women after . no trend was identified in the incidence of type diabetes in people aged ≥ years. the present study is limited by its reliance on electronic medical records for identification of people with diabetes, which may result in incomplete capture of diabetes cases. the differentiation of type and type diabetes was based on an algorithm subject to potential misclassification. conclusions: there was an increase in incidence of type diabetes in people aged < years and stabilisation in people aged ≥ years. incidence of type diabetes continued to climb in people aged < years but remained constant in other age groups. the hong kong diabetes surveillance database hosts clinical information on people with diabetes receiving care under the hong kong hospital authority, a statutory body that governs all public hospitals and clinics. sex-specific incidence rates were standardised to the age structure of the world health organization population. joinpoint regression analysis was used to describe incidence trends. a total of , cases of incident diabetes (type diabetes [n = , ]: mean age at diagnosis is . years, . % men; type diabetes [n = , ]: mean age at diagnosis is . years, . % men) were included. among people aged < years, incidence of both type and type diabetes increased. for type diabetes, the incidence increased from . ( % ci . - . ) to . ( % ci . - . ) per , person-years (average annual percentage change [aapc] . % [ % ci . - . ], p < . ) in boys and from . ( % ci . - . ) to . ( % ci . - . ) per , person-years (aapc . % [ % ci . - . ], p < . ] in girls; for type diabetes, the incidence increased from . ( % ci . - . ) to . ( % ci . - . ) per , person-years (aapc . % [ % ci . - . ], p < . ) in boys and from . ( % ci . - . ) to , p < . ) in women. in people aged to < years, incidence of type diabetes increased until / and then flattened. in people aged � years, incidence was stable in men and declined in women after . no trend was identified in the incidence of type diabetes in people aged � years. the present study is limited by its reliance on electronic medical records for identification of people with diabetes, which may result in incomplete capture of diabetes cases. the differentiation of type and type diabetes was based on an algorithm subject to potential misclassification. there was an increase in incidence of type diabetes in people aged < years and stabilisation in people aged � years. incidence of type diabetes continued to climb in people aged < years but remained constant in other age groups. why was this study done? • diabetes affects over million people worldwide, and over half of the diabetes population comes from asian countries. • most studies on the burden of diabetes in asia reported the diabetes prevalence, i.e., the proportion of the population with disease, but few have considered diabetes incidence, i.e., the rate at which new cases have developed in the population. • knowledge of disease incidence informs how population exposure to risk factors has changed over time and is useful for projection of future prevalence. what did the researchers do and find? • we identified , people with new-onset type or type diabetes occurring between and in the electronic medical record system of the hong kong hospital authority. • we calculated the incidence rates of diabetes according to age categories and gender and analysed incidence trends over time. • we found that the incidence of both type and type diabetes increased in children and adolescents (aged < years). • incidence of type diabetes also increased in people aged to < years but was stable in people aged � years. type diabetes mellitus is a complex progressive disease of rapidly growing prevalence. the global prevalence of type diabetes is currently estimated at million and is projected to escalate to million over the next years [ ] . more than half of the world's population of those with type diabetes comes from asia, where rapid industrialisation and urbanisation have contributed to an obesogenic living environment and increasing rates of overweight and obesity [ ] . the majority of published population-based studies on the epidemiology of type and type diabetes in asia, including china, reported prevalence data, but few examined the incidence of diabetes [ , ] . although the prevalence informs the disease burden, the incidence reflects how population exposure to risk factors has changed over time, and these estimates are necessary for the accurate projection of future prevalence. importantly, disease incidence communicates to the health policy makers whether strategies to prevent diabetes have been effective. recently, quan and colleagues reported the incidence and prevalence of diabetes in hong kong and detected a decline in the incidence of diabetes over a -year period between and [ ] . however, diabetes types were not differentiated, and trends among the paediatric population were not explored. we accessed the territory-wide database hosted by the hong kong hospital authority (ha) and described the secular trends in incidence of type and type diabetes from to . hong kong is a special administrative region of people's republic of china and has a population of . million. the hong kong ha is a statutory body formed in that governs all public hospitals and government outpatient clinics in hong kong. because of the wide cost differential between public and private healthcare sectors, around % of the local residents receive care for chronic illnesses in the ha [ ] . a territory-wide electronic medical record system, adopted in , captures demographic information, diagnostic and procedure codes, laboratory results, and drug prescriptions of people attending public hospitals and clinics. the hong kong diabetes surveillance database (hkdsd) is a population-based cohort of people with diabetes in hong kong identified from the ha electronic medical record system between january and december . the analysis was not prespecified and was planned after obtaining and reviewing the content of the database. diabetes was ascertained based on one or more of the following qualifying criteria: ( ) glycated haemoglobin (hba c) � . % ( mmol/mol) in any one available hba c measurement [ ] ; ( ) fasting plasma glucose (fpg) � . mmol/l in any one available fpg measurement [ ] ; ( ) prescription of noninsulin antihyperglycaemic drugs and/or ( ) prescription of insulin for at least days continuously, with or without ( ) recording of the diagnostic code of diabetes based on the international classification of diseases, ninth revision (icd- ) code ; and/or ( ) recording of the diagnostic code of diabetes according to the revised edition of the international classification of primary care, world organization of national colleges, academics, and academic associations of general practitioners/family physicians code t or code t . to minimise misclassification of normal individuals as having diabetes, people who received diagnostic coding of diabetes but did not fulfil any of the laboratory or drug criteria throughout the observation are not considered. to avoid inclusion of women with gestational diabetes, we removed episodes occurring within months prior to or months after delivery (icd- codes - ) or occurring within months before or after any pregnancy-related encounter (icd- codes - ). however, women with subsequent episodes that met the criteria for diabetes occurring outside the context of any obstetric events would be included. the separation of diabetes types is not reliable based on coding alone in administrative databases. in the hkdsd, a small subset of people received icd- coding for both type and type diabetes. for the purpose of this analysis, we developed and validated an algorithm to delineate type from type diabetes using another database, the hong kong diabetes register (hkdr). in brief, the hkdr contains clinical information on people with physician-diagnosed diabetes who were referred to two public hospitals (prince of wales hospital and alice ho miu ling nethersole hospital) for assessment of diabetes complications. in the hkdr, diabetes subtype was determined by the referring physician and was independently confirmed with chart review by one of the investigators (ck) of the present study. type diabetes was defined as clinical presentation with diabetic ketoacidosis and/or continuous requirement of insulin within year of diagnosis. positivity for islet autoantibodies was not used to define autoimmune diabetes because antibodies were not routinely measured. of , patients in the hkdr, we excluded people with onset dates outside of january to december (n = , ) and those with missing data on diabetes subtype (n = ). of the remaining , people with newly diagnosed diabetes, type diabetes was confirmed in patients. the cohort was separated in a : ratio for derivation and validation of the algorithm, respectively. we considered icd- codes (type diabetes: icd- .x or .x , type diabetes: icd- .x or .x ) and types of insulin treatment in the algorithms, and we tested combinations for sensitivity, specificity, positive predictive value (ppv) and negative predictive value (npv) for identifying type diabetes. of these combinations, ratios of type to type diabetes codes � or prescription of a combination of short-and long-acting insulin and no noninsulin antihyperglycaemic treatment within the first year of diagnosis yielded the optimal sensitivity of . % ( % ci . - . ), specificity of . % ( % ci . - . ), ppv of . % ( % ci . - . ), and npv of . % ( % ci . - . ) in the hkdr for people aged < years in the validation cohort. the sensitivity and ppv of this algorithm for identifying type diabetes decreased to . %- . % and . %- . %, respectively, in people aged � years, which is expected because of the rarity of type diabetes at older ages. an incident case of diabetes was identified by the first occurrence of any episode fulfilling the definition of diabetes and at least years of diabetes-free observation prior. at least year of surveillance from the date of diagnosis was required to enable discrimination of type and type diabetes. thus, although the database includes cases of diabetes from january to december , incidence of diabetes was described from january to december . the date of diagnosis was the date to first fulfil the qualifying event. we estimated sexspecific annual incidence rates of type and type diabetes. the numerator was the number of incident cases of diabetes in the hkdsd in each calendar year, and the denominator was the estimated hong kong population of the previous year at midyear as reported by the local census and statistics department. calculated rates were expressed per , person-years. we reported age-standardised rates (using the age structure of the world health organization standard population) for the entire population and for subgroups by sex and age categories (< years, to < years, to < years, � years). we calculated the annual percentage change (apc) and the average apc (aapc) in incidence rates with % ci by sex and by age bands. joinpoint regression analysis was used to describe incidence trends over time. the optimal number of line segments that best fit the pattern was identified, and apcs were computed for the slope before and after each joinpoint using regression analysis. because of an observed irregularity in the number of new cases of type diabetes in , trend analysis including reporting of apc and aapc for both type and type diabetes (for consistency) was restricted to the period between january and december . in a sensitivity analysis, we conducted trend analysis for type diabetes using restricted cubic spline, which allowed for a more flexible fitting of the change in incidence rates, and results are presented in the supporting information. statistical significance was set at p-value of < . . analysis was performed using sas version . (cary, nc) and joinpoint regression program version . . . (national cancer institute, bethesda, md). of , people captured in the hkdsd, we excluded , people who had diabetes codes but did not fulfil other criteria of diabetes. we further excluded , cases of prevalent diabetes (episodes fulfilling diabetes between january and december ) and , cases of diabetes that occurred between january and december . the remaining , people with incident type or type diabetes occurring between and were included in the analysis (s fig, s table) . demographic and clinical characteristics of the cohort at diagnosis are detailed in s table. trends in incidence of type diabetes using the derived algorithm, , people with newly diagnosed diabetes were classified as having type diabetes, among whom ( . %) were aged < years and ( . %) and ( . %) were aged to < years and � years, respectively (s table) . age-standardised incidence of type diabetes increased from to in people aged < years in both sexes, from . ( % ci . - . ) to . ( % ci . - . ) per , person-years (aapc . % [ % ci . - . ], p < . ) in boys and from . ( % ci . - . ) to . ( % ci . - . ) per , person-years (aapc . % [ % ci . - . ], p < . ) in girls (table ) . broken down into narrower age bands, the incidence of type diabetes peaked in the age groups of - years in the female sex and - years in the male sex and declined with increasing age. among those aged to < years and � years, incidence of type diabetes remained stable over time (table ). sex differences in the risk of type diabetes were detected in the people aged < years but not in people aged � years. the rate ratio for type diabetes was . ( % ci . - . , p < . ) in girls compared with boys. among , incident cases of type diabetes, , ( . %) presented below years of age, , ( . %) presented in people aged to < years, and , presented ( . %) in people aged � years (s table) . the incidence rose with age and peaked in the age groups of - years in both men and women (fig ) . table, the majority ( . %) of the new episodes of type diabetes in was identified by first-time use of noninsulin antihyperglycaemic drugs alone, suggesting that these occurrences could be prevalent cases initially diagnosed and followed outside of ha and later presenting to ha for continuation of existing treatment. incident cases captured in or before were excluded from subsequent trend analysis. an increase in the incidence of type diabetes was observed in the < years and to < years age groups (table , fig a and b) . for people < years old, the age-standardised incidence of type diabetes increased from . ( % ci . - . ) to . ( % ci . - . ) per , person-years ( ) in men and women, respectively, for the entire period (p < . ). the sex disparity in incidence of type diabetes varied by age. for people < years old, the incidence was disproportionately higher in girls than in boys, with an overall rate ratio of . ( % ci . - . , p < . ). upon reaching young adulthood, the risk reversed, and men had higher incidence rates than women. in people aged to < years, we detected an initial increase in incidence followed by stabilisation in both sexes (table , fig c) . in men, the incidence of type diabetes increased from . ( % ci . using the territory-wide hkdsd, we conducted time trend analyses on the incidence of type and type diabetes in people in hong kong. between and , the incidence of type diabetes increased in people aged < years and to < years, increased and then stabilised in people aged to < years, and was unchanged in those aged � years. incidence of type diabetes continued to climb in people aged < years but remained constant in other age groups. although the incidence of type diabetes was the highest in those < years old, adult-onset type diabetes accounted for over two-thirds of newly diagnosed cases during the surveillance period. among those < years old, type diabetes contributed to more than half of the newly presented cases. the key strengths of this study were the use of population-level data through access to the electronic medical record system of the hong kong ha and its low susceptibility to selection bias. given the scarcity of population-level data on secular changes in diabetes incidence in asia, our results are timely in providing insights into the contemporary diabetes epidemic in this region. importantly, this study highlights the emerging problem of young-onset diabetes and calls for effective strategies to reduce modifiable risk factors for diabetes in this group. the multinational european diabetes (eurodiab) register, which included , incident cases of type diabetes in youth aged < years recorded between and , revealed regional differences in the trend of diabetes incidence [ ] . in areas with a high burden of type diabetes, such as the nordic countries, the united states, and australia, the incidence appeared to have stabilised over the past decade [ , , ] . conversely, the incidence of type diabetes has continued to rise in places of low disease prevalence, including east asia [ , ] . based on a registry of newly diagnosed cases of type diabetes in children aged < years in shanghai, zhao and colleagues observed an increase in incidence from . per , person-years in - to . per , person-years in - [ ] . from the - national health insurance service database containing physician-reported cases of type diabetes in children aged < years in south korea, kim and colleagues reported an incidence of . per , person-years, which was . -fold higher compared with the rate recorded in the earlier period of - [ ] . in the present study, we also detected an increase in the incidence of type diabetes in children and adolescents, and the increase occurred in both sexes. in hong kong, the incidence of type diabetes has not been determined since the last report decades ago. based on retrospective retrieval of paediatric cases of newly diagnosed diabetes between and , huen and colleagues recorded an incidence of . per , person-years for type diabetes in children aged < years in hong kong, which was considerably lower than our updated estimates of . - . per , person-years in a comparable age group [ ] . our observations, together with others' observations showing a similar rise in incidence, remain unexplained, although environmental factors, including obesity, nutrition, climate, and infection, have been implicated in the induction and acceleration of beta cell destruction [ ] . from surveys of hong kong school children, the prevalence of overweight or obesity rose from . % in the mid- s to . % in [ ] , and the prevalence was . % in primary school students and . % in secondary school students in the last estimation in [ ] . it is noteworthy that although the incidence was the highest in the paediatric population, adults aged > years accounted for two-thirds of the newly diagnosed cases in our database. in contrast to the rising trend of childhood-onset type diabetes, the incidence of adult-onset type diabetes has not increased. the growing number of young people acquiring type diabetes is a major health concern that is now seen globally. in the present study, % of incident cases of diabetes in people aged < years were type diabetes. the search for diabetes in youth registry, which systematically identified and followed youth with diabetes in the us, reported an annual increase of . % in the incidence of type diabetes in people aged < years between and , from . to . per , person-years in boys and from . to . per , person-years in girls [ ] . furthermore, the increase was larger in asians and pacific islanders (annual increase . %) than europeans (annual increase . %). similarly, wu and colleagues reported an increase in incidence from . to . per , person-years using a registry of newly diagnosed cases of young-onset diabetes presented between and in zhejiang, china [ ] . among those aged < years in hong kong, the average annual increase in incidence was . %- . %, and the last recorded crude incidence rates in were . and . per , person-years in boys and girl, respectively, which were lower than the us figures but higher than rates in china. similarly, we detected an increase in incidence of type diabetes in people aged to < years in both sexes. besides exposure to an increasingly obesogenic environment, intrauterine effects from gestational diabetes and/or maternal obesity and exposure to present-day endocrine disruptors may be responsible [ ] [ ] [ ] . it is also possible that the observed incidence trend of young-onset type diabetes was the result of people seeking medical attention earlier in their disease trajectory rather than an actual fall in the age of diabetes development. in this regard, people who would develop diabetes were diagnosed progressively earlier, thus removing people from the undiagnosed pool who would have otherwise presented at an older age. in people aged to < years, the incidence of type diabetes in both sexes initially increased and then flattened from / onward. in women aged � years, the incidence was stable until and declined thereafter, whereas in men aged � years, the incidence remained constant. the absence of a rise might reflect levelling off in exposure to risk factors. in hong kong, the prevalence of obesity has been stable in men and has declined in women since the mid- s [ , ] , which is attributable to a series of government-led health promotion initiatives targeting healthier lifestyles, such as the eatsmart programme and mandatory nutrition labelling during this period [ ] . women are generally more receptive to health information and ready to adopt a healthy lifestyle compared with men, which might in part account for the sex difference in trends of obesity and type diabetes [ ] . cigarette smoking is linked to the development of type diabetes [ ] . antismoking advertising campaigns and government policy to ban smoking in many public areas have resulted in a reduction in smoking rates over the past years, from . % in to . % in in hong kong [ ] . lastly, saturation effect could be an explanatory factor, wherein increased health awareness and improved screening efforts during earlier periods have captured most of the high-risk individuals, thus depleting the pool of undiagnosed type diabetes over time. in support of this, we observed a significant decline in hba c values at diagnosis of type diabetes from to , possibly reflecting proactive case finding and earlier detection. although screening programmes for diabetes have not been formally introduced in hong kong, governmental policies to enhance primary care, including the dissemination of a reference framework for diabetes care, could have an effect toward improving diabetes detection [ , ] . the increasing number of outpatient and inpatient attendances to public healthcare facilities over time also supports an increasing opportunity for diagnosing diabetes [ ] . limited studies indicated interethnic variation in the risks for type diabetes. alangh and colleagues reported -year secular trends in diabetes incidence in ontario, canada, and found that incidence increased moderately by % in the european population as compared with a -fold increase in the chinese population, in which the absolute incidence rate was twice that of europeans ( . versus . per , person-years) by [ ] . from contemporary studies, the ageand sex-adjusted diabetes incidence rates among adults were per , person-years in the united kingdom [ ] , per , person-years in sweden [ ] , and per , person-years in the us during the - period [ ] . over a comparable reporting period, the rates were , and per , person-years in men and women in our study, per , person-years in korea [ ] , and per , person-years in taiwan [ ] . differences in methods used to capture incident cases and diagnostic intensities would have influenced the estimates. allowing for these factors, available data, including those from the present study, suggest that the incidence of type diabetes may be higher in east asians than europeans. although chinese people are leaner than europeans, for the same bmi, the former have more visceral fat, greater insulin resistance, and more metabolic complications [ ] . moreover, low body weight is linked to poorer pancreatic beta cell reserve, and chinese people are more vulnerable to external factors, such as glucolipotoxicity, that trigger progressive decline in beta cell function [ ] . a sharp peak in the incidence of type diabetes was recorded in , and this could be connected to the severe acute respiratory syndrome epidemic in in hong kong [ ] . the excess of , new cases of type diabetes in compared with might be due to heightened health vigilance at the aftermath of the epidemic, which prompted increased doctor visits. the modest dip in the number of new cases in the ensuing years (from to ) compared with numbers between and could support earlier identification of cases during the transitory period, leaving fewer cases to be detected in subsequent years. the excess might also represent an injection of prevalent cases from the private sector to the public system in response to economic adversity. we acknowledge the following limitations in the study. an electronic medical record database was used to identify patients with diabetes, and we cannot exclude the possibility of incomplete capture. the data source included patients who attended only public healthcare facilities, and those receiving care in the private sector-who represent about % of the entire disease population-were not included. as such, the incidence rates as reported were likely underestimations of the actual figure by up to %. however, except for , there was no evidence that the ratio of medical care in private sector versus public sector has changed over time, and therefore, this is unlikely to affect assessment of incidence trends. two-hour plasma glucose by an oral glucose tolerance test (ogtt) was not included as one of the qualifying criteria for diabetes, which could also underestimate incidence rates, especially in the paediatric population in whom hba c alone has low sensitivity of diagnosing type diabetes [ ] . the categorisation of type and type diabetes was based on an algorithm rather than direct inspection of the clinical notes. although the algorithm was developed in an independent data set in which the diagnosis of type diabetes was verified by reviewing medical records, the absence of confirmatory tests such as anti-islet cell antibodies or c-peptide levels could lead to incorrect differentiation of type and type diabetes, affecting the accuracy of the algorithm. misclassifying type as type diabetes would significantly inflate the incidence of type diabetes, whereas the impact on incidence of type diabetes was probably minimal. the probability of incorrect grouping would be greater in older patients in whom the algorithm performed less well, and estimates of incidence of type diabetes in adults will require confirmation in other cohorts. because of limitations in details of the extracted information, we could not discern patients with other aetiologies of diabetes, such as maturity-onset diabetes of the young and secondary diabetes. owing to the asymptomatic nature of diabetes, diagnosis is often delayed. despite the good quality of the surveillance database, undiagnosed cases could not be captured, leading to underreporting of case burden. the recorded incidence rates were also sensitive to secular changes in people's health-seeking behaviour, screening practice, and prescription behaviour. in this report on the secular trend of the incidence of diabetes in hong kong, we revealed that the incidence of type diabetes increased in people aged < years and was stable in other age groups. the incidence of type diabetes also increased in people aged < years and accounted for over half of new cases of diabetes among people aged < years. in people aged � years, the incidence of type diabetes remained constant. these observations provide an impetus for scaling up measures to prevent development of diabetes in people at risk. (a) incidence trends of type diabetes in boys aged < years using restricted cubic spline. (b) incidence trends of type diabetes in men aged to < years using restricted cubic spline. (c) incidence trends of type diabetes in men aged to < years using restricted cubic spline. (d) incidence trends of type diabetes in men aged � years using restricted cubic spline. (e) incidence trends of type diabetes in girls aged < years using restricted cubic spline. (f) incidence trends of type diabetes in women aged to < years using restricted cubic spline. (g) incidence trends of type diabetes in women aged to < years using restricted cubic spline. (h) incidence trends of type diabetes in women aged � years using restricted cubic spline. international diabetes federation china noncommunicable disease surveillance group. prevalence and control of diabetes in chinese adults china national diabetes and metabolic disorders study group. prevalence of diabetes among men and women in china diabetes incidence and prevalence in hong kong, china during - thematic household survey report no. . hong kong sar: census and statistics department standards of medical care in diabetes- report of the expert committee on the diagnosis and classification of diabetes mellitus solté sz g; eurodiab study group. incidence trends for childhood type diabetes in europe during - and predicted new cases - : a multicentre prospective registration study norwegian childhood diabetes study group. incidence of type diabetes in norway among children aged - years between and : has the incidence stopped rising? results from the norwegian childhood diabetes registry 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diabetes and obesity in the offspring of pima indian women with diabetes during pregnancy glucose intolerance and cardiometabolic risk in adolescents exposed to maternal gestational diabetes: a -year follow-up study arsenic exposure and prevalence of type diabetes in us adults epidemiology of cardiovascular risk factors in hong kong worsening trend of central obesity despite stable or decline body mass index in hong kong chinese between and the government of the hong kong special administrative region. promoting health in hong kong: a strategic framework for prevention and control of non-communicable diseases a demographic perspective on gender, family and health in europe cigarette smoking is an independent risk factor for type diabetes: a four-year community-based prospective study tobacco and alcohol control office. department of health. the government of the hong kong special administrative region the government of the hong kong special administrative region. our partner for better health the government of the hong kong special administrative region. hong kong reference framework for diabetes care for adults in primary care settings. food and health bureau the government of the hong kong special administrative region rapid increase in diabetes incidence among chinese canadians between examining trends in type diabetes incidence, prevalence and mortality in the uk between incidence, prevalence and mortality of type diabetes requiring glucose-lowering treatment, and associated risks of cardiovascular complications: a nationwide study in sweden prevalence and incidence trends for diagnosed diabetes among adults aged to years trends in diabetes incidence in the last decade based on korean national health insurance claims data time trend analysis of the prevalence and incidence of diagnosed type diabetes among adults in taiwan from to : a populationbased study asians have lower body mass index but higher percent body fat than do whites: comparisons of anthropometric measurements diabetes in asia: epidemiology, risk factors, and pathophysiology a major outbreak of severe acute respiratory syndrome in hong kong utility of hemoglobin a( c) for diagnosing prediabetes and diabetes in obese children and adolescents we acknowledge the hong kong hospital authority for providing the data for research. key: cord- -wc k sm authors: feng, mingqian; bian, hejiao; wu, xiaolin; fu, tianyun; fu, ying; hong, jessica; fleming, bryan d; flajnik, martin f; ho, mitchell title: construction and next-generation sequencing analysis of a large phage-displayed v(nar) single-domain antibody library from six naïve nurse sharks date: - - journal: antib ther doi: . /abt/tby sha: doc_id: cord_uid: wc k sm background: shark new antigen receptor variable domain (v(nar)) antibodies can bind restricted epitopes that may be inaccessible to conventional antibodies. methods: here, we developed a library construction method based on polymerase chain reaction (pcr)-extension assembly and self-ligation (named “easel”) to construct a large v(nar) antibody library with a size of . × ( ) from six naïve adult nurse sharks (ginglymostoma cirratum). results: the next-generation sequencing analysis of . million full-length v(nar)s revealed that this library is highly diversified because it covers all four classical v(nar) types (types i–iv) including % of classical type i and % of classical type ii. about % of the total v(nar)s could not be categorized as any of the classical types. the high variability of complementarity determining region (cdr) length and cysteine numbers are important for the diversity of v(nar)s. to validate the use of the shark v(nar) library for antibody discovery, we isolated a panel of v(nar) phage binders to cancer therapy-related antigens, including glypican- , human epidermal growth factor receptor (her ), and programmed cell death- (pd ). additionally, we identified binders to viral antigens that included the middle east respiratory syndrome (mers) and severe acute respiratory syndrome (sars) spike proteins. the isolated shark single-domain antibodies including type i and type ii v(nar)s were produced in escherichia coli and validated for their antigen binding. a type ii v(nar) (pe -b ) has a high affinity (k(d) = . nm) for its antigen. conclusions: the naïve nurse shark v(nar) library is a useful source for isolating single-domain antibodies to a wide range of antigens. the easel method may be applicable to the construction of other large diversity gene expression libraries. classical immunoglobin g (igg) is widely used in many biotechnologies and therapeutics [ ] . igg is best described as a heterodimeric homodimer, consisting of two copies of disulfide-bonded heavy (h) and light (l) chains. the h and l chain variable (v) domains (vh and vl, respectively) combine to form the antigen-binding region. when these two v domains are synthesized as a dual-domain singlechain v fragment (scfv), the minimum size of the fragment is - kda. in recent years, single-domain immunoglobulins such as the shark v nar (new antigen receptor variable domain) and the camelid heavy-chain variable domain (v h h) antibodies have been explored, both of which can be isolated as soluble, stable, monomeric binding domains [ ] [ ] [ ] . these v nar and v h h domain antibodies range from to kda in size, roughly half the size of a scfv binding domain. cartilaginous fish (sharks, rays, skates, and chimaeras) are phylogenetically the oldest living organisms that use antibodies as part of their adaptive immune system [ , ] . they produce three different antibody isotypes that function in their humoral immune responses, immunoglobulin m (igm), immunoglobulin w (igw), and immunoglobulin new antigen receptor (ignar) [ ] [ ] [ ] [ ] . ignar antibodies are homodimeric proteins composed of heavy chains with an antigen-binding region at the end of each chain. they serve as a major component in humoral responses [ , ] . a number of camelid v h h domains are being evaluated in phase i, ii, and iii clinical trials [ , ] . even though the shark v nar is less known, it has the potential to be used in biological therapeutics based on (i) their small size and ability to penetrate dense tissues inaccessible to igg [ ] , (ii) their ability to bind in protein clefts and buried functional sites (e.g. enzyme pocket sites for substrate) [ ] , (iii) their solubility and robustness in harsh conditions [ ] , and (iv) their ability for high-affinity (including sub-nanomolar) binding. additionally, these antibodies have the potential to bind a wide range of antigens, despite the nature of their single-domain architecture [ ] . the v nar domain is an ig superfamily domain with two β sheets held together by two canonical cysteine residues in framework regions (frs) and . in addition to these canonical cysteines, complementarity determining region (cdr ) can have one or two additional cysteines forming additional disulfide bonds within the v domain. ignars are classified into four types based on the number and position of non-canonical cysteines in the v nar domain [ ] . type i v nar contains two non-canonical cysteine residues in cdr encoded by the diversity region or by n-nucleotide additions that form two disulfide bonds with fr and . interestingly, type i has only been reported in nurse sharks, ginglymostoma cirratum [ ] . type ii v nar domains form disulfide bonds between one dencoded non-canonical cysteine in cdr and another non-canonical cysteine in cdr . type iii is similar to type ii except there is a highly conserved tryptophan residue in cdr positioned adjacent to the disulfide bond. type iv has no non-canonical disulfide bonds as found in other three v nar types. both type i and type ii v nar s have protruding cdr s that enable binding to pockets and grooves [ , ] . classical igg and camelid v h h contain a cdr loop that is not present in shark v nar s and are replaced with highly diverse amino acids, termed hypervariable region (hv ) [ ] . additionally, there is a second hypervariable region, named hv , which is inserted in the middle of fr , therefore separating fr into fr a and fr b. shark v nar domains may have advantageous properties over conventional igg. first, sharks are evolutionarily distant from mammals on the phylogenetic tree, therefore can generate high-affinity binders to structurally conservative mammalian drug targets. these may include highly conserved heparan sulfate proteoglycans, g-protein coupled receptors, and ion channels that may exhibit poor immunogenicity in mice and rabbits [ , ] . second, the elongated cdr in shark and camel antibodies has the ability to seek out buried epitopes and enzyme functional sites [ , , , ] . the shark ignar cdr regions are relatively longer (ranging from to a.a. and including various numbers of cysteine residues) compared to mouse or human counterparts. this can potentially lead to a larger diversity of structures that can interact with more diversified antigens [ ] . similarly, the longer cdr region in shark antibodies possesses the extraordinary capacity to form long finger-like extensions that can probe proteins for hidden epitopes [ ] . third, conventional antibodies may have poor tissue penetration ability due to their large size [ ] . whole igg is kda and scfv fragments - kda. v nar domains can be as small as - kda. finally, shark v nar domain antibodies have structural advantages and are easily expressed in escherichia coli systems [ ] . sharks enrich their blood with urea to maintain osmotic balance in the marine environment, so shark antibody structure has evolved to become particularly stable [ ] . phage display technology has been used to isolate shark v nar antibodies. in one study, two shark v nar libraries with a size of clones were constructed from both naïve spiny dogfish (squalus acanthias) and smooth dogfish (mustelus canis) sharks [ ] . phage antibodies were isolated from immunized sharks for specific antigens such as hen egg white lysozyme [ ] [ ] [ ] [ ] . synthetic shark v nar singledomain libraries were also pursued [ ] [ ] [ ] [ ] [ ] . however, none of these approaches has generated a large shark v nar single-domain library that covers the wide range of diversity required (> ) commonly for therapeutic antibody discovery. in this study, we developed a pcr-extension assembly and self-ligation-based method (named easel) to make a large phage-displayed v nar single-domain library from six nurse sharks. nurse sharks were chosen to maximize the diversity of the v nar library because they exclusively have been reported to contain type i v nar domains. to assess its diversity and analyze the v nar sequences on the largest scale in the field, we conducted next-generation sequencing (ngs) analysis on . million unique full-length v nar s. the unique sequences were then analyzed using unbiased methods to investigate their cysteine numbers and cdr lengths for all v nar sequences together as well as type i and type ii/iii v nar s separately. to validate the potential of this library as a new platform for therapeutic antibody discovery, we conducted phage panning to identify shark antibody therapeutics, v nar binders to a panel of tumor and viral antigens. these included antigens associated with liver and breast cancers, as well as antigens against viral proteins associated with sars and mers. these selected binders including type i and ii v nar s were produced successfully in e. coli as soluble proteins for antigen-binding validation. this work validated the large diversity of the nurse shark v nar library and the utility of the shark v nar library as a platform for therapeutic antibody discovery. to construct a large shark v nar library, we developed a method called easel. as illustrated in figure a , we isolated peripheral blood leukocytes from six adult nurse sharks (g. cirratum) and amplified shark v nar sequences with pcr primers. the reverse primers contain a -nucleotide sequence on the pcomb x backbone. extension overlap pcr was conducted to combine the v nar and phagemid backbone (fig. b) . finally, after gel purification we performed a self-ligation with t ligase to circularize the assembled v nar pcomb x plasmids. our shark v nar library size (∼ . × ) was determined by titration based on the number of individual tg bacteria colonies on agar plates. this high-efficiency method is a significant improvement over the conventional phage library construction method, which usually requires many rounds of restriction enzyme digestion and ligation, making the previous method labor intensive and time consuming. moreover, the sequence diversity of the shark v nar is preserved and represented in our method to a maximum degree due to highly efficient pcr-based amplification and ligation. to analyze the diversity of the library, we performed deep sequencing of the whole library using ngs. each sequencing was done twice in both forward and reverse direction for paired-end reads. the merging of paired-end reads was then performed with high stringency by combining the forward and reverse sequencing results to ensure accuracy for each unique sequence. nearly . million full-length v nar sequences with in-frame translation were used for further analysis with a focus on cysteine numbers, cdr length, amino acid variability, and v nar type counts. this is the largest scale shark v nar sequence analysis reported thus far. as shown in figure a , the presence of two canonical cysteines located at both amino acid and are used as a key criterion to characterize type i-iv v nar s. the sequences that do not contain one or both of these cysteines are considered as other types (n = ; ∼ % of the total v nar s) because they do not fit in with the four known v nar type families. the sequences that have both c and c (n = ) are further categorized based on their placement of additional cysteines. type iv v nar s contain only two canonical cysteines found at position and (n = ). type i v nar s contain an extra cysteine at position (n = ; ∼ % of the total v nar s). this group can be further divided into subtypes based on how many additional cysteines they contain. the classical definition of a type i v nar describes an antibody that has a total of six cysteines. about % of the total v nar s are classical type i. type ii and iii v nar s have at least one extra cysteine at amino acid (n = ; % of the total v nar s). among them, ∼ % of the total v nar s are classical type ii. unique full-length v nar s are defined as having one differing amino acid in sequence. as shown in figure b , % ( ) of the . million sequences only appeared once in ngs results, and . % of the . million sequences appeared twice. only % of the . million sequences appeared more than times. the most frequently repeated clone appeared times in ngs results. the percentage of the different types of v nar is plotted in the pie chart in figure c . based on the number of extra cysteines in these sequences, the type i (six cysteines in total) and type ii (four cysteines in total) v nar s are considered classical to others (other numbers of cysteines) as shown in figure d and e, respectively. representative sequences of type i-iv shark v nar s with different numbers of cysteines were randomly picked from ngs data and shown here as examples (fig. ) . the frs and cdr , cdr , hv , and hv are marked based on stanfield et al. [ ] and fennell et al. [ ] in figure . these sequences were aligned to sequences on imgt database for cdr determination. as shown in figure , part of the hv sequence was identified as "cdr " in the imgt database. these v nar s may have multiple cysteine residues in cdr based on the sequences from ngs data. we analyzed the total number of cysteines, the number of cysteines found in cdr , the length of cdr , and the amino acid sequence variability. due to the high variability of cdr lengths, we defined cdr to be the sequence between conserved framework sequences tyrc (end of fr b) and xxxgtxxtvn (fr ). the total cysteines in v nar sequences can vary from to (fig. a ) and the cdr can have - cysteines (fig. b ). the cdr length varies greatly as well, and it can be between - amino acids according to the ngs data (fig. c ). the separate analysis of type i and type ii/iii v nar s showed type i v nar s (shown as blue lines) have more total cysteines and in cdr than type ii/iii (shown as red lines) ( fig. a and b). the cdr lengths for type i v nar s are also slightly longer compared to type ii/iii (fig. c ). these findings are consistent with published small-scale sequence analysis: type i v nar s have more even numbers of cysteines in cdr ( , , or ) (fig. b) [ , ] . the high variability of cdr length and cysteine numbers are crucial to the diversity of v nar s since binding diversity is dependent on the cdr structure diversity. amino acid sequence variation analysis showed the sequence diversity is mainly contributed to cdr with minimal variation in cdr , hv , and hv (fig. ) . taken together, we designed a pcr-based method to establish a large shark v nar singledomain antibody library with the size of . the library contains all types (i-iv) of shark v nar sequences as well as many other previously undefined types. to evaluate the library's potential for therapeutic development, we chose a variety of human tumor biomarkers and virus antigen proteins as selection targets. these include glypican- (gpc ), her and pd , the spike proteins of the mers and sars viruses, and pseudomonas exotoxin (pe ). after four rounds of panning, specific binders to the listed targets were identified by monoclonal phage elisa (fig. a ). the binders were assigned the following names based on their targets and well numbers, which include gpc -f , her -a , pd -a , mers-a , mers-a , mers-a , mers-b , mers-b , sars- , and pe -b . only one binder was isolated for all antigens, except mers spike protein. five binders were isolated for mers spike protein. sequence analysis showed that most of these binders were type ii v nar except for one type i and two undefined types. one type ii binder (pe -b ) targeting pe fragment was produced in e. coli hb strain as a single-domain protein. it had . nm k d binding affinity for its antigen as measured by octet kinetic assay ( fig. b and c) . the affinity is high as a monomeric single-domain soluble protein isolated from a naïve shark library without immunization. we also produced the type i binder (mers a ) and type ii binder mers a in e. coli. the protein yield varied based on protein sequences. pe -b has a relatively low yield ( mg from l e. coli culture). mers a binder yielded . mg and mers a yielded . mg of single-domain soluble protein from ml e. coli culture. nickel-charged histrap columns (ge healthcare, chicago, il) were used to purify these proteins from the polymyxin b lysed bacteria pellet supernatants. the elution profile showed the protein elution in figure a and d. sodium dodecyl sulfate polyacrylamide gel electrophoresis (sds-page) of the elution peaks in figure b and e showed the later peak eluted by higher imidazole concentrations had over % purity of the target soluble v nar s. both mers a and a had specific binding to the phage panning antigen mers spike protein in figure c and f. the type i binder (mers a ) had higher signals. tsk size exclusion column purification for the type ii binder pe -b -his soluble single-domain protein showed this protein is monomeric. the . kda protein was eluted with one peak that is slightly earlier than . kda control protein peak ( fig. g and h) . taken together, binders to tumor and viral antigens, including type i and type ii single domains containing multiple cysteine residues, can be selected from the library and produced as functional single-domain proteins in e. coli. our data indicate that the new shark phage library is a valuable platform for discovery of v nar binders. in the present study, we developed the easel method to construct phage display libraries based on pcr extension assembly followed by self-ligation. by using this easel method, we successfully constructed a large phagedisplayed v nar library with a size of from six antigen-antibody therapeutics, we also demonstrated that type i and type ii shark v nar binders can be produced from e. coli as functional single-domain proteins. the large nurse shark v nar singledomain phage library described here provides an attractive single-domain antibody platform for drug discovery. in this study, we use two canonical cysteines located at both amino acid and as a key criterion to characterize type i-iv v nar families. about % of the total v nar s do not fit in any one of the four known v nar type families. we have identified ∼ % of the total v nar s are classical type i and % classical type ii. while a majority of the v nar s are either classical type i or type ii, ∼ % of the v nar s do not fit any of the four classical types. most of these nonclassical v nar s have various numbers of cysteines (fig. ) . future structural and functional analysis of the binders isolated from the library for various antigens will be needed to understand the role of these non-classical v nar s in therapeutic antibody discovery and engineering. the key for a successful isolation of antibodies is the phage library used for the selection [ ] . various methods have been employed to make antibody phage display libraries. most widely used methods consist of pcr amplification of antibody fragments, followed by enzymatic digestion and ligation with the vector. these methods are time consuming and have a low efficiency in the ligation and transformation step. in the present study, we developed the easel method to construct a large shark v nar phage library. we used a labeling pcr to add homologue regions to the v nar pool. then an overlap extension pcr was used to assemble v nar and phagemid dna, followed by self-ligation of the whole library dna. it only took several weeks to make the shark v nar library while conventional approaches would require several months. our protocol has significantly shortened the time required to construct a large gene library. another contemporary method used for gene cloning is "gibson assembly". this method is based on enzymatic assembly for joining multiple overlapping dna fragments into a single reaction system [ ] . our method may be more efficient than gibson assembly. gibson assembly requires -exonuclease to nick the -terminals of the dna fragments to make them complementary followed by annealing together. dna polymerase was then used to fill the gap, and dna ligase was used to seal the gap. therefore, the two termini that antibody therapeutics, are being ligated have to be identical for each other for ∼ nucleotides. the gibson assembly reaction may fail due to complications related to total repeat density (direct, inverse, and palindromic repeat elements), extremes in g/c content, and secondary structures near the ' and ' termini of the sequence. to our best knowledge, there is no report using the gibson assembly for the development of a large phage-displayed antibody library. in our method, we only need the conventional t dna ligase to run a standard ligation at • c for - h. the two ends of the dna are blunt and do not need to be identical for blunt-end ligation. therefore, our method is similar to traditional blunt-end ligation, and the terminal part of the dna fragment sequence can be varied. during the preparation of this manuscript, our laboratory has applied the easel method to successfully make large camel v h h singledomain phage libraries. taken together, the new easel library construction method is robust, comprehensive, and quick. the new easel method also maximized the sequence diversity of cdr represented in the phage library to increase the selection of high-affinity binders. a previous study produced a shark phage library with diversity using the conventional cloning method. our library has diversity and contains all known four types of v nar single domains. the majority of the sequences are type ii and type i v nar as shown in figure c . interestingly, type iv v nar sequences are significantly under-represented as shown in figure c . the shark v nar sequences in the ncbi database are mostly derived from bamboo shark, dogfish shark, wobbegong shark, and other types of smaller sharks [ ] [ ] [ ] [ ] [ ] [ ] . the percentage of v nar types can be significantly different between various species of sharks. interestingly, our deep sequencing analysis showed that of the nurse shark v nar sequences in our library were not categorized in any one of the known v nar types (types i-iv). whether these novel types of v nar s possess unique confirmations and biophysical properties should be analyzed structurally and functionally when binders for cancer or viral antigens are found from these novel types of v nar s in the future. since the method we used to assemble the unique v nar sequences for analysis is highly accurate and the number of the unique sequences analyzed is large, we used unbiased methods to analyze all the sequences for cysteine number and cdr length without further subdividing them into the four known v nar types. a significant percentage of the sequences are not classical v nar as defined in the known four types. analyzing the data sequences altogether would provide more comprehensive picture of the sequence patterns in the naïve nurse shark without having to fit the sequences into a defined v nar type. the extra cysteines in both type i and type ii v nar s are important for stabilizing the antigen-binding regions [ ] . extra disulfide bonds formed by these cysteines are essential for forming diverse antigen-binding surfaces. it may be one strategy for increasing antigen-binding region diversity in heavy chain-only antibodies. interestingly, the sequence variation mostly lies in cdr with minimal variation in cdr , hv , and hv . previous literature suggested there are sequence variability in hv and hv during in vivo affinity maturation [ ] . it was also shown that somatic mutations within hv / can contribute to antigen binding [ ] . however, ngs results in the present study showed there is minimal sequence variability in cdr , hv , and hv in our naïve shark library. it is possible that these naïve sharks used in our study have been kept in captivity for a long time, therefore do not have as much sequences diversity in antibody therapeutics, cdr , hv , and hv . another major type of heavy chain-only domain antibody v h h from camels also has highly diversified cdr . the lengths of v h h cdr are much longer than conventional vh from camel igg. the median cdr length of v h h is amino acids (similar with shark v nar ), whereas median length for camel vh cdr is only amino acids [ ] . however, there is no comprehensive study on the cysteine numbers and locations in camel v h h sequences for comparison with shark v nar . we hypothesize that the diversity of this large naïve shark library can be further increased significantly by randomizing the cdr sequences and keeping the highly diverse cdr [ , ] . for example, the eight amino acids in cdr can be completely randomized to further diversify our v nar library. furthermore, affinity maturation of the binders in figure can be performed by randomizing cdr . in summary, we believe this shark library has a suitable size and diversity for antibody discovery and can potentially be used as a large single-domain antibody discovery platform. the potential immunogenicity of shark antibodies could be a concern in clinical applications. these concerns could be addressed by identifying the b-cell and t-cell immunogenic epitopes and silence them by specific site mutations [ , ] . furthermore, durable immune tolerance to highly immunogenic proteins (e.g. bacterial toxins) can be induced by nanoparticles containing rapamycin in vivo [ ] . in conclusion, we have built a large, highly diverse phage-displayed v nar library using b cells isolated from nurse sharks. it provides a new platform to discover single-domain antibodies for therapeutic and diagnostic applications. the easel library construction method described here may be applicable to the construction of other large gene expression libraries including antibody libraries derived from other species and t-cell receptors. the gpc -positive human hepatocellular carcinoma cell line hepg was maintained as adherent monolayer cultures in dmem medium (invitrogen, carlsbad, ca) supplemented with % fetal bovine serum (hyclone, logan, ut), % l-glutamine, and % penicillin-streptomycin (invitrogen) in a % co incubator at • c. gpc -negative a cells (human epithelial carcinoma cell line) were engineered in our laboratory to express high levels of human gpc by transfection with a plasmid encoding full-length gpc [ , ] . both a and the stably transfected cells (g ) [ ] were maintained in dmem with supplements. the gpc peptide (a.a. - ) was synthesized. the recombinant extracellular domain of her and pd , the s-protein of mers and srs were purchased from sino biological (beijing, china). the recombinant pe and mpe were made in our laboratory according to the pub-lished methods [ ] . the recombinant gpc -hfc was generated as previously published [ ] . six naïve nurse sharks (three males and three females, ranging from . to ft long) were bled for ml of blood in phosphate buffered saline (pbs)/ iu/ml heparin. the buffy coat was collected, spun, and resuspended in . ml of trizol for rna preparation. total rna was isolated using the trizol reagent (thermo fisher scientific, grand island, ny) according to the manufacturer's instruction. five micrograms of total rna were reverse-transcribed into cdna in a total of μl volume using the superscript iii first-strand synthesis system (thermo fisher scientific) according to the manufacturer's instruction. one forward primer and two reverse primers were synthesized to pcr amplify the v nar sequence from the cdna product. forward primer ignar-f: gctc-gagtgaccaaacaccg, reverse primer ignar-r : ggtggccggcctggccactattcacagtcacgg cagtgccat, reverse primer ignar-r : ggtggc-cggcctggccactattcacagtcacgacagtgc-cacc. primer pairs of ignar-f/ignar-r , ignar-f/ignar-r were used to amplify the v nar fragment in a μl of pcr volume that contains μl of cdna product. the pcr cycling parameters were the following: initial denaturation at • c for min, cycles of denaturation at • c for s, annealing at • c for s, and elongation at • c for s using primestar (clonetech). in the meantime, the linear vector backbone fragment was prepared by pcr using forward primer ignarcom x-f: agtggccaggccggccacc, and reverse primer ignarcom x-r: ggccgcctgggccacggta. five nanograms of the plasmid pcomb x was used as the template in a total of μl of pcr reaction volume. the primers to amplify the vector backbone were forward ignarcom x-f: agtggccaggccggccacc and reverse ignarcom x-r: ggccgcctgggccacg-gta. the pcr cycling parameters were the following: initial denaturation at • c for min, then cycles of denaturation at • c for s, annealing at • c for s, and elongation at • c for min using primestar (takara, shiga, japan). to assemble the v nar and the amplified vector backbone, ng of vector backbone was mixed with ng of v nar pcr products in a μl of pcr reaction volume, the overlapping extension pcr was primed by primers of ignar-f/ignarcom x-r using primestar. twenty micrograms of the assembled pcr product were circularized by intra-molecular self-ligation in a ml of ligation buffer using t dna ligase (new england biolabs, ipswich, ma). the ligation products were cleaned up by removing the enzymes and transformed into μl of electroporation competent tg cells (lucigen, middleton, wi) to make the library. this method is referred to as easel method in this study. ngs of the library was generated from shark v nar insert dna digested out of plasmid library using xhoi and msci restriction enzymes (new england biolabs). the shark v nar library was excised out from the vector with restriction enzyme (xhoi and msci) and gel purified. the insert fragments were ligated to illumina adaptor and sequenced on illumina miseq with × bp paired-end reads using both nebnext ultra ii dna library preparation kit and kappa hyper prep library preparation kit. paired-end reads were merged to cover the full length of the insert using flash [ ] . merged sequence reads were then analyzed with custom perl scripts. all dna sequences were oriented and translated to protein sequences. sequences with stop codons were removed from further analysis. we also required the amino acid sequences to start with "rv" at the n-terminus and end with "xxxgtxxtvns" at the c-terminus to be considered as full-length v nar s. after the selection with these criteria, there are almost million v nar amino acid sequences from this experiment, with more than million unique v nar sequences. we aligned all v nar sequences by anchoring the constant regions and allow cdr regions to have variable lengths from to amino acids. we then calculated the variability according to methods described in wu and kabat [ ] . we further classified v nar sequences into different subtypes based on residue numbers and positions. type i and type ii/iii sequences were also analyzed separately for the cdr lengths, total cysteine numbers, and cdr cysteine numbers. library tg bacterial stock was inoculated into . l of yt media containing % glucose, μg/ml ampicillin, and cultured at • c with shaking ( rpm). when the cells reached mid-log phase (od between . - . ), super-infection was performed by adding helper phage m ko at × pfu/ml. after h of continued growth, the tg cells were pelleted and resuspended in . l of yt media containing μg/ml ampicillin and μg/ml kanamycin, and incubated at • c overnight. after the cells were centrifuged and filtered with a . μm membrane, the supernatant was stored at • c for panning. the phage panning protocol has been described previously [ , ] . briefly, a well maxisorp elisa plate (nunc/thermo fisher scientific, rochester, ny) was used to capture various antigens ( μg/ml) in pbs buffer at • c overnight. after the coating buffer was decanted, the plate was treated with blocking buffer ( % bovine serum albumin in pbs) at room temperature for h. then μl pre-blocked phage supernatant (typically contained - cfu) in μl blocking buffer was added per well for h at room temperature to allow binding. after four washes with pbs containing . % tween- , bound phages were eluted with mm triethylamine. after four rounds of panning, single colonies were picked and identified by using phage elisa. the antigenic proteins were used to coat a well plate at μg/ml in pbs buffer, μl/well, at • c overnight. the irrelevant antigen used was μg/ml bsa in pbs. after the plate was blocked with % bsa in pbs buffer, μl pre-blocked phage supernatant (typically - cfu) were added to the plate. binding was detected by hrp conjugated mouse anti-m antibody (ge healthcare life sciences, pittsburg, pa). the cut-off value for positive binder was set as × higher signal of antigen binding compared to background noise. the pcomb x phagemids containing the v nar binders were transformed into hb e. coli cells. the formed colonies were pooled for culture in l yt media containing % glucose, μg/ml ampicillin at • c until the od reaches . - . culture media was then replaced with yt media containing mm iptg (sigma), μg/ml ampicillin, and shook at • c overnight for soluble protein production. bacteria pellet was spun down and lysed with polymyxin b (sigma) for h at • c to release the soluble protein. the supernatant was harvested after lysis and purified using histrap column (ge healthcare) using akta. the shark single-domain soluble protein pe -b -his was buffer exchanged in pbs buffer after purification. the binding kinetics of pe -b -his was measured with fortebio octet red located at the biophysics core in national heart, lung and blood institute. pe -b -his diluted in assay buffer pbs supplemented with . % tween- and % (w/v) bsa. the octet red program was as follows: min presoak, s baseline establishment, s antigen loading, s baseline re-establishment after antigen loading, s pe -b -his association, min dissociation. a total of μg/ml pe -b -his was used to load the ni-nta biosensor, and serial diluted antigen protein pe was used for binding assay. the binding kinetics was calculated with fortebio octet red software. all statistical analyses were conducted using graphpad prism (graphpad software, inc., la jolla, ca). differences between groups were analyzed using the two-tailed student's t-test of means. antibody-based immunotherapy of cancer single domain antibodies: promising experimental and therapeutic tools in infection and immunity antibody repertoire development in cartilaginous fish the structural analysis of shark ignar antibodies reveals evolutionary principles of immunoglobulins diversity and repertoire of igw and igm vh families in the newborn nurse shark unprecedented multiplicity of ig transmembrane and secretory mrna forms in the cartilaginous fish structural analysis of the nurse shark (new) antigen receptor (nar): molecular convergence of nar and unusual mammalian immunoglobulins the development of nanobodies for therapeutic applications nanobodies as versatile tools to understand, diagnose, visualize and treat cancer ribosome display and affinity maturation: from antibodies to single v-domains and steps towards cancer therapeutics crystal structure of a shark single-domain antibody v region in complex with lysozyme dimerisation strategies for shark ignar single domain antibody fragments dissection of the ignar v domain: molecular scanning and orthologue database mining define novel ignar hallmarks and affinity maturation mechanisms structural insights and biomedical potential of ignar scaffolds from sharks shark novel antigen receptors--the next generation of biologic therapeutics? structural analysis, selection, and ontogeny of the shark new antigen receptor (ignar): identification of a new locus preferentially expressed in early development display scaffolds: protein engineering for novel therapeutics inaugural editorial: searching for magic bullets comparisons of the intraocular tissue distribution, pharmacokinetics, and safety of i-labeled full-length and fab antibodies in rhesus monkeys following intravitreal administration selection of cholera toxin specific ignar single-domain antibodies from a naive shark library maturation of shark single-domain (ignar) antibodies: evidence for induced-fit binding selection and characterization of naturally occurring single-domain (ignar) antibody fragments from immunized sharks by phage display shark immunity bites back: affinity maturation and memory response in the nurse shark, ginglymostoma cirratum first molecular and biochemical analysis of in vivo affinity maturation in an ectothermic vertebrate rapid isolation of ignar variable single-domain antibody fragments from a shark synthetic library isolation of anti-toxin single domain antibodies from a semi-synthetic spiny dogfish shark display library isolation of the new antigen receptor from wobbegong sharks, and use as a scaffold for the display of protein loop libraries isolation and characterization of an ignar variable domain specific for the human mitochondrial translocase receptor tom selection and affinity maturation of ignar variable domains targeting plasmodium falciparum ama construction of human naive antibody gene libraries enzymatic assembly of dna molecules up to several hundred kilobases comparative analysis of immune repertoires between bactrian camel's conventional and heavy-chain antibodies generation of semi-synthetic shark ignar single-domain antibody libraries construction of histidine-enriched shark ignar variable domain antibody libraries for the isolation of ph-sensitive vnar fragments recombinant immunotoxin with t-cell epitope mutations that greatly reduce immunogenicity for treatment of mesothelin-expressing tumors a recombinant immunotoxin against the tumor-associated antigen mesothelin reengineered for high activity, low off-target toxicity, and reduced antigenicity tolerogenic nanoparticles restore the antitumor activity of recombinant immunotoxins by mitigating immunogenicity inactivation of wnt signaling by a human antibody that recognizes the heparan sulfate chains of glypican- for liver cancer therapy high-affinity monoclonal antibodies to cell surface tumor antigen glypican- generated through a combination of peptide immunization and flow cytometry screening immunotoxin targeting glypican- regresses liver cancer via dual inhibition of wnt signalling and protein synthesis therapeutically targeting glypican- via a conformation-specific single-domain antibody in hepatocellular carcinoma flash: fast length adjustment of short reads to improve genome assemblies an analysis of the sequences of the variable regions of bence jones proteins and myeloma light chains and their implications for antibody complementarity in vitro antibody affinity maturation targeting germline hotspots in vitro antibody evolution targeting germline hot spots to increase activity of an anti-cd immunotoxin we thank dr gregory piszczek from biophysics core facility in national heart, lung, and blood institute for octet assay technical support. antibody therapeutics, key: cord- -x dq sy authors: wan, dongshan; jiang, wei; hao, junwei title: research advances in how the cgas-sting pathway controls the cellular inflammatory response date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: x dq sy double-stranded dna (dsdna) sensor cyclic-gmp-amp synthase (cgas) along with the downstream stimulator of interferon genes (sting) acting as essential immune-surveillance mediators have become hot topics of research. the intrinsic function of the cgas-sting pathway facilitates type-i interferon (ifn) inflammatory signaling responses and other cellular processes such as autophagy, cell survival, senescence. cgas-sting pathway interplays with other innate immune pathways, by which it participates in regulating infection, inflammatory disease, and cancer. the therapeutic approaches targeting this pathway show promise for future translation into clinical applications. here, we present a review of the important previous works and recent advances regarding the cgas-sting pathway, and provide a comprehensive understanding of the modulatory pattern of the cgas-sting pathway under multifarious pathologic states. pattern-recognition receptors (prrs) serve as innate cellular sensors of danger signals, such as pathogen-associated molecular patterns (pamps) or danger-associated molecular patterns (damps), and yield cellular-stress response. dna molecules are vital genetic components within cells, which are compartmentalized restrictively into specific regions. the occasionally misplaced dna is degraded rapidly by scavenger cells and extracellular or intracellular ribonucleases. aberrant accumulation of dna is relevant to tissue damage ( ) . in , several research teams discovered a new protein on the endoplasmic reticulum (er) which can be activated by immune-stimulatory dna (isd) and initiate type-i interferon (ifn) responses, which was named "stimulator of interferon genes" (sting, also known as mita, eris) ( ) ( ) ( ) . sting does not bind to dna directly, and bacteria-derived cyclic di-guanylate monophosphate (c-dgmp) or cyclic di-adenosine monophosphate (c-damp) were confirmed to be ligands for sting ( , ) . subsequently, it was found that some dna sensors can facilitate sting activation, such as interferon gamma inducible protein (ifi ) ( ) . however, sting activation could not be fully explained by the upstream factors/ligands that had been found. it was postulated that an unknown upstream regulator might be responsible for sting activation. in , wu and sun found that cyclic guanosine monophosphate-adenosine monophosphate (cgamp) was a novel secondary messenger serving as a ligand of sting ( ) . beside it, they purified a new protein named "cyclic-gmp-amp synthase" (cgas) that had cytosolic dna-sensing ability and can synthesize cgamp ( ) . also, they found that the cgas-cgamp-sting pathway was indispensable for host anti-viral immunity ( ) . their work filled in the gaps missing from upstream of sting. stimulator of interferon genes or cyclic-gmp-amp synthase is expressed widely in a broad spectrum of cells including immune, non-immune, cancer cells ( ) . mounting evidence has demonstrated that the cgas-sting pathway is important for mediating cellular immune sensing, and shows particular responses pattern to the isd distinguished from other nucleotide-sensing pathways. it is also regulated delicately by several molecules or feedback loops to maintain cellular homeostasis. nevertheless, cgas-cgamp-sting pathway itself has distinctive or even opposing effects under different conditions. in this review, we cover the roles of cgas-sting pathway in cellular type-i ifn immune response, and several cellular processes including autophagy, survival and senescence. we also summarize the literature on intrinsic cellular mechanisms modulating cgas-sting pathway as well as its cross-regulations with other dna-sensing pathways. moreover, the inflammationmodulation capacities of this pathway in infectious disease, inflammation and cancers have been elucidated too, and a pervasive pattern of this pathway has been described, which could provide a plausible explanation of the contradictory findings of studies. finally, current or prospective therapeutic strategies targeting the pathway, and issues that need to be addressed in the future, are discussed. cyclic-gmp-amp synthase belongs to the structurally conserved cgas/dncv-like nucleotidyltransferase (cd-ntases) superfamily. the latter is expressed universally in prokaryotes and eukaryotes, and can use purines or pyrimidines selectively as substrates for the production of linear or cyclic di-or even tri-nucleotide compounds, which act as secondary intracellular messengers ( ) . cyclic-gmp-amp synthase is distributed mainly in the cytosol (also nucleus in some specific conditions) ( ) . generally speaking, cgas is activated upon the recognition of b-type double-stranded dna (dsdna) without sequence-specificity but not a-type dsdna or rna ( , ) . hybrid dna:rna or stemlike single-stranded dna (ssdna) are also low-affinity ligands for cgas ( , ) . after binding with ligands, cgas undergoes an allosteric structural change, and subsequently catalyzes its substrates guanosine triphosphate (gtp) and adenosine triphosphate (atp) to produce a mixed phosphodiester-linked cyclic dinucleotide: g( - )pa ( - ) p cgamp (abbreviated as , -cgamp or cgamp) ( ) . cgas also catalyze the synthesis of linear dinucleotides such as amp- -atp, gmp- -gtp, and amp- -gtp as intermediate products ( ) . there are two major dsdna-binding sites on opposite sides of the catalytic pocket: a and b site. site a is the primary contact surface for dsdna, whereas site b is complementary, binding another dsdna. it allows for cgas to the formation of a : cgas:dsdna complex structure directed into two orientations with dsdna at least bp ( ) ( ) ( ) (figure a) . increased numbers of back-to-back dimers of cgas hold the two dsdna molecules together and permit successive recruitment of cgas which, consequently, forms a n: cgas:dsdna higherordered "ladder-like" oligomerization, with cgas arrayed "head to head/tail to tail" ( , ) . the dna-binding protein hu, mitochondrial transcription factor a (tfam), or bacterial high mobility group box protein (hmgb ) can bend the dsdna into a u-shaped structure and, thus, facilitate binding of cgas dimers to the same strand as it travels in opposite directions ( ) (figure b) . human cgas, unlike mouse cgas, is prone to formation of this ladder-like network with long dsdna, because of the human-specific residues k and l . these two dsdna-interfacing residues of site a loosen the interaction of dsdna with cgas, leading to dsdna curving and allowing more convenient binding for the next adjacent cgas ( , ) ( figure c) . finally, accumulated cgas-dsdna complexes can go through a liquid-phase separation and condense into gellike droplets as a reaction unit ( figure d) . this conformation requires a sufficiently long dsdna strand to form multivalent interaction positions, also requires the function of the n-terminal tail of cgas and a recently discovered dsdna-binding site in the catalytic domain of cgas (site c) ( , ) . meanwhile, the n-terminal tail of cgas mediates cgas localization onto the membrane by binding to phosphatidylinositol , bisphosphate (pi ( , ) p ) and prevents liberation of cgas and oligomerization, but can release cgas during cell stress ( ). the structure of cgas determines long strand dsdna (> - , bp) could potentially stimulate the enzyme activity and cgamp production of cgas ( ). the ability of human cgas to discriminate long dsdna strands from shorter dsdna may contribute to the specific sensing and recognition of the "danger dna" of pathogens, necrotic cells or cancer cells rather than irrelevant shorter dsdna, thereby enhancing the immunity against them specifically. double-stranded dna is restricted into the nucleus or mitochondria and is rarely present in the cytoplasm. extrinsic dsdna from pathogens such as viruses, bacteria, transcellular vesicles or rupture of dying cells can be internalized into the cytosol in several diverse ways ( - ). these extrinsic dsdna sources are engulfed by endosome through phagocytosis and digested immediately by dnaseii when fusing with lysosomes ( , ). however, some escaping mechanisms under certain conditions could help protect them from being degraded. for example, antimicrobial peptide ll could efficiently transports self-dna from endosome into cytosol of monocytes ( ). cell oxidative stress can lead to phagosomal acidification delay and probably release endosome context including dsdna owing to increased membrane permeability ( , ). the intrinsic self-dsdna can also be segregated inaccurately and released into the cytosol ( , ). for example, genomic dna (gdna) injury as a result of genotoxic stress and dna self-instability or replication errors leads to double-strand breaks (dsbs) and can be repaired by several ways ( ). impaired mediators of dna-damage repair response mediators, such as ataxia telangiectasia mutated (atm)-rad , poly adpribose polymerase (parp) and breast cancer / (brca / ) multiple cgas molecules can bind two double-stranded dnas (dsdna) to form a n: cgas:dsdna higher-ordered "ladder-like" oligomerization. mitochondrial transcription factor a (tfam) can bend the dsdna into a u-shaped structure and promote polymerization. (c) cgas can recognize b-type dsdna. in humans, the cgas dna-interfacing residue of site a loosens the interaction of dsdna to curve dsdna away for more convenient binding with next adjacent cgas. cgas can catalyze gtp and atp to synthesize cyclic guanosine monophosphate-adenosine monophosphate (cgamp). the n-terminal tail binds to the cell membrane, associating with phosphatidylinositol , -bisphosphate [pi ( , )p ]. (d) accumulation of cgas-dna complex goes through a liquid-phase separation and condenses into gel-like droplets. are associated with persisting dsbs and accumulation of cytosolic dna ( - ). extra-nuclear micronuclei formation during mitosis is a source of cytosolic dsdna caused by dsbs ( , ). followed by homologous recombination repair of collapsed replication forks, dna cleavage by methyl methanesulphonate (mms) and ultraviolet-sensitive (mus ) also lead to cytosolic dsdna presenting ( ). furthermore, manually cre/loxp recombination technology can induce dsdna damage during dna cleavage, which results in the accumulation of cytoplasmic dsdna ( ). in normal cellular mitotic processes, chromosomal dna can be exposed to the cytoplasm, while it is hard to bind and trigger cgas ( ). in addition, mitochondrial dna (mtdna) is also a considerable ligand of cgas and can be released into the cytosol under mitochondrial stress or dysfunction of proteins which participates in maintaining mitochondrial operations ( , , ) (figure a) . cells have several types of nucleases to restrict cytosolic dna to avoid cgas activation. for example, three-prime repair exonuclease (trex also known as dnaseiii) is a cytosolic dna exonuclease which removes unprotected dsdna from the cytosol ( ) . rnaseh locates to the nucleus and specifically degrades the rna in rna:dna hybrids participating in dna replication ( ) . dnaseii is a lysosomal dnase which degrades undigested dna in endosomes or autophagosomes to prevent their entry into the cytoplasm ( ). sam domain and hd domain-containing protein (samhd ) is characterized as a dntpase and restricts reverse transcription of the rna virus ( , ) . samhd can also stimulate the exonuclease (but not the endonuclease) activity of mre to degrade nascent cdns (including cgamp) might be exported by some ways. (c) inflammatory signaling mediated by the cgas-sting pathway. after sensing dna, cgas produces cgamp and extracellular cdns, promoting stimulator of interferon genes (sting) to undergo dimerization. sting can exit from the endoplasmic reticulum (er), and be translocated from the er to the er-golgi vesicle, and arrives at the golgi. sting and tank binding kinase (tbk ) can be oligomerized and cluster at the golgi. the sting-tbk /iκb kinase (ikk) signalosome forms a scaffold to phosphorylate interferon regulatory factor (irf ) and inhibitor of nf-κbα (iκbα). then, dimerized irf and the activated canonical nf-κb p /p complex can be translocated into the nucleus as transcription factors to promote transcription of type-i ifn. (d) autophagy initiation and degradation. sting activation on er triggers er stress and mechanistic target of rapamycin complex (mtorc ) dysfunction. er stress and mtorc dysfunction can stimulate the unc- like autophagy activating kinase (ulk ) complex and beclin -phosphatidylinositol -kinase catalytic subunit type (pi kc ) complex. autophagy-related protein (atg ) and light chain (lc ) are associated with genesis and elongation of the autophagosome. after autophagy initiation, cgas-sting is ubiquitinated and binds with p . then, they are packaged into autophagosomes and terminally sorted to lysosomes (bold arrows represent main signaling pathways, thinner arrows represent regulatory signaling pathways, and dashed arrows represent bypass or suspicious pathways). ssdna, and start dna-repair responses at stalled replication forks ( ) . depletion of samhd leads to the cleaving of nascent ssdna by the activity of mre endonuclease and cytosolic translocation of gdna ( ) . deficiency of any of these nucleases can lead to accumulation of self-dna in the cytoplasm, thereby activating the cgas-sting pathway against dna molecules ( ) (figure a ). the production of asymmetrically linked , -cgamp catalyzed by cgas has the highest affinity for sting to promotes sting dimerization ( , ) . cgamp as a second messenger can be also transferred among cells in several ways to pass danger signaling of frontiers in immunology | www.frontiersin.org cytosolic dna. intercellular gap junction consists of two docking hexamer channels formed by different connexins, which allows many small molecules, including cgamp, to pass bi-directionally through cells. and intercellular transfer of cgamp through gap junction is largely dependent on connexin ( ) ( ) ( ) . additionally, cgamp can be packaged into virons and pre-notify newly infected cells ( , ) . cell fusion is a distinct manner for intracellular transmission of the human immunodeficiency virus (hiv); cgamp also enter membrane-fused bystander cells in this way ( ) . extracellular vesicles such as exosomes can contain cgamp along with viral dna, host gdna or mtdna, and mediate cells communication ( , ) . there were no evidences that cgamp could be pumped out to extracellular space by a channel/transporter. however, it was found that slc a can transmit cyclic dinucleotides (cdns) into cell plasma ( , ) . notably, ectonucleotide pyrophosphatase/phosphodiesterase family member (enpp- ) can degrade extracellular cgamp ( ) (figure b ). besides triggering sting, these exogenous cgamp can directly bind to cgas and prompt its activation as well ( ) . after binding to cgamp, the "lid" region of the sting dimer undergoes a conformational change that converts sting from an inactive "open" formation to an active "closed" formation. following that, the sting dimer translocates from the er to perinuclear er-golgi intermediate compartment (ergic) vesicles, finally arriving at the golgi to form punctuate structures with downstream molecules ( , ) . er-retention of sting caused by mutations results in reduced ifn signaling ( , ) . the translocon-associated protein β (trapβ) recruited by inactive rhomboid protein (irhom ) initially forms the trap translocon complex that mediates sting exit from the er ( , ). they both assist cytoplasmic coat protein complex-ii (copii) to drive er-vesicle formation and carry the sting complex to the golgi ( , ) . trafficking sting can bind directly to and be phosphorylated by tank binding kinase (tbk ) dimer or iκb kinase (ikk) complex ( , , ) . the c-terminal tail (ctt) of sting is a linear unfolded segment, which determines the optimization of combination specificity. sting ctt in mammals tends to bind tbk , whereas in fish it tends to activate nuclear factor-kappab (nf-κb) signal ( ) . the sting phosphorylation site ser in the ctt cannot reach the kinase-domain active site of its directly bound tbk , instead can reach the kinase-domain active site of the next adjacent tbk binding with another sting and be phosphorylated, while tbk phosphorylate each other ( , ) . hence, sting and tbk can aggregate on the golgi to form the sting signalosome. clustering sting undergoes palmitoylation and full activation ( ) . it is also possible for sting-ikk to cluster and form the sting signalosome in this manner. the sting-tbk /ikk signalosome produces a scaffold to phosphorylate interferon regulatory factor (irf ) or inhibitor of nf-κbα (iκbα). activated irf undergo dimerization ( ) . the activation of iκbα leads to its polyubiquitination and degradation by the proteosome, thereby eliminating its inhibition of nf-κb. there is also evidence suggesting that nf-κb activation might not require sting trafficking from the er ( ) . then, the dimerized irf or activated nf-κb p /p (p is also known as rela) complex are translocated into the nucleus as transcription factors and bind to the promoter of type-i ifn to aid the transcription of type-i ifn ( , , ) . meanwhile, activation of nf-κb p /relb can prevent recruitment of p and inhibit the p /p signal ( ) (figure c ). expressed type-i ifn can propagate among cells in paracrine or autocrine manners. the binding of ifnα/β with its receptor triggers janus kinase (jak) and signal transducer and activator of transcription (stat) pathways, then induce transcription of type-i ifn-stimulated genes (isgs), which have ifn-sensitive response elements (isres) in their -untranslated regions (utrs) ( ) . irf can also bind partially to several isres alone ( ) . herein, the expression of some isgs including interferon-induced protein with tetratricopeptide repeats (ifit) and pro-inflammatory cytokines such as tumor necrosis factor α (tnfα), interleukin (il)- , c-x-c motif chemokine ligand (cxcl ) and c-c motif chemokine ligand (ccl ) is increased remarkably by the cgas-sting pathway ( ) . furthermore, cgas and sting are both isgs, suggesting a positive feedback loop in spreading of the ifn signal ( , ) . stimulator of interferon genes activation on the er also triggers an er stress response with an "unfolded protein response (upr) motif " on the c-terminus of sting, which leads to and er stress-mediated autophagy ( , ) . sting-tbk activation and er stress also induce mechanistic target of rapamycin complex (mtorc ) dysfunction ( ) . er stress or reduced mtorc signaling activates unc- -likeautophagy activating kinase (ulk ) complex and the beclin- -class iii phosphatylinositol -kinase (pi kc also known as vps ) complex, which promotes initiation of the classical autophagy path ( ) . cgas can also interact directly with the autophagy protein beclin- -pi kc complex and trigger autophagy ( ) . furthermore, cgas-dsdna polymer can form a liquid-phase condensate (as mentioned above), which could theoretically be an initiator of autophagy ( ) . after autophagy initiation, autophagy-related protein (atg ) undertakes the genesis of the autophagosome along with light chain (lc ) undergoing lipidation, thereby resulting in elongation of the autophagosome ( ) . lc can also be recruited directly by ergic-loading sting and bypass the classical autophagy pathway ( , ) . cgas-sting-mediated autophagy can spread to the whole cell and help the elimination of intracellular microorganisms, subcellular organelles or misfolded proteins, as well as the er itself that loads the sting signalosome ( - ) ( figure d ). cgas-sting-mediated autophagy is also indispensable for removing cytosolic dna and inflammatory signaling factors to restrict the inflammatory response raised by the pathway itself ( ) . excessive signaling of the autophagy cascade can lead to irreversible apoptosis termed "autophagic cell death" ( ) . consequently, oligomerized cgas or sting undergoes ubiquitination and is packaged into autophagosomes with the help of p , to be terminally sorted into lysosomes ( , , , ) . cgas or sting is digested immediately in the autophagolysosome after transient activation of downstream signaling ( , , , ) . autophagy functions as a negative feedback loop which ensures transient cgas-sting signaling and avoids consistent over-reaction of the pathway. thus, impairment of autophagy may give rise to destructive inflammatory diseases ( ). we cataloged factors in the literature that could potentially up-or down-regulate expression of cgas/cgamp/sting in pre-translational or post-translational stages (tables , ) . the regulatory mechanisms of tbk , irf, and nf-κb in signaling pathways associated with expression of type-i ifn are outside the scope of this review. pyhin family member absent in melanoma (aim ) is a cytoplasmic dsdna sensor. it can recruit apoptosis-associated speck-like protein containing a card (asc) by its pyhin domain and form the aim inflammasome. the inflammasome activates caspase- , which activates il- and trigger pyroptosis ( ) . the aim pathway could counteract the cgas-sting pathway ( ) . first, cgas is a target for caspase- cleavage ( ) . second, gasdermin d activated by caspase- can lead to potassium ion (k + ) efflux which inhibits cgas ( ) . conversely, the cgas-sting pathway can trigger the aim inflammasome or nlr family pyrin domain containing (nlrp ) by several means, and the process lags behind canonical ifn signaling ( , ) . in this way, the inhibitory nucleic-acid sensor nlr family card domain containing (nlrc ) can counteract sting by binding and occupying it, but viral dna as a possible nlrc ligand can reverse its occupation of sting ( ) (figure a) . another pyhin family member, ifi , is a dna sensor located in the nucleus. ifi can bind to viral dna sequences or damaged chromatin dna and be translocated to the cytoplasm to recruit sting cooperatively with tnf receptor associated factor (traf ) and p ( , ) . several studies have shown that ifi (which can stimulate the phosphorylation and recruitment of sting and tbk ) is required for the full response of sting ( , ) ( figure b) . conversely, cgas can partially enter the nucleus and interact with ifi to promote its stability ( ) . therefore, it is inferred that during viral infection, ifi can facilitate recognition of decapsidated viral dna in the nucleus, while cgas in the cytoplasm engages with viral gene transcription products ( , ) . however, sting signaling can trigger ifi degradation by tripartite motif-containing (trim ) ubiquitination ( ) . tlr is also an important prr for multiple pamps ( ) . tir domain-containing adaptor-inducing ifnβ (trif) is downstream of several subtypes of tlrs (including tlr ). trif may be responsible for interacting with sting and helping the dimerization of sting ( ) . during viral infection, the tlr -myeloid differentiation primary response (myd )-irf / pathway is necessary for mouse monocytes recruitment to lymph nodes, whereas the sting pathway is necessary for local production of type-i ifn ( ) . however, sting signaling can induce suppressor of cytokine signaling (socs ) expression, which can negatively regulate myd activity ( ) (figure c ). oxidized mtdna can be released into the cytoplasm during cell stress elicited by hypoxia, viral infection and mitochondrial damage, etc.; oxidized mtdna is resistant to degradation by the cytosolic nuclease trex ( ) . in addition, mtdna accompanied with tfam (a mtdna-binding protein that can bend mtdna) is also a reasonable target for recognition by cgas ( , ) . however, during regulated cell death (as represented by apoptosis), it undergoes mtdna release but has certain mechanisms to ensure a minimal cgas-stingmediated immune response. mitochondrial outer membrane permeabilization (momp) activation, which is executed by bcl- -associated x protein (bax) and bcl- antagonist or killer (bak), is a highly controlled conserved process in regulated cell figure | interaction of the cgas-sting pathway with other dna-sensing pathways and its role in cell survival. (a) absent in melanoma (aim ) pathway and pyroptosis and necroptosis. aim can be triggered by cgamp and form an inflammasome, consequently triggering interleukin (il)- production and pyroptosis. stimulator of interferon genes (sting) trafficking to the lysosome ruptures the lysosome membrane, resulting in k + efflux and activation of the nlrp inflammasome, leading to pyroptosis. cyclic-gmp-amp synthase (cgas) and interferon regulatory factor (irf ) can be a target for caspase- cleaving. gasdermin d can lead to k + efflux and inhibition of cgas. (b) interferon gamma inducible protein (ifi ). ifi can be transported to the cytoplasm to help to recruit sting and tank binding kinase (tbk ). ifi as a pyhin family protein may form the inflammasome only in theory. (c) toll-like receptor (tlr) pathway. tir domain-containing adaptor-inducing ifnβ (trif) may be responsible for helping the dimerization of sting. sting signaling can induce suppressor of cytokine signaling (socs ) expression, which negatively regulates myd activity. (d) apoptosis. mitochondrial outer membrane permeabilization (momp) formed by bax/bak induced by mitochondrial stress can release oxidized mitochondrial dna (mtdna) and cytochrome c into the cytosol. oxidized mtdna is a suitable ligand for cgas recognition and is resistant to dnaseiii (trex ) degradation. cytochrome c binds to apoptotic protease-activating factor (apaf ) and initiates the formation of an apoptosome cooperatively with caspase- to activate caspase- , which can induce apoptosis. caspase- can cleave cgas. death. bak and bax activated by apoptosis signals cooperatively form a pore-like conformation on the mitochondrial outer membrane, leading to a permeability change of outer and also inner membranes ( , ) . consequently, the mitochondrial matrix, including cytochrome c and oxidized mtdna-tfam, is released into the cytoplasm ( , ) . cytochrome c binds to apoptotic protease-activating factor (apaf ) and initiates the formation of the apoptosome cooperatively with caspase- , which further triggers the intrinsic apoptosis program ( ) . in vivo and in vitro studies have shown that an absence of caspase- is associated with greater release of type-i ifn ( , ) . this occurs because caspase- and its downstream caspase- can cleave cgas and irf to restrain deleterious inflammation ( ) (figure d) . the cgas-sting pathway can also initiate programmed cell death. activation of sting enhances phosphorylation and activation of receptor interacting serine/threonine kinase (rip ) and mixed lineage kinase domain-like pseudokinase (mlkl). proapoptotic bcl binding component (puma), a member of bh -only family, is subsequently activated in a rip /mlkl-dependent manner, which promotes leakage of mtdna by momp ( , ) . activated irf can bind directly to bax to form irf /bax complex and induce apoptosis ( ) . excessive cgas-sting-mediated autophagy signaling can cause "autophagic cell death" and prevent malignant transformation of cells through dna damage ( , ) . sting trafficking to the lysosome can broaden permeabilization of the lysosome membrane, thereby rupturing the lysosome and releasing its contents, resulting in "lysosomal cell death (lcd)". lcd further triggers k + efflux and nlrp activation, ultimately resulting in pyroptosis ( , ) (figure d) . moreover, stimulating sting-dependent type-i ifn and tnfα signals simultaneously can lead to necroptosis of tumor cells ( , ) . cell senescence is recognized as a permanent arrest of the cell cycle, and is common in aging, immunity, ontogenesis and infectious defense ( ) . it lacks a specific biomarker but can be identified by the expression of several antiproliferative molecules (representatively rb-p andp -p pathway) ( ) . during senescence, changes in the nuclear structure and loss of the nuclear lamina protein disrupt the integrity of the nuclear envelope, leading ultimately to dna damage and cytoplasmic chromatin fragments ( ) . cellular senescence can be accelerated by accumulation of cytoplasmic chromatin in turn ( ) . these senescent cells produce the senescence-associated secretory phenotype (sasp), which shapes an inflammatory microenvironment ( ) . the cgas-sting pathway has been reported to be involved in the recognition of cytoplasmic chromatin fragments from senescence-related dna damage, and mediate the expression of sasp genes ( ) ( ) ( ) ( ) . along with these actions, the expression of trex and dnaseii is inhibited by dna damage through the inhibition of e f/dp (a potential transcription factor of trex and dnaseii) ( ) . for hematopoietic stem cells (hscs), dna damage can promote excessive secretion of type-i ifn in the hsc niche and activate p pathway, both of which can lead to long-term senescence and exhaustion of hscs ( , ) . hscs expressing a circular rna named "cia-cgas" in the nucleus, however, are protected from this exhaustion as a result of cia-cgas having stronger affinity than that of self-dna, which prevents it from being sensed ( ) . it implied a novel target to manipulate the immune environment in bone marrow and help for finding treatment approaches for hematopoiesis-based diseases, such as aplastic anemia. utilizing cellular senescence to restrain tumor growth is discussed below. cgas-sting signaling has an essential role in defense against a broad spectrum of intracellular dna and rna viruses ( , , ) . hiv is a typical rna retrovirus: there is neither dsdna in its genome, nor production of nucleic acids ( ) . nevertheless, cgas can detect the presence of hiv. rna:dna hybrids synthesized during reverse transcription that can be sensed by cgas explain (at least in part) this phenomenon ( ) . cgas may be triggered by endogenous dna broken and released during hiv infection as well theoretically. however, some studies found the new mechanisms. the early reverse-transcription production of hiv- can flank short stem loops with paired base, which lead to the production of y-type dna containing unpaired guanosines that can activate cgas well ( ) . moreover, nucleolus protein non-pou domain-containing octamer-binding protein (nono), as a sensor of capsid components of hiv, can help cgas to be translocated to the nucleus and assist cgas to sense hiv dna accompanied by polyglutamine-binding protein (pqbp ) ( , ) . the assistance proffered by nono in assisting cgas to sense dna is also associated with its role in constructing a ribonuclear complex with dna-dependent protein kinase (dna-pk) subunits around hexamethylene bisacetamide-inducible protein (hexim ), termed as "hexim -dna-pk -paraspeckle components-ribonuclear protein complex (hdp-rnp), " which also has a role in repair of dna damage and transduction of genotoxic signals ( ) . this complex is also required to accompany cgas-pqbp in sensing dna virus, such as kaposi's sarcoma-associated herpes virus ( ) . in addition, during virus infection, sting activation can lead to global suppression of translation in cells, which restricts viral replication ( ) . compared with hiv- , hiv- is less infective because it can infect dendritic cells (dcs) and elicit an anti-virus immune response. as a result, hiv- can cross-protect against hiv- ( ). this phenomenon has been attributed to the fact that hiv- (instead of hiv- ) can encode protein vpx, which overcomes the samhd restriction of dntp in dcs ( , ) . hiv- can infect dcs via vpx presentation, nevertheless, hiv- still cannot be fully sensed and induce an efficient immune response owing to certain escape mechanisms. whether it is hiv- or hiv- , a completely robust ifn response is required at pre-and post-integration sensing stages ( ) . cgas in dcs can detect reverse-transcribed cdna of hiv- before and after integration, whereas hiv- sensing is after genome integration owing to its capsid protection ( , ) . it was suggested that during initial infection by hiv- , nucleotides are recruited into the intact capsid through the hexamer pores on the hiv- capsid. therefore, the capsid-coated hiv- virus prevents the encapsidated reverse-transcription production from being sensed by the cytosolic nucleic-acid sensors ( ) . hiv- capsids can be ubiquitinated and then degraded by the host e ubiquitin ligase function of trim , which leads to detection of viral dna, meanwhile hiv- could use some host protein like cyclophilins to evade the sensing ( , ) (figure a) . similarly, other viruses also have evasion mechanisms to escape cgas-sting pathway surveillance (table ) . therefore, identifying and preventing such viral-evasion factors could be a viable means to design novel anti-viral drugs. cgas-sting pathway is responsible to protect against intracellular or extracellular bacterial infection (especially hsv, herpes simplex virus; cmv, cytomegalovirus; irhom , inactive rhomboid protein ; trapβ, translocon-associated protein β; kshv, kaposi's sarcoma-associated herpes virus; hdp-rnp, hexim -dna-pk-paraspecklecomponents-ribonuclear protein complex; lana, latency-associated nuclear antigen; virf , viral interferon regulatory factor ; plpro, papain-like protease; lc , light chain ; mtor, mechanistic target of rapamycin; traf , tnf receptor associated factor ; hcv, hepatitis c virus; hpv, human papilloma virus; hbv, hepatitis b virus; hiv, human immunodeficiency virus; nf-κb, nuclear factor-κ b. intracellular infections). cdns (e.g., c-dgmp, c-damp, and cgamp) produced by bacteria are essential for the regulation of bacterial function, such as biofilm formation, colonization, and reproduction ( , ) . as ligands for sting, cdns can bind directly to and activate sting independently of cgas, which contributes to several immune responses from bacteria ( ) . usually, bacteria can enter or be engulfed by the cell through the endophagosome and be sequestered from the cytosolic sense receptor. some bacteria, such as mycobacterium tuberculosis (mtb), can survive in vacuoles, resulting in an insufficient cellular immune response to defend against it ( ) . in contrast, the esx- secretion system of the mycobacterium can translocate the phagosomal vacuolar matrix including bacterial genome molecules into the cytoplasm and trigger the cgas-sensing pathway ( ) . for other bacteria, such as legionella pneumophila or and brucella abortus, the host guanylate binding proteins (gbps) facilitate rupture of phagosome vacuoles and are indispensable for controlling their infection ( , ) . autophagy signaling mediated by cgas/sting is also involved in microorganism clearance mentioned above ( , ) . bacteria have evolved strategies to confront this pathway too. bacterial phosphodiesterase cdnp produced by mtb or group-b streptococci can degrade cdns ( , ) . cpsa (a type of mtb lytr-cpsa-psr domain-containing protein) can prevent autophagy responses for eliminating pathogens ( ) . chlamydia trachomatis inclusion membrane proteins can maintain the stability of the inclusion membrane and avoid inclusion lysis (leading to pathogen antigens leaking out and being detected by the host cell) ( , ) . yersinia outer protein j (yopj) deubiquitinates sting and impedes the formation of the sting signalosome ( ) . the cgas-sting pathway activation even impedes the elimination of listeria monocytogenes because bacterial dna can be packaged into evs and transferred into t cells, where it induces apoptosis of t cells ( , ) . several protozoans, such as toxoplasma gondii and malaria parasites, have an intracellular period in their lifecycle. t. gondii could engage cgas-sting exclusively ( ) . however, irf activation inducing isg expression promotes t. gondii development independently of ifn expression ( ) . p. falciparum can target erythrocytes, lacking a nucleus and unable produce ifn, but infected erythrocytes can secrete evs containing parasitic gdna to monocytes and trigger cgas ( ) . the immune system is regulated by a complicated network. disorder of immune signaling can elicit non-infectious inflammatory or autoimmune diseases. excessive, uncontrolled production of type-i ifn can lead to a spectrum of inflammation diseases termed "type-i interferonopathies, " which have some common manifestations ( ) . cgas-sting is the one of main sources of type-i ifn, acts as a cellular immune-sensing signaling axis, and is involved in type-i interferonopathies. stimulator of interferon genes -associated vasculopathy with onset in infancy (savi) is a typical sting-related hereditary inflammatory type-i interferonopathy, and is manifested by interstitial lung disease, dermatomyositis and arthritis. its pathology is featured by leukocytoclastic vasculitis and microthrombotic angiopathy of small dermal vessels ( , ) and patients can also suffer from lymphopenia ( ) ( ) ( ) ( ) . the etiology of savi is a gain of function (gof) mutant in sting which leads to constitutive sting activation without cdns stimulation ( ) . currently, several mutant amino acids residues have been found in or close to the dimerization domain (v m, n s, g e, v l, and v m) ( , , , ) , as well as r g, r s, r q, and c y in the cgamp-binding domain ( ) . other types of type-i interferonopathies, such as systemic lupus erythematosus (sle) and aicardi-goutières syndrome (ags), have relationships with defective clearance of cytosolic nucleic acids caused by congenital dysfunction of trex , rnaseh , and samhd . sle is a heterogeneous autoimmune disease which has prominent type-i and also -ii ifn signatures ( ) . ags comprises some systemic autoimmune syndromes overlapping with sle, and can be classified as a "lupuslike disease" ( ) . additionally, ags also causes severe developmental neurological disorders, including cerebral calcifications, encephalopathy and cerebral atrophy. systemic lupus erythematosus is a representative model for elucidating the mechanism of type-i interferonopathies. in sle, the level of self-dna which is packaged into apoptosis-derived membrane vesicles along with the level of anti-dsdna antibody is increased in the serum of patients ( ) . a study revealed increasing levels of isgs (including cgas/sting) as well as the cgamp-detected ratio in peripheral-blood mononuclear cells of sle patients ( ) . as innate immune cells, dcs have essential roles in antigen presentation, cytokine secretion, and priming the adaptive response of immune cells ( ) . plasmacytoid dcs (pdcs) can internalize and recognize self-dna and they are the main source of type-i ifn in serum during sle ( ) . ifnα/β is essential for complete function of immature pdcs ( ) . ifnα/β and il- can induce mitochondrial oxidative stress in dcs and decrease atp production, which blocks proton-pump function and increases ph of lysosomes. this process inhibits mitochondrial degradation and blocks mtdna clearance, which engages the cgas-sting pathway ( , ). moreover, monocytes may sense mtdna through cgas-sting and differentiate into dcs ( , ). neutrophil extracellular traps (nets) are complexes released by neutrophils exposed to stimuli or autoantibody immune complexes. nets comprise extracellularly released chromatin, myeloperoxidase enzymes, and also oxidized mtdna. in lupus-like diseases, nets can be induced by ifnα/β and may play a major part in priming pdcs ( , ) . all mechanisms stated above contribute to a more aggravated type-iifn response and exacerbate disease. a similar phenomenon can be observed in savi, ataxia telangiectasia (at) and artemis deficiency ( ) . however, compared with savi, dcs in sle can prime t-cell maturation significantly and increasing secretion of pro-inflammatory cytokines, such as il- and tnfα can also lead to activation of adaptive immunity ( figure a) . the cgas-sting pathway can mediate systemic inflammation as well as autoimmune activation. however, it is also involved in the local inflammation of multiple tissues. with regard to ischemic myocardial infarction (mi), cardiac macrophages can sense dying ruptured cells and lead to fatal post-mi cardiac inflammation, which is reversed by ablation of cgas/sting/irf ( , ) . in a non-alcoholic steatohepatitis (nash) model induced by a methionine-and choline-deficient or high-fat diet, lipotoxicity can cause mitochondrial damage and up-regulate sting/irf expression in hepatocytes, which in turn promotes lipid accumulation and inhibits glycogen synthesis. all above bring out hepatic inflammation and hepatocytes apoptosis ( ) . in this model, mice with deficiency of sting presents alleviated insulin resistance and lower levels of low-density lipoprotein in serum, and also decreased hepatic inflammation and fibrosis/steatosis, in which hepatic macrophages/kupffer cells may take a big part ( , ) . lipotoxicity can induce p to be phosphorylated through the cgas-sting-tbk pathway, which causes aggravated protein inclusions in hepatocytes and it indicates that p could be a biomarker for nash prognosis ( ) . mtdna-dependent inflammation induced by lipotoxicity also occurs in adipose tissue and endothelial cells of blood vessels, which contributes to tissue inflammation, insulin resistance, and cardiovascular diseases ( , ) . in traumatic brain injury (tbi), local injury initiates breakdown of the blood-brain barrier and global neuroinflammation ( ) . sting expression is up-regulated in tbi and can lead to increased expression of pro-inflammatory cytokines and enlargement of secondary injury ( ) . reduced autophagy-associated protein expression induced by sting may contribute to the dysfunction of autophagy and dampen the elimination of necrotic tissue, thereby intensifying inflammation ( ) . during silicosis, silica can yield cytosolic dsdna release and engage cgas-sting, which activates dcs and macrophages to cause severe lung inflammation. it also leads to death of epithelial cells through the nlrp pathway and pulmonary fibrosis ( ) . similarly, mtdna release in renal tubule cells has been found to be associated with acute kidney injury by cytotoxic drugs and chronic renal fibrosis ( , ) . neurodegenerative diseases are correlated with local inflammation ( ) . in the central nervous system (cns), microglia is considered to be the main source of cgas-sting-dependent ifn expression ( ) . in neurodegenerative diseases, levels of the marker of microglia activation-cluster of differentiation (cd ), and pro-inflammatory cytokines are increased ( ) . a significant feature of parkinson's disease (pd) is the neuronal loss of cerebral nuclei (especially dopaminergic neurons in the substantianigra). serine/threonine-protein kinase pink and e ubiquitin-protein ligase parkin are ubiquitinrelated factors that take part in removing damaged mitochondria by autophagy, and their dysfunction lead to the early onset of pd ( ) . parkin −/− and pink −/− mice, following exhaustive exercise, show inflammation and loss of dopaminergic neurons, which can be rescued by loss of sting ( ) . similarly, at is a genetic disease caused by missense mutation of a dna-repair protein: atm. at patients usually show neurodegenerative defects (especially ataxia) complicated with telangiectasia on their eyes or body, deficiency of adaptive immune cells, and predisposition to cancer ( ) . nevertheless, up-regulation of expression of type-i ifn can also be found in at patients and mice, causing them to be prone to autoimmune diseases ( , , ) . this syndrome is related to p -mediated senescence but also the chronic inflammation mediated by the cgas-sting pathway which engages cytosolic uncombined broken gdna caused by atm dysfunction ( ) . in addition, accumulation of cellular mtdna occurs in age-related macular degeneration characterized by retinal pigmented epithelium (rpe) degeneration. this can trigger chronic inflammation by cgas-sting pathway, in which nlrp inflammasomes, inflammatory/apoptotic caspases are also involved ( , ) . with regard to other diseases in which adaptive immune cells prime, cgas-sting has a different role. multiple sclerosis (ms) is a local inflammatory disease of cns. ms is characterized by over-reactive microglia, infiltration of self-reactive t cells, demyelization of nerve fibers and hyperplasia of gliocytes. autoantibodies against proteins expressed in immune-privileged regions of cns also contribute to its pathogenesis ( ) . in ms, ifnα/β can attenuate disease severity effectively. this implies a protective role for type-i ifn in cns, which is considered to counteract the pro-inflammatory ifnγ ( ) . using experimental autoimmune encephalitis (eae) as a ms model, sting was found to be indispensable for amelioration of type-i ifnmediated neuroinflammation, and it could be induced by a conventional anti-viral drug ganciclovir ( ) . ultraviolet (uv) radiation is a factor inversely related to the morbidity of ms ( ) . it was found that uv-b irradiation can recruit inflammatory monocytes and produce type-i ifn in a sting-dependent manner ( ) . all above indicate that cgas-sting-ifnα/β pathway may have a beneficial effect on some cns inflammatory diseases such as ms. a possible reason for the observed effect above is due to indoleamine , -dioxygenase (ido), which can catabolize tryptophan (trp) oxidatively. trp withdrawal or trpoxidative catabolites can interact with general control non-derepressible (gcn ) and mtor, of which both can control cellular aminoacid metabolism and suppress t helper (t h ) cells immunity ( ) . ifnα/β is a potent ido inducer to suppress proliferation of cd + t h cells and promote differentiation of foxp + regulatory t (t reg ) cells, which are believed to suppress cnsspecific autoimmunity ( , ) . in addition, dna released from dying cells can be internalized directly by t cells and sensed by cgas-sting pathway, which leads to enhancement of the t h transcription factor gata but suppression of the t h transcription factor t-bet. consequently, this process polarizes naive t cells toward t h differentiation ( ). studies mentioned above may (at least in part) explain why the cgas-sting signal is a negative regulator of ms. the inhibitory role of cgas-sting in inflammation is also attributed to its apoptosis-triggering role. in some subtypes of savi and mouse models, apoptosis of blood-vessel endothelial cells or bronchial epithelial cells and leucopenia can be observed (especially t-cell lymphopenia) ( , , ) . when the sting signal is stimulated, apoptosis occurs more frequently in normal or cancerous t cells ( ) . also, bone-marrow chimeras and gene-knockout studies have shown that t cells defect in savi are not associated with type-i ifn signaling or cgas ( , ) . localization of sting at the golgi can cause delay of t-cell mitosis and reduced proliferation independently of irf and tbk ( ) . furthermore, a "upr motif " on the c-terminus of sting can cause er stress and upr, resulting in ca + overloading and t-cell death ( ) . a controversial view is that b cells express sting variously and may undergo apoptosis through this way ( , , ) . however, simultaneous signaling by sting and the b-cell antigen receptor can promote b-cell activation and antibody production independently of type-i ifn ( ) (figure b) . as for some diseases with inflammatory responses involved, the acute phase of pancreatitis causes dying acinar cells to produce free dsdna, which activates cgas-sting signaling in macrophages, and exacerbates inflammation severity ( ) . however, in the chronic phase of pancreatitis, cgas-sting activation decreases pancreatic inflammation, which may be mediated by limiting t h response ( ). for gut mucosal immunity, transient stimulation of sting could strengthen the function of antigen-presenting cells (apcs) and promote t h and t h immune responses against microbes ( ) . chronic sting signaling, however, elicits an il- response to control the inflammation and avoids inflammatory enterocolitis such as bowel disease ( ) . sting knockout mice present reduced numbers of goblet cells, a decreased ratio of commensal versus harmful bacteria and compromised t reg cells in the gut, making it prone to enterocolitis ( ) . chromosomal instability (cin) is an intrinsic feature of cancer, and results spontaneously from errors in chromosome segregation during the mitosis of cancer cells. cin can also be induced manually by radiotherapy or chemotherapy, which causes dsbs. it results in micronuclei formation outside the nucleus, of which rupture brings out irrepressible accumulation of cytosolic self-dna and engages cgas ( , , ). however, normal mitotic processes involve exposure of chromosomal dna to the cytoplasm, but this cannot initiate a substantial inflammatory reaction or apoptosis because nucleosomes can suppress dsdna-cgas binding in a competitive manner ( ). an appropriate immune response against tumors via a type-i ifn plays an indispensable part in limiting tumors and prolonging host survival ( ) . it was found sting-deficient mice are prone to developing several types of cancer and have poor survival under a tumor burden, whereas stimulation of sting can elicit robust immunity to tumors ( ) ( ) ( ) . a mechanism is many cancer cells expressing cgas can recognize cytosolic dna and produce cgamp to stimulate secretion of type-i ifn through sting ( , ) . excessive expression of trex in cancer cells, which can be induced by radiotherapy, attenuates this progression ( ) . cgas-sting can also promote senescence of cancer cells through the p -p pathway ( ) . cgas-sting-mediated autophagy contributes to autophagic cell death if mitotic crisis occurs to avoid transformation of cancer cells ( ) . melanoma cells can also transfer cgamp produced by them to proximal non-cancerous host cells through gap-junction channels and activate sting in these cells, which contributes to the recruitment of tumor-infiltrating immune cells such as natural killer (nk) cells ( , ) . expression of the nk cellspecific ligand nkg d retinoic acid early transcript (rae ) on cancer cells is highly up-regulated by sting once nk cells permeate into tumor tissue ( ) . the activation of sting in the endothelium within the tumor microenvironment (tme) could contribute to the remodeling of tumor vasculatures, and may have positive effects on tumor regression ( ) . dendritic cells are the main source of type-i ifn in several types of tmes and are dependent on sting signaling ( ) . more preferentially than macrophages, infiltrating dcs take up tumor-derived dna or cgamp from dying cell fragments by phagocytosis ( , , , , ) . moreover, cancer cells can package dna into exosomes and transfer dna to dcs ( ) . produced cgamp by cancer cells can also be transferred to dcs through forming gap junction ( ) . by activating cgas-sting signal in dcs, cd α + subtype dcs secret chemokines such as ccl and cxcl and crossprime infiltrating anti-tumor cd + t cells ( , , ( ) ( ) ( ) . in contrast, numbers of immune-suppressing cells such as t reg cells, myeloid-derived suppressor monocytes and m macrophages have been reported to be decreased ( , , ) . expression of il- /il- rα complex is up-regulated in myeloid cells with the help of sting/type-i ifn and promotes tumor regression ( ) . tumor cells can evade intrinsic cellular surveillance in different ways. in various cancer cell lines, cgas, sting, tbk , and irf are mutated frequently and their decreased expressions are also related to the high level of methylation ( ) . sting expression has been shown to be suppressed by the alternative lengthening of telomeres (alt) pathway, which is responsible for prolonging the telomere length and maintaining the proliferation of tumor cells ( ) . a hypoxic environment in tumor cells can lead to accumulation of lactic acid and is associated with the inhibition of tumor-conditional dcs and reduced expression of ifn signaling molecules ( ) . in breast cancer, functional up-regulation of expression of human epidermal growth factor receptor (her ), a ligand-independent receptor tyrosine kinase (rtk), can arrest the expression of rac-alpha serine/threonineprotein kinase (akt ) (a key factor in the mtor pathway), which is reported to inhibit the activation of cgas and tbk ( , ) . patients with lung adenocarcinoma have a low probability of survival if they have reduced expression of cgas ( ) . thus, expression of the cgas-sting and dna-damage marker histone γh ax in tumor cells could be considered as independent prognostic factors to predict therapy response and clinical outcome, and could be superior to that of traditional markers like immunogenic cell death and t cells number ( ) . however, some scholars have arrived at opposite conclusions. when dsbs occur in cancer cells, cgas can be relocated to the nucleus and obstruct the formation of the parp -timeless complex, thereby inhibiting homologous recombination repair and maintaining cin, which potentiates tumor evolution ( , ) . it has also been reported that cgas recognizing cin activates non-canonical nf-κb signaling and potentiates cellular metastasis programs ( ) . furthermore, sting −/− mice are resistant to skin carcinogenesis in a , -dimethylbenz(a)anthracene (dmba)-treatment model. it has been demonstrated that when dmba-induced nuclear dna leaks into the cytoplasm, sting can induce chronic inflammatory stimulation that contributes to cancer development ( ) . during brain metastasis, cgamp transferred to bystander cells (e.g., astrocytes) can also produce ifnα and tnfα in the tme but, in this context, it will support tumor development and chemoresistance ( ) . coordinating with myeloid cells penetrating into the tumor, myeloidderived suppressor cells can also be recruited through the c-c chemokine receptor type (ccr ) ( ) . another study found that microparticles yielded by tumor cells can turn macrophages into the m type through cgas-sting-tbk , contrary to previous findings ( ) . immune-system interactions with tumor cells are complicated. the effect of cgas-sting on cancer is dependent on the type of tumor, host immune state, activated cell types, therapeutic intervention, and the magnitude of cgas-sting activation. like inflammation generated by cgas-sting, a time-dependent inflammatory anti-tumor response mediated by cgas-sting may be present. temporary activation of cgas-sting in innate immune cells could enhance the anti-tumor effect, whereas sustained activation of cgas-sting might induce immune tolerance of the tumor. more investigations are necessary to ascertain the exact role of cgas-sting in oncology, and elucidate the specific advantages and adverse effects of targeting the cgas-sting pathway in cancer therapy ( figure ). considering the pivotal role of the cgas-sting pathway in infection, inflammation and cancer, positive modulation of the pathway signaling is a promising way to enhance the immune state and restrict microorganisms or heterogeneous cells, whereas negative modulation can control aberrant inflammation. radiotherapy or chemotherapy drugs such as cisplatin or cyclophosphamide can induce dsbs and micronuclei, then trigger the cgas-sting pathway to enhance tumor immunogenicity ( ) ( ) ( ) . in addition, parp inhibitors such as olaparib have promising effects on cancer cells lacking brca because of their cooperative dna-repair functions ( ) . although cgas activation is inhibited by nucleosomes, taxol can induce mitotic cell-cycle arrest and sustain divided chromatin in the cytosol to activate the cgas-sting pathway slowly, and accumulation of signaling could stimulate apoptosis of cancer cells ( ). inhibitors of topoisomerase or used conventionally as chemotherapy drugs trigger minor damage to dna and accumulation of cytosolic dna, which can engage cgas and enhance the anti-tumor or anti-infection responses of cells ( ) ( ) ( ) . cgas-sting is essential on anti-tumor immune checkpoints therapies. for example, blockade of cd -signal regulatory protein α (sirpα) signaling on dcs can activate nadph oxidase (nox ) and increase the ph in phagosomes along with incomplete degradation of mtdna, which can trigger cgas-sting ( ) . sting deficiency in mice abrogates the anti-tumor effect of cd blockade ( ) . a similar phenomenon also can be seen in anti-programmed cell death- (pd- ) therapy ( ) . there is greater infiltration of ifnγ + cells and cd + t cells and pd- /pd- ligand (pd-l ) expression in tme treated by sting-ligand derivatives ( ) . therefore, in several types of tumors, combined administration of a sting agonist and immune-checkpoint antibody could elicit a more curative outcome compared with one therapy alone ( , ) . viruses can infect cells lacking cgas-sting more effectively, and have higher oncolytic activity compared with virus therapy alone. hence, the use of oncolytic viruses such as talimogene laherparepvec is beneficial for treating tumors with low expression of cgas/sting. sting expression can be regarded as a prognostic measurement for such therapy ( ) . some artificial analog molecules of cdns, such as , dimethylxanthenone- -acetic acid (dmxaa) and -carboxymethyl- ( h) acridone (cma) can bind the cdn pocket of mouse-specific sting dimers and promote conformational transition of sting from inactive "open" to an active "closed" state ( , ) . dmxaa showed convincing efficacy in restraining tumors in mice ( ) . however, dmxaa is restricted in activating mouse-specific sting but not humanspecific sting, which could be an explanation for the failure of dmxaa in treating non-small-cell lung cancer patients in a phase-iii clinical trial (nct ) ( ) . nonetheless, with three substitutions (g i, q i, and s a), human sting can also be induced by dmxaa to undergo conformational transition ( ) . another new compound, amidobenzimidazole, has been found to be an agonist of sting without lid closure and has potential therapeutic value ( ) . cyclic dinucleotides and their derivates that can stimulate sting directly are candidate adjuvants to restrain tumors. intratumoral administration of c-damp, c-dgmp, or cgamp analogs alone or combined with other adjuvants or tumor antigens have shown anti-tumor effects ( , ) ; phase-i or ii clinical trials (nct , nct , and nct ) of dithio-(rp,rp)-c-damp (known as adu-s ) are ongoing ( ) . to avoid the degradation and ensure maximal phagocyte internalization of cdns, endosomolytic nanoparticles have been designed to package and deliver cdns. for example, ph-sensitive nanoparticles (e.g., stingnanoparticles) can release their contents if located in acidic endosomal environments ( ) . for treatment of type-i interferonopathies, lessons can be taken from the treatment of canonical autoimmune disease such as sle, but there are several differences. for example, it was found that corticosteroid pulse therapy, γ-immunoglobulins, disease-modifying anti-rheumatic drugs, anti-cd , and some immunosuppressants (e.g., methotrexate) have limited efficacy against savi ( , ) . jak inhibitors such as ruxolitinib, tofacitinib and baricitinib that reduce type-i ifn downstream signaling have shown therapeutic value against savi, but further verification of their efficacy is needed ( ) . moreover, novel immune therapies, such as anti-ifnα and anti-ifnar immunoglobin, are in clinical trials for sle. these could also be tested against savi in the future ( ) . pharmaceutically screening has revealed that some antimalaria drugs, such as suramin, have an inhibitory effect on cgas by blockade of interaction between dna and cgas ( ) . in addition, novel small molecules such as ru or g can occupy the enzymatic pocket of species-specific cgas to abrogate cgamp synthesis ( , ) . recently, a study found that aspirin can acetylate cgas at three lysine residues and block cgas activity ( ) . with regard to sting, the cyclopeptide astin c can block irf recruitment onto the sting signalosome ( ) . the molecule h- can block the palmitoylation of human-sting ( ) . all of these agents are potential candidates for alleviating type-i interferonopathies. the cgas-sting pathway is primarily responsible for the modulation of immune response in cells when facing cytosolic dsdna challenge. moreover, it is complicatedly cross-regulated by other cellular processes or cellular signaling networks. the exact fundamental mechanism of the pathway in cells and the effect on the whole organism in specific states is not completely clear and requires further investigation. in conclusion, the cgas-sting pathway has dichotomous roles in the immune system. in general, cgas-sting-type-i ifn signaling can promote the innate immune response in myeloid cells but alleviate the adaptive immune response exerted by t cells and b cells. cgas shows high expression in apcs such as macrophages and dcs, but sting is expressed in most cells ( ) . cgas-sting signaling corresponding to cytoplasmic dsdna in apcs can boost innate and adaptive immunity transiently. in this situation, the dna challenge signal is limited to only macrophages and dcs. their pro-inflammatory and antigenpresenting functions to adaptive immune cells are promoted in the short-term. if the signal spreads to other bystander cells, such as t cells, b cells, local resident cells by means of cgamp transfer, or just aberrant sting activation by gof, it causes apoptosis in bystander cells or adaptive immune cells and immune tolerance in the long-term. therefore, it is reasonable to conclude that the intrinsic function of the cgas-sting pathway is essential for the innate immune system responses of the host immediately after pathogen invasion or abnormal cell appearing. once the challenge persists, the cgas-sting pathway controls the adaptive immune system to avoid chronic, detrimental inflammatory reactions or autoimmune diseases. the inflammatory response exists universally in almost all physiologic and pathologic progressions. cgas-sting is pivotal in modulating cellular inflammation, so it is promising to extend our conception of the cgas-sting pathway onto more diseases with inflammatory responses involved, especially cns-based diseases such as stroke, in which the inflammatory reaction exists but was recognized less. moreover, for targeting the cgas-sting pathway for therapeutic purposes, drugs should be optimized to augment the desirable effect and prevent its unwanted effects. for example, to eliminate tumor cells or infectious agents, agonists of cgas-sting would be a rational option if designed to target apcs exclusively but not t cells or b cells. in this scheme, the antitumor immune response is enhanced while avoiding apoptosis of adaptive immune cells and infiltration of immune suppressor cells. also, most research on the cgas-sting pathway has focused on its ifn-expressing role but overlooked autophagy and cell-death roles, which are also main downstream signaling of the pathway. therefore, some drugs, such as emricasan, are potential apoptosis inhibitors that may have a complementary effect on ameliorating apoptosis of blood-vessel endothelial cells or bronchial epithelial cells, and lymphopenia, in savi. until now, studies of the cgas-sting pathway have been done mainly in the laboratory but it has large space to be explored in clinical or translational fields. additionally more prrs and cellular immune-surveillance pathways may remain to be discovered to piece together the molecular puzzles of the cell. dw drafted the manuscript and drew the figures. wj and jh conceived and revised the review. frontiers in immunology | www.frontiersin.org recognition of endogenous nucleic acids by the innate immune system sting is an endoplasmic reticulum adaptor that facilitates innate immune signalling the adaptor protein mita links virus-sensing receptors to irf transcription factor activation an endoplasmic reticulum ifn stimulator, activates innate immune signaling through dimerization c-di-amp secreted by intracellular listeria monocytogenes activates a host type i interferon response sting is a direct innate immune sensor of cyclic di-gmp ifi is an innate immune sensor for intracellular dna cyclic gmp-amp synthase is a cytosolic dna sensor that activates the type i interferon pathway pivotal roles of cgas-cgamp signaling in antiviral defense and immune adjuvant effects an expression atlas of human primary cells: inference of gene function from coexpression networks bacterial cgas-like enzymes synthesize diverse nucleotide signals structural mechanism of cytosolic dna sensing by cgas structure of human cgas reveals a conserved family of second-messenger enzymes in innate immunity cytosolic rna:dna hybrids activate the cgas-sting axis sequence-specific activation of the dna sensor cgas by y-form dna structures as found in primary hiv- cdna cgas produces a - -linked cyclic dinucleotide second messenger that activates sting the catalytic mechanism of cyclic gmp-amp synthase (cgas) and implications for innate immunity and inhibition the cytosolic dna sensor cgas forms an oligomeric complex with dna and undergoes switch-like conformational changes in the activation loop cyclic gmp-amp synthase is activated by double-stranded dna-induced oligomerization structure of the human cgas-dna complex reveals enhanced control of immune surveillance cgas senses long and hmgb/tfam-bound u-turn dna by forming protein-dna ladders dna-induced liquid phase condensation of cgas activates innate immune signaling human cgas catalytic domain has an additional dna-binding interface that apoptotic caspases suppress mtdna-induced sting-mediated type i ifn production obsessive-compulsive disorder is associated with broad impairments in executive function: a meta-analysis ribonuclease h mutations induce a cgas/sting-dependent innate immune response restriction by samhd limits cgas/sting-dependent innate and adaptive immune responses to hiv- host restriction factor samhd limits human t cell leukemia virus type infection of monocytes via sting-mediated apoptosis samhd acts at stalled replication forks to prevent interferon induction cyclic gmp-amp containing mixed phosphodiester linkages is an endogenous high-affinity ligand for sting cyclic gmp-amp synthase is an innate immune sensor of hiv and other retroviruses cell intrinsic immunity spreads to bystander cells via the intercellular transfer of cgamp connexin-dependent transfer of cgamp to phagocytes modulates antiviral responses cancer-cell-intrinsic cgas expression mediates tumor immunogenicity viruses transfer the antiviral second messenger cgamp between cells transmission of innate immune signaling by packaging of cgamp in viral particles cgas-mediated innate immunity spreads intercellularly through hiv- envinduced membrane fusion sites priming of dendritic cells by dna-containing extracellular vesicles from activated t cells through antigen-driven contacts cells infected with herpes simplex virus export to uninfected cells exosomes containing sting, viral mrnas, and micrornas slc a is an importer of the immunotransmitter cgamp slc a transports immunoreactive cyclic dinucleotides hydrolysis of -cgamp by enpp and design of nonhydrolyzable analogs cgas facilitates sensing of extracellular cyclic dinucleotides to activate innate immunity sting regulates intracellular dna-mediated, type i interferon-dependent innate immunity activation by translocation from the er is associated with infection and autoinflammatory disease atg a controls dsdna-driven dynamic translocation of sting and the innate immune response irhom is essential for innate immunity to dna viruses by mediating trafficking and stability of the adaptor sting tmed potentiates cellular ifn responses to dna viruses by reinforcing mita dimerization and facilitating its trafficking autophagy induction via sting trafficking is a primordial function of the cgas pathway phosphorylation of innate immune adaptor proteins mavs, sting, and trif induces irf activation sting specifies irf phosphorylation by tbk in the cytosolic dna signaling pathway modular architecture of the sting c-terminal tail allows interferon and nf-kappab signaling adaptation structural basis of sting binding with and phosphorylation by tbk a conserved plplrt/sd motif of sting mediates the recruitment and activation of tbk activation of sting requires palmitoylation at the golgi ubiquitination of sting at lysine controls irf activation non-canonical nf-kappab antagonizes sting sensor-mediated dna sensing in radiotherapy immunomodulatory functions of type i interferons dna-binding landscape of irf , irf and irf dimers: implications for dimer-specific gene regulation attenuation of cgas-sting signaling is mediated by a p /sqstm -dependent autophagy pathway activated by tbk positive feedback regulation of type i ifn production by the ifn-inducible dna sensor cgas positive feedback regulation of type i interferon by the interferon-stimulated gene sting sting-mediated disruption of calcium homeostasis chronically activates er stress and primes t cell death sting senses microbial viability to orchestrate stress-mediated autophagy of the endoplasmic reticulum chronic innate immune activation of tbk suppresses mtorc activity and dysregulates cellular metabolism mtor signaling in growth, metabolism, and disease crosstalk between the cgas dna sensor and beclin- autophagy protein shapes innate antimicrobial immune responses a brief history of autophagy from cell biology to physiology and disease autophagy in the renewal, differentiation and homeostasis of immune cells sting directly activates autophagy to tune the innate immune response mycobacterium tuberculosis is protected from nadph oxidase and lc -associated phagocytosis by the lcp protein cpsa extracellular m. tuberculosis dna targets bacteria for autophagy by activating the host dna-sensing pathway upr, autophagy, and mitochondria crosstalk underlies the er stress response dnase a deficiency uncovers lysosomal clearance of damaged nuclear dna via autophagy self-consumption: the interplay of autophagy and apoptosis trafficking-mediated sting degradation requires sorting to acidified endolysosomes and can be targeted to enhance anti-tumor response the dna inflammasome in human myeloid cells is initiated by a sting-cell death program upstream of nlrp aim recognizes cytosolic dsdna and forms a caspase- -activating inflammasome with asc antagonism of the sting pathway via activation of the aim inflammasome by intracellular dna inflammasome activation triggers caspase- -mediated cleavage of cgas to regulate responses to dna virus infection gasdermin d restrains type i interferon response to cytosolic dna by disrupting ionic homeostasis a noncanonical function of cgamp in inflammasome priming and activation viral dna binding to nlrc , an inhibitory nucleic acid sensor, unleashes sting, a cyclic dinucleotide receptor that activates type i interferon non-canonical activation of the dna sensing adaptor sting by atm and ifi mediates nf-kappab signaling after nuclear dna damage nuclear ifi induction of irf- signaling during herpesviral infection and degradation of ifi by the viral icp protein ifi is required for dna sensing in human macrophages by promoting production and function of cgamp ifi and cgas cooperate in the activation of sting during dna sensing in human keratinocytes cgas-mediated stabilization of ifi promotes innate signaling during herpes simplex virus infection viral dna sensors ifi and cyclic gmp-amp synthase possess distinct functions in regulating viral gene expression, immune defenses, and apoptotic responses during herpesvirus infection sting-mediated ifi degradation negatively controls type i interferon production assembly and localization of toll-like receptor signalling complexes sting requires the adaptor trif to trigger innate immune responses to microbial infection sequential activation of two pathogen-sensing pathways required for type i interferon expression and resistance to an acute dna virus infection cross-regulation of two type i interferon signaling pathways in plasmacytoid dendritic cells controls anti-malaria immunity and host mortality oxidative damage of dna confers resistance to cytosolic nuclease trex degradation and potentiates sting-dependent immune sensing mitochondria and cell death: outer membrane permeabilization and beyond mitochondrial inner membrane permeabilisation enables mtdna release during apoptosis apoptotic caspases prevent the induction of type i interferons by mitochondrial dna apoptotic caspases suppress type i interferon production via the cleavage of cgas, mavs, and irf signalling strength determines proapoptotic functions of sting puma amplifies necroptosis signaling by activating cytosolic dna sensors autophagic cell death restricts chromosomal instability during replicative crisis induction of necroptotic cell death by viral activation of the rig-i or sting pathway tnfalpha and radioresistant stromal cells are essential for therapeutic efficacy of cyclic dinucleotide sting agonists in nonimmunogenic tumors cellular senescence: aging p ink a induces an age-dependent decline in islet regenerative potential autophagy mediates degradation of nuclear lamina extranuclear dna accumulates in aged cells and contributes to senescence and inflammation senescenceassociated secretory phenotypes reveal cell-nonautonomous functions of oncogenic ras and the p tumor suppressor cytoplasmic chromatin triggers inflammation in senescence and cancer downregulation of cytoplasmic dnases is implicated in cytoplasmic dna accumulation and sasp in senescent cells innate immune sensing of cytosolic chromatin fragments through cgas promotes senescence cgas is essential for cellular senescence dna-damage-induced type i interferon promotes senescence and inhibits stem cell function bacterial c-di-gmp affects hematopoietic stem/progenitors and their niches through sting a circular rna protects dormant hematopoietic stem cells from dna sensor cgas-mediated exhaustion pqbp is a proximal sensor of the cgas-dependent innate response to hiv- nono detects the nuclear hiv capsid to promote cgas-mediated innate immune activation p induces formation of neat lncrna-containing paraspeckles that modulate replication stress response and chemosensitivity hexim and neat long non-coding rna form a multi-subunit complex that regulates dna-mediated innate immune response stingdependent translation inhibition restricts rna virus replication inhibition of hiv- disease progression by contemporaneous hiv- infection samhd is the dendritic-and myeloid-cell-specific hiv- restriction factor counteracted by vpx reshaping of the dendritic cell chromatin landscape and interferon pathways during hiv infection the capsids of hiv- and hiv- determine immune detection of the viral cdna by the innate sensor cgas in dendritic cells hiv- activation of innate immunity depends strongly on the intracellular level of trex and sensing of incomplete reverse transcription products hiv- uses dynamic capsid pores to import nucleotides and fuel encapsidated dna synthesis restriction of hiv- and other retroviruses by trim hiv- evades innate immune recognition through specific cofactor recruitment cyclic di-gmp: second messenger extraordinaire cyclic gmp-amp signalling protects bacteria against viral infection a bacterial cyclic dinucleotide activates the cytosolic surveillance pathway and mediates innate resistance to tuberculosis tuberculosis pathogenesis and immunity mycobacterium tuberculosis differentially activates cgas-and inflammasome-dependent intracellular immune responses through esx- brucella abortus triggers a cgas-independent sting pathway to induce host protection that involves guanylate-binding proteins and inflammasome activation beneficial effects of yogasanas and pranayama in limiting the cognitive decline in type diabetes group b streptococcus degrades cyclic-di-amp to modulate stingdependent type i interferon production inhibition of innate immune cytosolic surveillance by an m. tuberculosis phosphodiesterase absence of specific chlamydia trachomatis inclusion membrane proteins triggers premature inclusion membrane lysis and host cell death the chlamydia trachomatis inclusion membrane protein cpos counteracts sting-mediated cellular surveillance and suicide programs yersinia yopj negatively regulates irf -mediated antibacterial response through disruption of sting-mediated cytosolic dna signaling intracellular bacteria engage a sting-tbk -mvb b pathway to enable paracrine cgas-sting signalling sting-dependent type i ifn production inhibits cell-mediated immunity to listeria monocytogenes induction of interferon-stimulated genes by irf promotes replication of toxoplasma gondii malaria parasite dna-harbouring vesicles activate cytosolic immune sensors type i interferon in rheumatic diseases activated sting in a vascular and pulmonary syndrome inherited sting-activating mutation underlies a familial inflammatory syndrome with lupus-like manifestations stimulator of interferon genes-associated vasculopathy with onset in infancy: a mimic of childhood granulomatosis with polyangiitis severe combined immunodeficiency in stimulator of interferon genes (sting) v m/wild-type mice sting-associated vasculopathy develops independently of irf in mice disease-associated mutations identify a novel region in human sting necessary for the control of type i interferon signaling personalized immunomonitoring uncovers molecular networks that stratify lupus patients aicardi-goutieres syndrome and the type i interferonopathies apoptosisderived membrane vesicles drive the cgas-sting pathway and enhance type i ifn production in systemic lupus erythematosus expression of cyclic gmp-amp synthase in patients with systemic lupus erythematosus. arthritis rheumatol the role of dendritic cells in autoimmunity human plasmacytoid dentritic cells elicit a type i interferon response by sensing dna via the cgas-sting signaling pathway interferon priming is essential for human cd + cell-derived plasmacytoid dendritic cell maturation and function interleukin- beta induces mtdna release to activate innate immune signaling via cgas-sting induction of dendritic cell differentiation by ifn-alpha in systemic lupus erythematosus neutrophil extracellular traps enriched in oxidized mitochondrial dna are interferogenic and contribute to lupus-like disease netting neutrophils are major inducers of type i ifn production in pediatric systemic lupus erythematosus type i ifnrelated netosis in ataxia telangiectasia and artemis deficiency cytosolic dna sensing promotes macrophage transformation and governs myocardial ischemic injury irf and type i interferons fuel a fatal response to myocardial infarction activation of the sting-irf pathway promotes hepatocyte inflammation, apoptosis and induces metabolic disorders in nonalcoholic fatty liver disease expression of sting is increased in liver tissues from patients with nafld and promotes macrophage-mediated hepatic inflammation and fibrosis in mice sting-mediated inflammation in kupffer cells contributes to progression of nonalcoholic steatohepatitis lipotoxicity induces hepatic protein inclusions through tank binding kinase -mediated p /sequestosome phosphorylation sting-irf triggers endothelial inflammation in response to free fatty acid-induced mitochondrial damage in diet-induced obesity priming the inflammatory pump of the cns after traumatic brain injury stingmediated type-i interferons contribute to the neuroinflammatory process and detrimental effects following traumatic brain injury sting-dependent sensing of self-dna drives silica-induced lung inflammation mitochondrial damage and activation of the sting pathway lead to renal inflammation and fibrosis mitochondrial damage causes inflammation via cgas-sting signaling in acute kidney injury how neuroinflammation contributes to neurodegeneration sensing of hsv- by the cgas-sting pathway in microglia orchestrates antiviral defence in the cns chronic neurodegeneration induces type i interferon synthesis via sting, shaping microglial phenotype and accelerating disease progression the roles of pink , parkin, and mitochondrial fidelity in parkinson's disease parkin and pink mitigate sting-induced inflammation atm and the molecular pathogenesis of ataxia telangiectasia autoimmune phenomena in ataxia telangiectasia mitochondrial dna has a pro-inflammatory role in amd cgas drives noncanonical-inflammasome activation in age-related macular degeneration multiple sclerosis type i/ii interferon balance in the regulation of brain physiology and pathology activation of the sting-dependent type i interferon response reduces microglial reactivity and neuroinflammation modulation of the immune system by uv radiation: more than just the effects of vitamin d? ultraviolet b irradiation causes stimulator of interferon genes-dependent production of protective type i interferon in mouse skin by recruited inflammatory monocytes. arthritis rheumatol engineering dna nanoparticles as immunomodulatory reagents that activate regulatory t cells activation of the sting adaptor attenuates experimental autoimmune encephalitis nucleic acid sensing by t cells initiates th cell differentiation sting-associated lung disease in mice relies on t cells but not type i interferon hierarchy of clinical manifestations in savi n s and v m mouse models intrinsic antiproliferative activity of the innate sensor sting in t lymphocytes agonist-mediated activation of sting induces apoptosis in malignant b cells b cell-intrinsic sting signaling triggers cell activation, synergizes with b cell receptor signals, and promotes antibody responses sting signaling promotes inflammation in experimental acute pancreatitis habtezion a. sting signalling protects against chronic pancreatitis by modulating th response stingactivating adjuvants elicit a th immune response and protect against mycobacterium tuberculosis infection sting-dependent signaling underlies il- controlled inflammatory colitis the cytosolic sensor sting is required for intestinal homeostasis and control of inflammation the multifaceted role of chromosomal instability in cancer and its microenvironment immunogenic cell death in cancer and infectious disease sting contributes to antiglioma immunity via triggering type i ifn signals in the tumor microenvironment sting-dependent cytosolic dna sensing mediates innate immune recognition of immunogenic tumors suppression of sting associated with lkb loss in kras-driven lung cancer dna exonuclease trex regulates radiotherapy-induced tumour immunogenicity growing tumors induce a local sting dependent type i ifn response in dendritic cells tumorderived cgamp triggers a sting-mediated interferon response in nontumor cells to activate the nk cell response rae ligands for the nkg d receptor are regulated by sting-dependent dna sensor pathways in lymphoma sting activation reprograms tumor vasculatures and synergizes with vegfr blockade dendritic cells but not macrophages sense tumor mitochondrial dna for cross-priming through signal regulatory protein alpha signaling exosomes shuttle trex -sensitive ifn-stimulatory dsdna from irradiated cancer cells to dcs host type i ifn signals are required for antitumor cd + t cell responses through cd {alpha}+ dendritic cells sting-dependent cytosolic dna sensing promotes radiation-induced type i interferondependent antitumor immunity in immunogenic tumors targeting dna damage response promotes antitumor immunity through sting-mediated t-cell activation in small cell lung cancer intratumoral sting activation with t-cell checkpoint modulation generates systemic antitumor immunity sting signaling remodels the tumor microenvironment by antagonizing myeloid-derived suppressor cell expansion il- is a component of the inflammatory milieu in the tumor microenvironment promoting antitumor responses suppression of sting signaling through epigenetic silencing and missense mutation impedes dna damage mediated cytokine production extrachromosomal telomere repeat dna is linked to alt development via cgas-sting dna sensing pathway downregulation of membrane trafficking proteins and lactate conditioning determine loss of dendritic cell function in lung cancer her recruits akt to disrupt sting signalling and suppress antiviral defence and antitumour immunity akt kinase-mediated checkpoint of cgas dna sensing pathway dna damage predicts prognosis and treatment response in colorectal liver metastases superior to immunogenic cell death and t cells chromosomal instability drives metastasis through a cytosolic dna response inflammation-driven carcinogenesis is mediated through sting carcinomaastrocyte gap junctions promote brain metastasis by cgamp transfer host stingdependent mdsc mobilization drives extrinsic radiation resistance tumor cellderived microparticles polarize m tumor-associated macrophages for tumor progression parp inhibition enhances tumor cell-intrinsic immunity in ercc -deficient non-small cell lung cancer parp inhibitor efficacy depends on cd (+) t-cell recruitment via intratumoral sting pathway activation in brca-deficient models of triple-negative breast cancer radiotherapy combined with novel sting-targeting oligonucleotides results in regression of established tumors topoisomerase inhibition promotes cyclic gmp-amp synthasedependent antiviral responses topoisomerase ii inhibitors induce dna damage-dependent interferon responses circumventing ebola virus immune evasion cgas/sting axis mediates a topoisomerase ii inhibitor-induced tumor immunogenicity cd blockade triggers t cell-mediated destruction of immunogenic tumors sting activation of tumor endothelial cells initiates spontaneous and therapeutic antitumor immunity a sting agonist given with ox receptor and pd-l modulators primes immunity and reduces tumor growth in tolerized mice magnitude of therapeutic sting activation determines cd (+) t cellmediated anti-tumor immunity recurrent loss of sting signaling in melanoma correlates with susceptibility to viral oncolysis species-specific detection of the antiviral small-molecule compound cma by sting bindingpocket and lid-region substitutions render human sting sensitive to the species-specific drug dmxaa the sting agonist dmxaa triggers a cooperation between t lymphocytes and myeloid cells that leads to tumor regression randomized phase iii placebo-controlled trial of carboplatin and paclitaxel with or without the vascular disrupting agent vadimezan (asa ) in advanced non-small-cell lung cancer design of amidobenzimidazole sting receptor agonists with systemic activity sting ligand c-di-gmp improves cancer vaccination against metastatic breast cancer endosomolytic polymersomes increase the activity of cyclic dinucleotide sting agonists to enhance cancer immunotherapy efficacy of the janus kinase / inhibitor ruxolitinib in the treatment of vasculopathy associated with tmem -activating mutations in children suramin potently inhibits cgamp synthase, cgas, in thp cells to modulate ifnbeta levels small molecule inhibition of cgas reduces interferon expression in primary macrophages from autoimmune mice development of human cgas-specific small-molecule inhibitors for repression of dsdnatriggered interferon expression the cyclopeptide astin c specifically inhibits the innate immune cdn sensor sting targeting sting with covalent small-molecule inhibitors trim -mediated monoubiquitination of cgas for cytosolic dna sensing the ubiquitin ligase trim regulates innate immune responses to intracellular doublestranded dna the e ubiquitin ligase rnf facilitates the cgas-mediated innate immune response rnf temporally regulates virus-triggered type i interferon induction by two distinct mechanisms the e ubiquitin ligase amfr and insig bridge the activation of tbk kinase by modifying the adaptor sting trim inhibits cgas degradation mediated by selective autophagy receptor p to promote innate immune responses p inhibition provides anti-dna virus immunity by regulation of usp phosphorylation and sting activation usp recruits usp to promote innate antiviral response through deubiquitinating sting/mita the deubiquitinase cyld is a specific checkpoint of the sting antiviral signaling pathway sumoylation promotes the stability of the dna sensor cgas and the adaptor sting to regulate the kinetics of response to dna virus senp potentiates cgas activation by relieving sumo-mediated inhibition of cytosolic dna sensing g bp promotes dna binding and activation of cgas zcchc is a cosensor of cgas for dsdna recognition in innate immune response manganese increases the sensitivity of the cgas-sting pathway for double-stranded dna and is required for the host defense against dna viruses tmem is a binding partner and regulator of sting-mediated inflammatory signaling in macrophages the erassociated protein zdhhc is a positive regulator of dna virus-triggered, mita/sting-dependent innate immune signaling association of abnormal elevations in ifit with overactive cyclic gmp-amp synthase/stimulator of interferon genes signaling in human systemic lupus erythematosus monocytes s k-sting interaction regulates cytosolic dna-mediated activation of the transcription factor irf glycogen synthase kinase beta regulates irf transcription factor-mediated antiviral response via activation of the kinase tbk trim short isoform preferentially promotes dna and rna virus-induced production of type i interferon by recruiting gsk beta to tbk lsm a plays a critical role in antiviral immune responses by regulating mita level in a cell-specific manner trim promotes dna virus infections by inhibiting innate immune response trim alpha is a negative-feedback regulator of the intracellular dna and dna virustriggered response by targeting sting the ubiquitin ligase rnf regulates antiviral responses by mediating degradation of the adaptor protein mita usp negatively regulates antiviral responses by deubiquitinating sting oligoadenylatesynthetase-family protein oasl inhibits activity of the dna sensor cgas during dna virus infection to limit interferon production interactome and proteome dynamics uncover immune modulatory associations of the pathogen sensing factor cgas sensing of bacterial cyclic dinucleotides by the oxidoreductase recon promotes nf-kappab activation and shapes a proinflammatory antibacterial state the ca( +) sensor stim regulates the type i interferon response by retaining the signaling adaptor sting at the endoplasmic reticulum nitro-fatty acids are formed in response to virus infection and are potent inhibitors of sting palmitoylation and signaling nlrc , a member of the nlr family of proteins, is a negative regulator of innate immune signaling induced by the dna sensor sting ppm a regulates antiviral signaling by antagonizing tbk -mediated sting phosphorylation and aggregation ptpn / -mediated dephosphorylation of mita/sting promotes its s proteasomal degradation and attenuates innate antiviral response nlrx sequesters sting to negatively regulate the interferon response, thereby facilitating the replication of hiv- and dna viruses nlrx promotes immediate irf -directed antiviral responses by limiting dsrnaactivated translational inhibition mediated by pkr mitigating sox -potentiated immune escape of head and neck squamous cell carcinoma with a sting-inducing nanosatellite vaccine mediates hypoxia-induced immunosuppression by repressing cgas nrf negatively regulates sting indicating a link between antiviral sensing and metabolic reprogramming kdm histone demethylases repress immune response via suppression of sting herpes simplex virus abrogates the cgas/sting-mediated cytosolic dna-sensing pathway via its virion host shutoff protein, ul herpes simplex virus tegument protein vp abrogates cgas/sting-mediated antiviral innate immunity species-specific deamidation of cgas by herpes simplex virus ul protein facilitates viral replication evasion of the sting dna-sensing pathway by vp / of herpes simplex virus herpes simplex virus gamma . protein inhibits sting activation that restricts viral replication human cytomegalovirus protein ul inhibits dna sensing of cgas to mediate immune evasion type i interferon production by inactivating the dna sensor cgas without affecting sting essential role of hcmv deubiquitinase in promoting oncogenesis by targeting anti-viral innate immune signaling pathways human cytomegalovirus tegument protein ul inhibits sting-mediated signaling to evade antiviral immunity the herpesviral antagonist m reveals differential activation of sting-dependent irf and nf-kappab signaling and sting's dual role during mcmv infection human cytomegalovirus-encoded us targets mavs and sting signaling to evade type i interferon immune responses inhibition of cgas dna sensing by a herpesvirus virion protein cytoplasmic isoforms of kaposi sarcoma herpesvirus lana recruit and antagonize the innate immune dna sensor cgas modulation of the cgas-sting dna sensing pathway by gammaherpesviruses sars coronavirus papain-like protease inhibits the type i interferon signaling pathway through interaction with the sting-traf -tbk complex poxviruses evade cytosolic sensing through disruption of an mtorc -mtorc regulatory circuit viral and metazoan poxins are cgamp-specific nucleases that restrict cgas-sting signalling zika virus elicits inflammation to evade antiviral response by cleaving cgas via ns -caspase- axis denv inhibits type i ifn production in infected cells by cleaving human sting dengue virus ns b protein targets cgas for degradation and prevents mitochondrial dna sensing during infection dna tumor virus oncogenes antagonize the cgas-sting dna-sensing pathway influenza a virus targets a cgas-independent sting pathway that controls enveloped rna viruses hepatitis c virus ns b blocks the interaction of sting and tbk to evade host innate immunity hepatitis c virus ns b protein targets sting and abrogates rig-i-mediated type i interferon-dependent innate immunity hepatitis b virus polymerase disrupts k -linked ubiquitination of sting to block innate cytosolic dnasensing pathways hiv- /siv vpx targets a novel functional domain of sting to selectively inhibit cgas-stingmediated nf-kappab signalling we thank k. wood from barrow neurological institute for discussions and editing. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © wan, jiang and hao. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -u gt fh authors: teijaro, j.r. title: pleiotropic roles of type interferons in antiviral immune responses date: - - journal: adv immunol doi: . /bs.ai. . . sha: doc_id: cord_uid: u gt fh since isaac's and lindenmann's seminal experiments over years ago demonstrating a soluble factor generated from heat killed virus-stimulated chicken embryos could inhibit live influenza virus replication, the term interferon has been synonymous with inhibition of virus replication. while the antiviral properties of type interferon (ifn-i) are undeniable, recent studies have reported expanding and somewhat unexpected roles of ifn-i signaling during both acute and persistent viral infections. ifn-i signaling can promote morbidity and mortality through induction of aberrant inflammatory responses and recruitment of inflammatory innate immune cell populations during acute respiratory viral infections. during persistent viral infection, ifn-i signaling promotes containment of early viral replication/dissemination, however, also initiates and maintains immune suppression, lymphoid tissue disorganization, and cd t cell dysfunction through modulation of multiple immune cell populations. finally, new data are emerging illuminating how specific ifn-i species regulate immune pathology and suppression during acute and persistent viral infections, respectively. systematic characterization of the cellular populations that produce ifn-i, how the timing of ifn-i induction and intricacies of subtype specific ifn-i signaling promote pathology or immune suppression during acute and persistent viral infections should inform the development of treatments and modalities to control viral associated pathologies. many viruses harbor viral proteins with specific functions geared toward preventing ifn-i production and/or signaling, highlighting the evolutionary selective pressure exerted by ifn-i during viral replication (devasthanam, ) . the absence of ifn-i signaling during acute virus infection in vivo increases virus replication, dissemination, and lethality during multiple viral infections in animal models. global deletion of ifnar results in enhanced mortality during vesicular stomatitis virus (vsv), vaccinia virus (vv), west nile virus (wnv), and lymphocytic choriomeningitis virus (lcmv) infections (muller et al., ) . moreover, infection of ifnar ko mice with acute lcmv armstrong (arm) (nakayama et al., ; zhou, cerny, fitzgerald, kurt-jones, & finberg, ) and treatment of arm-infected mice with an ifnar neutralizing antibody elevated viral loads and promoted virus persistence (teijaro et al., ; wilson et al., ) . dendritic cell-specific deletion of ifnar results in elevated virus replication and systemic persistence of the cw strain of murine norovirus (mnov) despite increased cell-mediated and humoral adaptive immune responses (nice et al., ) . ifn-i signaling has been shown to be essential for controlling wnv infection and restricting viral pathogenesis (sheehan, lazear, diamond, & schreiber, ) . mice deficient in ifnar signaling display increased susceptibility to wnv infection (pinto et al., ; samuel & diamond, ) . during infection with the coronavirus, mouse hepatitis virus (mhv-a ), the magnitude of the ifn-i and -ii responses directly correlated with viral loads (raaben, koerkamp, rottier, & de haan, ). moreover, ifn-i produced by plasmacytoid dendritic cells (pdcs) was essential to control virus replication and prevent mortality following mhv-a infection in mice (cervantes-barragan et al., ) . during experimental infection of mice and nonhuman primates with the lassa hemorrhagic fever virus, delayed or reduced induction of ifn-i and downstream gene signatures correlated with high viral loads and fatal outcome (baize et al., ; yun et al., ) . deletion of ifn-i related signaling pathways during respiratory virus infections in animal models results in diverse effects depending on the virus strain and genetic background (durbin et al., ; price, gaszewska-mastarlarz, & moskophidis, ) . in the context of respiratory viral infection, genetic deletion of stat reduced virus control, enhanced pathology, and mortality during sars-cov and influenza virus infection (durbin et al., ; frieman et al., ) . interestingly, stat -deficient animals were highly susceptible to influenza virus infection, displaying elevated viral titers and increased pathology compared to stat -sufficient mice. studies in mouse models of influenza virus have revealed conflicting evidence for the role of ifnar in controlling influenza virus replication, morbidity, and mortality. infection of ifnar À/À mice with the pr strain of influenza virus resulted in altered recruitment of ly c hi vs ly c int monocytes in the lung, translating into increased production of the neutrophil chemoattractant, kc (cxcl ), elevated numbers of neutrophils in the lung and increased morbidity and mortality (seo et al., ) . therefore, modulation of type interferon signaling and production needs to be balanced to have enough to control virus infection but not promote excessive inflammation. the discrepancy between influenza pathogenicity in ifnar and stat -deficient mice was later clarified when animals lacking both ifnar /ifn-λ were unable to control influenza virus replication. this is further supported in humans where null mutations in the human interferon regulatory factor- gene results in reduced ifn-i and -iii production from myeloid dcs and pdcs and life-threatening seasonal influenza virus infection (ciancanelli et al., ) . exposure of bone marrow cells to ifn-i prior to their recruitment to lung endows these cells with an antiviral program that protects from virus infection after entry into the infected lung (hermesh, moltedo, moran, & lopez, ) . deletion of the ifn-β or ifnar genes in mice with a functional mx gene increased virus replication and reduced the ld -fold (koerner, kochs, kalinke, weiss, & staeheli, ) . infection of ifnar -deficient mice with low dose mouse adapted h n influenza viruses resulted in mortality, elevated viral loads, exacerbated lung pathology, and reduced numbers of il- -producing cells as compared to ifnar -sufficient controls (arimori et al., ) . moreover, exogenous administration of il- to ifnar -deficient animals following influenza virus infection partially restored survival and ameliorated lung pathology. thus, ifn-i can be protective during influenza virus infection either through suppressing virus spread or prompting induction of immune-suppressive cytokines to reign in excessive inflammation. in addition to directly inhibiting virus propagation, ifn-i also has potent immune stimulatory functions which support the resolution of virus infection. ifn-i promotes upregulation of mhc-i expression in multiple cell lineages (lindahl, gresser, leary, & tovey, a , b , which is required for optimal t cell stimulation, differentiation, expansion, and killing of virus-infected cells. autocrine signaling of ifn-i on dendritic cells promotes their activation and t cell stimulatory capacity (montoya et al., ) . ifn-i signaling during virus infection promotes conversion of pdcs into myeloid derived dcs and impairs hematopoietic differentiation of bone marrow progenitors into dcs (sevilla, mcgavern, teng, kunz, & oldstone, ; zuniga, mcgavern, pruneda-paz, teng, & oldstone, ) . following exposure to ifn-i, metallophilic macrophages induce expression of the usp protein which prevents jak phosphorylation and inhibits ifn-i signaling in these cells. in turn, repression of ifn-i signaling allows for restricted virus replication in these macrophages, promoting the production of viral antigens which are recognized by b cells, the final result is the facilitation of antiviral antibody generation and enhanced virus control (honke et al., ) . ifn-i also exerts potent costimulatory effects directly on cd t cells, enhancing cd t cell proliferation upon ifnar signaling (curtsinger, valenzuela, agarwal, lins, & mescher, ; kolumam, thomas, thompson, sprent, & murali-krishna, ) . the timing of cd t cell exposure to ifn-i significantly influences the differentiation and magnitude of the response (welsh, bahl, marshall, & urban, ) . exposure of naïve cd t cells to apc and ifn-i prior to antigenic stimulation promotes the maintenance of a naïve phenotype with reduced proliferation despite production of effector cytokines. direct ifn-i signaling on naïve and memory t cells promotes rapid apoptosis, inhibits proliferation, and promotes early effector differentiation of memory cells upon exposure. blockade of ifn-i signaling during wnv infection has significant effects on t cell expansion, cytokine production, and differentiation when administered during the maturation phase of the t cell response, however, had no effect when given prior to infection (pinto et al., ) . moreover, low dose priming with the vv ankara strain had little effect on effector or memory t cell recall in ifnar À/À mice (volz, langenmayer, jany, kalinke, & sutter, ) . in addition to t cells, ifn-i signaling is known to be important for nk cell function. ifn-i signaling promotes nk cell cytolytic capacity and survival during acute viral infection (hwang et al., ; martinez, huang, & yang, ; nguyen et al., ) and was recently reported to protect antiviral cd t cells from nk cell lytic effects (crouse et al., ; xu et al., ) . reconstitution of ifnar À/À mice with ifnar +/+ nk cells restored early control of vv infection in vivo (martinez et al., ) , suggesting that nk cell intrinsic ifnar signaling is important for early control of vv replication. moreover, direct ifn-i signaling on nk cells was required to induce nk cell ifn-γ production during acute lcmv infection. early ifn-γr signaling was required for promoting initial virus control in the peritoneum (mack, kallal, demers, & biron, ) , suggesting that ifn-i signaling directly on nk cells promotes virus control during acute lcmv infection. ifn-i signaling during viral infection can also signal to regulatory t cells and subsequently alter their suppressive functions. it was recently demonstrated that ifnar signaling on foxp + tregs limits their suppressive function during acute lcmv infection, thus promoting virus control (srivastava, koch, pepper, & campbell, ) . deletion of ifnar on foxp + cells blunted virus-specific t cell responses and elevated virus loads. thus, ifn-i signaling on suppressive t cell populations temporarily suspends suppressive function and allows for optimal antiviral t cell responses during an ongoing viral infection. similar to effects on t cells, ifn-i signaling has both positive (le bon et al., ) and negative effects on antiviral b cell responses. the survival and maturation of immature b cells can be inhibited by ifn-i signaling (lin, dong, & cooper, ) . in contrast to immature b cells, ifn-i signaling promotes b cell activation, antibody production, and isotype switch following influenza, vsv, and wnv infection (coro, chang, & baumgarth, ; fink et al., ; purtha, chachu, virgin, & diamond, ; rau, dieter, luo, priest, & baumgarth, ). however, it was also reported that influenza virus-specific antibody levels were elevated at later time points following influenza virus challenge in ifnar -deficient mice compared to ifnar -sufficient controls (price et al., ) . during acute lcmv infection, blockade of ifn-i signaling in both wild-type and stat -deficient mice enhanced t follicular helper cell (t fh ), germinal center b cell differentiation, and anti-lcmv antibody responses (ray et al., ) . elevated antibody responses during acute viral infections following ifnar blockade suggest that, in certain circumstances, ifn-i signaling can restrain optimal antiviral antibody responses. the correlation of an aggressive immune response and severe disease following influenza virus infection in humans and animal models has been discussed previously (la gruta, kedzierska, stambas, & doherty, ). an aggressive innate response, with elevated recruitment of inflammatory leukocytes to lung, likely contributed to the morbidity of the influenza infection (ahmed, oldstone, & palese, ; kobasa et al., ) . in fact, lung injury during infection of macaques with the h n influenza virus strain directly correlated with early dysregulated inflammatory gene expression, including elevated ifn-i signatures (cilloniz et al., ; kobasa et al., ) . more recently, clinical studies on avian h n -infected humans documented a significant association between excessive early cytokine responses and immune cell recruitment as predictive of poor outcome (de jong et al., ). an aberrant cytokine/chemokine response was observed in patients with severe disease during the most recent h n pandemic in (arankalle et al., ) . type i interferon signaling is well known to inhibit influenza virus replication and spread (garcia-sastre & biron, ) . the production of the ns protein, one of viral proteins, acts to inhibit type interferon production and signaling (hale, randall, ortin, & jackson, ) , suggesting that ifn-i signaling exerts substantial selection pressure on virus fitness. deletion or mutation of the ns gene results in significant increases in the levels of type interferon in infected cells and significantly lower virus titers both in vitro and in vivo (garcia-sastre, egorov, et al., ; jiao et al., ; kochs, garcia-sastre, & martinez-sobrido, ) . despite strong evidence demonstrating extensive antiviral properties of ifn-i, several studies also suggest pathogenic roles for ifn-α during influenza virus infection. the production of several proinflammatory cytokines and chemokines is known to be amplified by ifn-i receptor signaling. in addition to protective effects of ifn-i signaling, pathogenic roles for ifn-i have been reported during influenza virus infection ( fig. a) . appearance of ifn-α in lavage fluid directly coincides with symptom onset during human experimental influenza virus infection (hayden et al., ) , suggesting that ifn-i signaling and pathological responses in humans temporally coincide. recently, it was paradoxically reported that deletion of ifnar or depletion of pdcs in svev mice inhibited pulmonary pathology and improved survival following lethal influenza virus challenge (davidson, crotta, mccabe, & wack, ) . reduced immune pathology and enhanced survival in mice deficient in ifn-i signaling transpired without significant increases in viral loads or impediment of eventual viral clearance (fig. b) . in contrast to deletion of ifn-i signaling, treatment of influenza virus-infected mice with ifnα resulted in enhanced morbidity and mortality; thus, ifn-i can promote pathological consequences during acute influenza virus infection. over the past years, we identified that therapeutic administration of sphingosine phosphate (s p) analogs early during influenza virus infection in mice resulted in reduced morbidity and mortality . s p is a lipid metabolite converted from ceramide precursors to sphingosine. fig. ifn-i signaling enhances cytokine/chemokine amplification, innate immune cell recruitment, and immune pathology during respiratory viral infections. (a) viral infection in the lung with influenza or sars-cov promotes the induction of delayed ifn-i production which enhances cytokine/chemokine production, recruitment of nk cells, and neutrophils and inflammatory macrophage/monocytes all which contribute to lung immune-mediated pathology. (b) blockade or genetic deletion of ifnar blunts cytokine/chemokine amplification, inhibits recruitment of nk cells, neutrophils, and inflammatory macrophages/monocytes resulting in reduced immunopathology, and improved survival. treatment of mice with s p r agonists early during influenza virus infection suppresses ifn-i amplification from plasmacytoid dendritic cells which lowers ifn-i levels. the end result is blunting of cytokine/chemokine amplification, inhibition of nk cell, neutrophil, and inflammatory macrophage/monocyte recruitment into the lung, reduced immunopathology, and improved survival. the subsequent phosphorylation by sphingosine kinase and produces bioactive s p in vivo where it acts on s p-specific g-protein couples receptors (gpcrs) (chalfant & spiegel, ) . the levels of bioactive s p are regulated through the actions of s p phosphatases and lyases which dephosphorylate and degrade s p, respectively. highest levels of s p are found in the blood and lymph with significantly lower levels maintained in peripheral tissues (cyster, ) . s p binds and signals through five gpcrs denoted as s pr - which couple to various g-protein signaling effectors. the expression of s p receptors is heterogeneous, being found on both hematopoietic and nonhematopoietic lineages (im, ) . the functional coupling to multiple heterotrimeric g-proteins promote the diverse cellular functions associated with s p receptor signaling. signaling through these five receptors is known to modulate multiple cellular processes including: cell adhesion, migration, survival, proliferation, endocytosis, barrier function, and cytokine production (rivera, proia, & olivera, ) . recently, we identified a novel regulatory function of s pr signaling in blunting early cytokine amplification and innate immune cell recruitment following influenza virus infection (fig. b) . early administration of a promiscuous s pr agonist, aal-r, or an s p r-selective agonist (cym- ) significantly blunted production of multiple pro-inflammatory cytokines and chemokines following infection with either wsn or human pandemic h n influenza virus walsh et al., ) . further, both aal-r-and cym- -mediated reduction of early innate immune cell recruitment and cytokine/chemokine production correlated directly with reduced lung pathology and improved survival during h n influenza virus infection. while these s pr agonists clearly inhibited innate immune responses, significant inhibition of activated t cell recruitment into the lung at various times post infection occurred in mouse adapted (marsolais et al., ) and human pathogenic strains of influenza virus . the above findings were extended using genetic and chemical tools to probe functions of the s p receptor (s p gfp knockin transgenic mice, s p receptor agonists and antagonists), revealing that pulmonary endothelial cells modulate innate immune cell recruitment and cytokine/chemokine responses early following influenza virus infection . importantly, s p r agonist treatment blunted cytokine/chemokine production and innate immune cell recruitment in the lung independently of endosomal and cytosolic innate sensing pathways (teijaro, walsh, rice, rosen, & oldstone, ) . further, s p r signaling suppression of cytokine amplification was independent of multiple innate signaling adaptor pathways but required the myd adaptor for cytokine amplification following influenza virus challenge. immune cell infiltration and cytokine production were found to be distinct events, both orchestrated by signaling through the s p r. suppression of early innate immune responses through s p r signaling also reduced mortality during infection with human pathogenic strains (h n / swine) of influenza virus in a ferret model, demonstrating that s pr -mediated blunting of influenza virus pathogenesis in mice could be extended to a model more closely resembling human disease. the link between s pr and ifn-α amplification following influenza virus infection was striking. in fact, the absence of ifnar abolished cytokine amplification and the capacity of s p r agonists to further blunt cytokine/chemokine responses (teijaro et al., . to understand how s pr signaling regulates ifn-α and cytokine amplification, we assessed the pulmonary cell subsets that produce ifn-α and cytokines/chemokines following influenza virus challenge. expression of s p r was quickly observed in purified pdcs; moreover, s p r agonists suppressed ifn-i induction/amplification from both mouse and human pdcs following influenza virus simulation (teijaro et al., ) . further mechanistic studies revealed that s p r agonist-mediated suppression was independent of gi/o signaling and required signaling through the s p r c-terminus. biochemically, s p r agonists accelerated the turnover of ifnar and promoted trafficking to lysosomes for degradation, abrogating stat phosphorylation, blunting the ifn-i autoamplification loop. the fact that ifn-i production/signaling can down modulate s pr expression/activity indirectly through upregulation of cd which promotes internalization of s pr in t cells is significant (shiow, ) and suggests that s p r and ifn-i signaling are closely linked and capable of counter regulating one another. an additional study also reported ifn-i modulation in pdcs via other s prs (dillmann et al., ) , suggesting that this phenomenon could be more promiscuous than originally thought. similar to influenza virus infection, aberrant innate cytokine/chemokine responses and immune cell recruitment into lungs correlate with disease severity in human patients (huang et al., ) . ifn-i signaling during murine sars-cov infection appears to be dispensable for virus control while also potentiating immune pathology. however, the role ifn-i signaling plays in this pathology has only recently been systematically addressed. deletion of ifnar in mice does not mirror the enhanced viral loads or pathological consequences observed in stat À/À mice in sars-cov infection, suggesting an ifnar -independent stat- -dependent pathway is necessary for controlling sars-cov (frieman et al., ) . this study provocatively suggests that ifn-i signaling is dispensable for controlling sars-cov replication in vivo. recently, an important study was published where the authors further highlighted the importance of ifn-i signaling in respiratory virus pathology by reporting that delayed ifn-i induction and signaling during sars-cov infection in mice promoted the development and infiltration of inflammatory monocyte-macrophages into the lung, resulting in exacerbated lung pathology and lethal pneumonia (channappanavar et al., ) . attenuation of ifn-i signaling either through genetic deletion or through antibody neutralization of ifnar prevented inflammatory monocyte-macrophage infiltration into the lung, abrogated lung immune pathology, and resulted in mild clinical disease. importantly, genetic deletion or blockade of ifn-i signaling resulted in control of viral loads similar to control animals, reinforcing that ifn-i signaling is dispensable for control of sars-cov infection in vivo. one possibility is that in the absence of ifn-i signaling, induction of an ifn-iii (ifn-λ) antiviral program may effectively limit viral replication. the results found in this study were strikingly similar to those found in influenza virus-infected svev mice and suggest that strategic modulation of ifn-i signaling could ameliorate pathologies associated with severe respiratory virus infection. collectively, the studies above suggest that ifn-i signaling is essential to cytokine and chemokine amplification and innate immune cell recruitment and can promote excessive immunopathology during acute respiratory viral infections (fig. ) . importantly, that ifn-i production and signaling can be blunted without enhancing virus propagation following acute respiratory viral infection suggests that this pathway can be modulated without compromising host antiviral responses. the correlation between blunting ifn-i signaling, lessened immune pathology, and improved survival during multiple respiratory viral infections highlight the need to mechanistically dissect how ifn-i promotes immune pathology during these infections. the role of ifn-i signaling in restraining chronic/persistent viral infection is well documented. inhibition of ifn-i signaling by antibody blockade of ifnar results in elevated virus replication early following lcmv cl infection and treatment of mice with ifn-i during the early stages of persistent lcmv infection promotes rapid virus control (wang et al., ) . mechanistically, ifn-i therapy increased expansion of virus-specific cd t cells and prevented t cell exhaustion; however, whether this was due to ifn-i-mediated immune stimulatory effects, lowering of antigen levels, or both was not systematically addressed. an additional study reported that deletion of the - oligoadenylate synthetase-like gene prior to lcmv cl infection facilitated sustained ifn-i production/signaling, promoted t cell expansion, reduced t cell exhaustion, and promoted rapid virus control (lee, park, jeong, kim, & ha, ) . similar to persistent lcmv infection, ifn-i administration can exert protective effects through slowing siv replication and disease progression if administered early following infection (sandler et al., ) and has shown some efficacy in patients with persistent hiv infection (asmuth et al., ; azzoni et al., ) . moreover, treatment with pegylated ifn-α in conjunction with the antiviral drug ribavirin was the standard of care for treating patients with chronic hepatitis c virus (hcv) infection until recently (heim, ; moreno-otero, ) . however, despite success in hcv therapy, the modest efficacy observed following ifn-α administration requires ribavirin and, even in combination, only a slim majority of patients respond. moreover, patients who fail to control hcv following ifn-i therapy were reported to express a higher ifn-i gene signature prior to treatment (sarasin-filipowicz et al., ) . similar trends were observed following ifn-i administration during hiv and siv infections, where ifn-i administration had only the modest effects if given during established persistent infection (asmuth et al., ; hubbard et al., ) . the reasons for the discrepancies observed in human persistent viral infections, where ifn-i therapy can promote control ( - % of hcv patients) while in others (during established hiv infection) minimal benefit is observed, remain unknown. one could imagine a scenario where in some persistently infected hcv patients, elevated ifn-i signatures persist, and addition of pegylated ifn-α provides minimal benefit while patients with lower ifn-i signatures respond to the therapy. whether treatment with pegylated ifn-α earlier during infection (prior to sustained ifn-i signatures) would be beneficial would be interesting to discern. a similar profile appears to exist in persistent siv infection, where early administration of ifn-i promotes control of viral loads and pathogenesis, while later administration has modest effects on viral titers and disease outcome. during infection with a model gamma herpesvirus, mhv , the lack of ifn-i signaling exacerbated virus replication, increased reactivation from latency, and resulted in enhanced morbidity and mortality (barton, lutzke, rochford, & virgin, ; dutia, allen, dyson, & nash, ) . taken together, ifn-i therapy may be beneficial during the early stages of persistent, latent chronic viral infection, or infections with lower ifn-i signatures; however, blocking ifn-i signaling either alone or in conjunction with antiviral or immune checkpoint therapies may prove more effective once virus persistence and elevated ifn-i signatures are established. however, the ultimate outcome will likely depend on the persistent virus studied, genetic susceptibilities of individuals, and subtype and timing of ifn-i species produced; all which require further investigation. moreover, given the undesirable side effects of ifn-i administration, ifn therapy can do as much harm as good during viral infection, highlighting the need for developing alternative approaches to treat persistent viral infections. during persistent viral infections, chronic immune activation, negative immune regulator expression, an elevated interferon signature, and lymphoid tissue destruction correlate with disease progression. elevated ifn-i signatures have been observed during lcmv infection in mice (hahm, trifilo, zuniga, & oldstone, ) and hiv and hcv infections in humans and nonhuman primates (bosinger et al., ; jacquelin et al., ; wieland et al., ) . chronic immune activation following hiv infection has been reported, and suppression of this hyperactivated state has been proposed as a potential strategy to alleviate hiv-associated pathologies (boasso, hardy, anderson, dolan, & shearer, ; d'ettorre, paiardini, ceccarelli, silvestri, & vullo, ) . disease following experimental siv infection in rhesus macaques correlates with elevated ifn-i production and inflammatory signatures (jacquelin et al., ; manches & bhardwaj, ). in contrast, siv infection in sooty mangabeys and african green monkeys, which develop modest pathology despite equivalent viral loads as macaques, correlate with reduced ifn-i and inflammatory gene signatures (bosinger et al., ) . similar correlations with respect to reduced immune activation exist in hiv-infected elite controllers, although whether reduced immune activation follows virus control is uncertain (deeks & walker, ; saez-cirion et al., ) . blockade of pd- signaling during chronic siv infection reduces hyperimmune activation and microbial translocation in rhesus macaques and lowers ifn-i signatures in the blood and colon (dyavar shetty et al., ) . moreover, an elevated interferon signature is observed in hcv-infected patients despite limited control of virus replication and development of liver pathology (guidotti & chisari, ; su et al., ; wieland et al., ) . in fact, hcv infection in culture blocks isg protein expression through activation of rna-dependent protein kinase (garaigorta & chisari, ) , creating a paradoxical ifn-i-dependent viral advantage. thus, ifn-i signaling pathways have the potential to aid viral fitness and promote pathology during persistent viral infection. these studies further highlight the viability of the ifn-i signaling system as a target to promote control of persistent viral infection. while the literature suggests a causative role for ifn-i in contributing to pathogenesis of persistent virus infections, definitive studies assessing how ifn-i neutralization affects the outcome of virus persistence were lacking until recently. two laboratories assessed the role ifn-i signaling plays during persistent infection using the lcmv clone- (cl ) strain of virus. during their investigation, they found that blockade of ifn-i signaling using an ifnar neutralizing antibody reduced immune system activation, decreased expression of negative immune regulatory molecules il- and pd-l and restored lymphoid architecture in mice persistently infected with lcmv (fig. ) . importantly, blockade of ifnar both prior to and following established persistent lcmv infection promoted faster virus clearance and required an intact cd t cell compartment (teijaro et al., ; wilson et al., ) . blockade of ifn-i signaling significantly enhanced cd t cell differentiation into th effectors as well as increased t fh cell differentiation (osokine et al., ) . the above studies demonstrate for the first time a direct causal link between ifn-i signaling, immune activation, negative immune regulator expression, lymphoid tissue disorganization, and long-term virus persistence. more recently, it was reported that during cl infection, both type i and ii interferon promoted the induction and suppressive capacity of cd + cd + immune regulatory dcs (iregdcs), respectively (cunningham et al., ) . while ifn-γ promoted the differentiation of iregdcs from monocytes, ifn-i promoted the suppressive functions of iregdcs. genetic deletion of ifnar prevented the expression of pd-l and production of il- from iregdcs, relieving their suppressive capabilities. in addition to modulating the suppressive capacity of iregdcs, ifn-i signaling also limited their generation/expansion. during mnov infection, selective genetic deletion of ifnar in dcs increased expression of the cellular activation markers cd , cd , and mhcii, suggesting that direct ifn-i signaling on dcs may be responsible for restraining dc function in vivo (nice et al., ) . generation of elevated numbers of iregdcs was also observed during hiv and mycobacterium tuberculosis infections as well as cancer, suggesting that iregdc generation is common in immunosuppressive environments. the ifn-i-driven immune-suppressive state during persistent lcmv infection also inhibits macrophage function. a recent study found that mice infected with the persistent docile strain of lcmv have impaired humoral immune responses to a superinfecting vsv infection (honke et al., ) . the absence of virus replication in cd + macrophages was not due to antiviral cd t cell-mediated killing of cd + macrophages but instead the result of sustained ifn-i responses and an elevated ifn-i antiviral gene program. in turn, reduction in vsv replication and antigen production in cd + macrophages reduced antigen production in these cells which was essential for antiviral antibody generation. the existence of multiple ifn-i subspecies ( ifn-α species in mice and in humans in addition to ifn-β) suggests that either the ifn-i system requires redundancy to be effective or that individual ifn-i species evolved to execute specific functions. certainly, different ifn-α species and β display varying degrees of affinity for the ifnar / receptor complex (ng, mendoza, garcia, & oldstone, ; thomas et al., ) , with ifn-β displaying the highest binding affinity. lcmv persistence was influenced more by ifn-β than ifn-α signaling as treatment of mice infected with lcmv cl with an ifn-β neutralizing antibody displayed accelerated virus clearance compared to a polyclonal ifn-α antibody which had minimal effects on virus control (ng et al., ) . ifn-β neutralization did not exacerbate early virus replication, improved lymphoid architecture, and enhanced virus-specific cd and cd t cell responses. however, while ifn-β neutralization clearly promoted faster virus clearance as compared to neutralization with a polyclonal ifn-α antibody, the contribution of ifn-α species not neutralized by the polyclonal antibody used was not investigated. nevertheless, neutralizing ifn-β may promote adaptive immune control of virus without significantly affecting virus replication and thus may represent a safer approach to promoting control of persistent virus infection in vivo. the dichotomy between ifn-α and β was further highlighted upon infection of new zealand black (nzb) mice with lcmv cl . infection of nzb mice with cl resulted in early lethality that was found to be due to cd t cell-dependent thrombocytopenia and pulmonary endothelial cells loss (baccala et al., ) . interestingly, despite upregulation of pd- /pd-l expression and il- production, t cell function remained intact. moreover, this enhanced pathology correlated with elevated ifn-i protein levels and gene signatures; however, unlike infection in c bl/ j mice, the pathology required ifn-α signaling and was ifn-β independent. it was recently reported that ifn-β signaling required binding to ifnar but was independent of ifnar . deletion of ifnar ameliorated lps-induced sepsis induction, while ifnar À/À mice were unaffected (de weerd et al., ) ; thus, it would be interesting to test how ifnar À/À nzb mice respond to cl infection. the above studies demonstrate that ifn-α and -β species can differentially modulate immune responses in various viral infections, highlighting the importance of future investigation into how different ifn-i subtypes modulate viral control and disease pathogenesis. several important questions still remain that provide exciting avenues for investigating the roles of ifn-i signaling during viral infection in the future. although ifn-i signaling can trigger various downstream effector pathways, how signaling via select ifn-i species dictate specific outcomes following viral infections remain incompletely understood. specifically, there is a great need to understand the roles individual ifn-i-α and -β subsets play in restraining viral replication or promoting immune inflammatory/suppressive programs in vivo. further, how ifn-i signaling in specific cellular subsets in vivo regulates immune pathological and immunesuppressive responses will be interesting to dissect. the ifnar -floxed mouse strain which was generated recently will be instrumental in future studies to investigate this question. illuminating what cell types require ifn-i signaling in vivo should pave the way for generating a detailed understanding of the cellular and molecular mechanisms by which ifn-i signaling acts to promote immune pathology and suppression in acute and persistent viral infections. the capacity of ifn-i signaling to promote immune pathology during acute respiratory viral infection appears in animal models of both influenza and sars-cov infection. the necessity of ifn-i signaling to restrain viral spread during acute viral infection suggest that targeting the ifn-i signaling pathway may be ill advised. however, one wonders whether targeting specific ifn-i species to suppress detrimental inflammation can be achieved without compromising virus clearance during acute respiratory viral infections. moreover, the production of ifn-λ during respiratory viral infection may be sufficient to control viral loads while ifn-i signaling is inhibited. recent results in mouse models suggest this may be possible; however, further studies are needed. moreover, whether the effects observed in mice will translate to human respiratory viral infections is unknown and should be investigated with caution. in the context of the immune-suppressive programs elicited by ifn-i signaling during persistent virus infection, the recent demonstration that blockade of ifn-β enhanced virus control by inducing improved lymphoid architecture and enhanced virus-specific cd and cd t cell responses, suggest that targeting selective ifn-i species can redirect immune responses sufficiently to promote immune-mediated virus control. importantly, relief of the immune-suppressive environment in this case was not accompanied by elevated viral loads following treatment with ifn-β-neutralizing antibody, suggesting that more selective modulation of specific ifn-i species can allow for preservation of some antiviral functions. the mechanisms by which the different ifn-i species interact with the ifnar and ifnar receptors to induce differential downstream signaling suggests this pathway could be manipulated pharmacologically. it is interesting to postulate whether small molecules or biologics could be developed to block binding/signaling of specific ifn-i species (i.e., ifn-β or specific α-species). for example, could ifn-β signaling be selectively inhibited without altering ifn-α species engagement with the ifnar / receptor complex during ongoing viral infection using a small molecule or antibody therapeutic? could a small molecule be designed to reverse aspects of the immune-suppressive environment and promote virus control without compromising virus replication? on the contrary, could selective ifn-i agonists be developed to increase ifn-i signaling in a productive way to lower viral loads and bring persistent/chronic viral infection under control? a similar question could be posited during acute viral infections where ifn-i signaling promotes aberrant inflammation and immune pathology. moreover, it would be interesting to investigate whether selective biological or pharmacological modulation of ifn-i signaling may translate to treat autoimmune disease states associated with elevated and sustained ifn-i signaling. however, any therapy that enhances or blocks ifn-i signaling will need to be approached carefully, given the delicate balancing act required for controlling virus replication while safely modulating immune responses. protective immunity and susceptibility to infectious diseases: lessons from the influenza pandemic role of host immune response and viral load in the differential outcome of pandemic h n ( ) influenza virus infection in indian patients type i interferon limits influenza virus-induced acute lung injury by regulation of excessive inflammation in mice pegylated interferon-alpha a treatment of chronic siv-infected macaques safety, tolerability, and mechanisms of antiretroviral activity of pegylated interferon alfa- a in hiv- -monoinfected 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endothelial cells are central orchestrators of cytokine amplification during influenza virus infection mapping the innate signaling cascade essential for cytokine storm during influenza virus infection. proceedings of the national academy of sciences of the united states of america structural linkage between ligand discrimination and receptor activation by type i interferons rapid expansion of cd + t cells in wild-type and type i interferon receptor-deficient mice correlates with protection after low-dose emergency immunization with modified vaccinia virus ankara suppression of cytokine storm with a sphingosine analog provides protection against pathogenic influenza virus timing and magnitude of type i interferon responses by distinct sensors impact cd t cell exhaustion and chronic viral infection type interferons and antiviral cd t-cell responses simultaneous detection of hepatitis c virus and interferon stimulated gene expression in infected human liver blockade of chronic type i interferon signaling to control persistent lcmv infection type i interferon protects antiviral cd + t cells from nk cell cytotoxicity functional interferon system is required for clearance of lassa virus role of interferon regulatory factor in t cell responses during acute lymphocytic choriomeningitis virus infection bone marrow plasmacytoid dendritic cells can differentiate into myeloid dendritic cells upon virus infection key: cord- -b no yrv authors: davies, robert l; maccorquodale, roslyn; caffrey, bridget title: diversity of avian pasteurella multocida strains based on capsular pcr typing and variation of the ompa and omph outer membrane proteins date: - - journal: vet microbiol doi: . /s - ( ) - sha: doc_id: cord_uid: b no yrv one hundred avian pasteurella multocida isolates recovered from cases of fowl cholera and related infections in england and wales over a -year period were characterised by capsular pcr typing and analysis of outer membrane protein (omp) profiles. sixty-eight percent of the strains were of capsular type a, % were type f, % were type d, % were type b and % were untypable. nineteen distinct omp profiles (omp-types) were identified based mainly on molecular mass heterogeneity of the heat-modifiable (ompa) and porin (omph) proteins. fifty-six percent of the isolates were represented by omp-types, whereas % of the isolates were associated with four omp-types. the extensive molecular mass heterogeneity of the ompa and omph proteins supports previous findings that avian p. multocida strains are very diverse. furthermore, the isolates studied were associated with different clinical symptoms and were recovered from a wide range of lesions and tissues. the high degree of strain diversity together with the wide variety of clinical symptoms suggest that certain avian strains of p. multocida are opportunistic pathogens of relatively low virulence. strains of capsular types b, d and f, as well as the untypable isolates, were associated exclusively with specific omp-types and represent distinct and widely disseminated clonal groups. these observations support the view that avian strains of p. multocida have a clonal population structure. based on previous studies, the molecular mass heterogeneity of the ompa and omph proteins might provide a selective advantage to p. multocida by generating antigenic variation. pasteurella multocida is the aetiological agent of fowl cholera, a widely distributed and economically important disease of poultry, particularly chickens, turkeys, ducks and geese rimler and glisson, ) . the organism is also responsible for disease in wild birds, commercially raised game birds and caged birds . four capsular serogroups are recognised among avian strains of p. multocida, namely a, b, d and f rimler, , ; rimler and rhoades, ) . strains of serogroup a are recognised as the primary cause of fowl cholera, whereas isolates of serogroups b, d and f are less frequently associated with disease rimler, , ; wilson et al., ) . in addition, some avian strains of p. multocida are non-encapsulated and are not serogroupable wilson et al., ) . sixteen somatic serotypes ( - ) are also recognised in p. multocida , a and most of these have been demonstrated in avian capsular serogroup a strains . there is considerable evidence, based on a wide range of molecular studies (snipes et al., ; carpenter et al., ; christiansen et al., ; wilson et al., wilson et al., , blackall et al., blackall et al., , gunawardana et al., ; petersen et al., ) , that avian strains of p. multocida are extremely diverse. in particular, a study of the population genetics of australian strains using multilocus enzyme electrophoresis (mlee) identified electrophoretic types among only field isolates (blackall et al., ) . based on dna-dna hybridisation and sugar fermentation patterns p. multocida has been subdivided into three subspecies, subsp. multocida, subsp. gallicida, and subsp. septica (mutters et al., ) and all of these have been isolated from birds (snipes et al., ; hirsh et al., ; fegan et al., ) . however, conflicting results from ribotyping and s rrna sequence data (petersen et al., ) suggest that the precise phylogenetic relationships of strains representing each of these subspecies is complex and has yet to be satisfactorily resolved. control of fowl cholera is primarily by good management practice and vaccination in areas where the disease is endemic (rimler and glisson, ) . both whole-cell bacterins and live vaccines composed of attenuated strains are currently available but neither is entirely satisfactory. bacterins only induce serotype-specific protection, whereas live vaccines sometimes cause disease (bierer and derieux, ; schlink and olson, ; prantner et al., ) and there is increasing interest in the development of subunit vaccines (kasten et al., ; luo et al., ) . outer membrane antigens that might be considered as potential vaccine candidates include the heat-modifiable or ompa and the porin or omph proteins mittal, , ; luo et al., luo et al., , . both of these proteins are expressed in high copy number, are surface exposed and immunogenic (hancock, ; tagawa et al., ; yi and murphy, ; zeng et al., ; neary et al., ) . the omph protein has been shown to be heterogeneous in strains of p. multocida representing somatic serotypes - (luo et al., ) and there is evidence that anti-omph antibodies are protective in chickens (luo et al., ) and mice . there is less information available about the ompa protein of p. multocida, but this protein also exhibits variation in other bacterial species (duim et al., ; webb and cripps, ) . the aim of the study was to investigate capsular and outer membrane protein (omp) diversity among avian p. multocida strains isolated from diseased poultry in england and wales. in particular, heterogeneity of the ompa and omph proteins was examined and used as the basis for an omp classification scheme. since ompa and omph are important surface-exposed components of the outer membrane, analysis of their diversity in avian p. multocida strains will contribute to our understanding of host-pathogen interactions in fowl cholera, including the role of these proteins in immune evasion, and to the development of improved vaccines against this pathogen. one hundred avian field isolates of p. multocida were investigated. these were obtained from regional laboratories of the veterinary laboratories agency (vla) and originated from widespread geographic locations within england and wales over a -year period ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . the isolates were recovered predominantly from cases of fowl cholera and related acute disease conditions such as septicaemia and pneumonia. however, some isolates were associated with chronic conditions such as conjunctivitis, sinusitis, swollen head, arthritis, etc. properties of the isolates and details of the clinical symptoms of the birds of origin are provided in table . the capsular reference strains x (a), m (b), p (d), p (e) and p (f) were kindly provided by dr. r. rimler, national animal disease center, ames, ia. the isolates were stored at À c in % (v/v) glycerol in brain heart infusion broth (bhib). from À c stock cultures, bacteria were streaked onto blood agar (brain heart infusion agar containing % (v/v) defibrinated sheep's blood) and incubated overnight at c. for preparation of dna, a few colonies were inoculated into ml volumes of bhib and grown overnight at c at rpm. for preparation of outer membranes, . ml of overnight growth in bhib was inoculated into ml volumes of bhib in l erlenmeyer flasks and incubated for h at c at rpm. cells from . ml of overnight cultures were harvested by centrifugation for min at ; Â g and washed once in sterile, distilled h o. dna was prepared with the instagene matrix (bio-rad) according to the manufacturers' instructions and stored at À c. the capsular types were determined by multiplex capsular pcr typing with the capsule-specific primer pairs (capa, capb, capd, cape and capf) described by townsend et al. ( ) . isolates that were negative for all five capsular types were confirmed as p. multocida with a p. multocida-specific primer set (kmt t and kmt sp ) (townsend et al., ) in separate pcr reactions (see section ) and classified as untypable. all primers were synthesised by sigma-genosys (cambridge, uk) and the capsular gene fragments were amplified with a taqdna polymerase kit (boehringer mannheim) according to the manufacturers instructions. pcrs were carried out in a geneamp pcr system (applied biosystems) thermal cycler using the following amplification parameters: denaturation at c for s, annealing at c for s and extension at c for min. thirty cycles were performed and a final elongation step of c for min was used. production of pcr amplicons of the expected size was confirmed by electrophoresis in % agarose gels. pooled pcr amplicons of capsular type a, b, d, e and f reference strains were used as standards in each gel. omps were prepared by sarkosyl extraction as previously described (davies et al., ; davies and donachie, ) . protein concentrations were determined by the modified lowry procedure (markwell et al., ) and adjusted to . mg/ml. omps were separated by sds-page in % (w/v) resolving gels (hoefer se electrophoresis apparatus) using the sds discontinuous system of laemmli (laemmli, ) as previously described (davies et al., ; davies and donachie, ) . unless otherwise stated all samples were heated at c for min prior to electrophoresis. twenty micrograms of protein were loaded per lane and the proteins were visualised by staining with coomassie brilliant blue. protein molecular mass standards (pharmacia) consisted of phosphorylase b ( kda), bovine serum albumin ( kda), ovalbumin ( kda), carbonic anhydrase ( kda), trypsin inhibitor ( . kda) and a-lactalbumin ( . kda). the molecular masses of individual proteins were calculated with the labworks tm image acquisition and analysis computer software. the capsular types of the avian p. multocida isolates were determined by capsular pcr typing and typical results are shown in fig. . the distribution of capsular types among the isolates is summarised in table . sixty-eight ( %) isolates were of capsular type a, ( %) were of type f, five ( %) were of type d, four ( %) were of type b and nine ( %) isolates were untypable. capsular type e was not detected among the population sampled. the p. multocida-specific primers were omitted from the capsular primer mixture because they interfered with the capsule-specific primers and resulted in a reduction of capsular pcr product (i.e. reduced band intensity). therefore, isolates that were negative for capsular typing were confirmed as untypable p. multocida in separate pcr assays with the p. multocida-specific primers (townsend et al., ) . microscopic examination of the untypable isolates after indian ink staining indicated that they were non-encapsulated. the stability of the omp profiles was examined by comparing the profiles of two isolates after repeated subculture and at different stages of the growth cycle. the profiles of these isolates were identical after , , and rounds of subculture on blood agar and after , , and h of growth in bhib (results not shown). the omp profiles of the isolates were analysed by sds-page and provisionally assigned to omp-types based on profile similarity (described below). isolates assigned to the same omp-type were subsequently rerun on up to three or four occasions such that isolates of the same omp-type were directly compared on the same gel. an omp classification scheme was devised-based, firstly, on molecular mass variation of the two major proteins, ompa and omph (omp-type , , etc.), and, secondly, on variation of minor protein patterns (omp-type . , . , etc.). the ompa and omph proteins have overlapping molecular mass ranges ( - kda) and were distinguished on the basis of their different behaviours in sds-page gels after heat-treatment. the omph porin protein is tightly associated with peptidoglycan and is not released unless heated at a temperature of approximately c or higher (rosenbusch, ) . therefore, the omph protein does not migrate into the gel unless heated at c or higher prior to sds-page. in contrast, the ompa protein is not associated with peptidoglycan and freely migrates into the gel after heat-treatment at temperatures below c prior to sds-page. however, the ompa protein undergoes a characteristic conformational change when heated at c that results in an increase in its apparent molecular mass in sds-page gels (beher et al., ) . therefore, to identify ompa and omph, one isolate representing each omp-type was subjected to heat-treatment at , , , , and c prior to sds-page. the results for two isolates of omp-types . and . are shown in fig. . the ompa (a) and omph (h) proteins for each omp-type are indicated in fig. and the results are described below. the isolates consisted of major omp groups that were classified as omp-types - based on variation of ompa and omph (described above). based on variation of minor proteins isolates of omp-types , and could be further subdivided into omptypes . - . , . and . , and . - . , respectively. profiles representing each of these omp-types (with the exception of omp-types . and . ) are shown in fig. . the molecular mass of ompa (a) varied from . to . kda and that of omph (h) varied from . to . kda. the distribution of omp-types among the avian isolates is shown in table . isolates of omp-types . ( %), . ( %), . ( %) and . ( %) were the most numerous and accounted for % of the total. a smaller number of isolates, ranging from to , were associated with each of the other omp-types but these accounted for % of the total number of isolates. there was a strong correlation between certain capsular types and specific omp-types (table ). the frequently occurring capsular type a was associated with isolates representing of the omp-types. in contrast, capsular type b was associated exclusively with the four isolates of omp-type . ; these isolates originated from four different regional laboratories. capsular type d was associated with / isolates of omptype . and with the four isolates of omp-type . ; three of the four isolates of omptype . originated from different regional laboratories and the single omp-type . isolate came from a fourth laboratory. capsular type f was associated with the two isolates of omp-type . , with / isolates of omp-type . and with / isolates of omp-type . . all seven of the isolates representing omp-types . - . were untypable, as were / isolates of omp-type . . the seven untypable/omp-type isolates originated from six different regional laboratories; the two omp-type . isolates also came from different laboratories. overall, the majority of omp-types were represented by a single capsular type, but isolates of omp-types . and . were associated with capsular types a and f, and isolates of omp-type . were either untypable or possessed capsular type d. there are a number of difficulties associated with conventional capsular serotyping of p. multocida (chengappa et al., ; rhoades, , ) . however, townsend et al. ( ) described an alternative and highly specific multiplex capsular pcr assay that is based on nucleotide sequence variation within the five capsular biosynthetic loci. this pcr-based capsular typing method was used in the present study and found to be a reliable and rapid method for capsular typing large numbers of p. multocida isolates. reference strains were used as internal standards and no cases of ambiguity occurred. the observed incidence of capsular serotypes in our sample was very similar to that described in the study of isolates by rhoades and rimler ( ) . in the latter investigation, capsular types a, f, b and d were associated with , , and % of isolates, respectively, whereas % of strains were untypable. the significantly higher incidence of serotype a strains with respect to isolates of serotypes b, d and f in this and previous studies wilson et al., ) suggests that the various serotypes differ in their virulence characteristics. although virulence studies have shown that strains of serotypes b, d and f are potentially pathogenic rimler, , b) , there is very little information about the comparative virulence of strains representing the different serotypes. the omp profiles of the avian p. multocida isolates were very diverse. the isolates could be classified into distinct omp-types based on variation of ompa and omph and, to a lesser extent, of the minor proteins. fifty-six percent of the isolates were represented by omp-types, whereas % of the isolates were associated with four omp-types. the high degree of heterogeneity observed in the omp profiles, and of ompa and omph in particular, was not unexpected because previous studies have shown that avian p. multocida strains are extremely diverse (snipes et al., ; christiansen et al., ; wilson et al., wilson et al., , blackall et al., blackall et al., , petersen et al., ) . in particular, blackall et al. ( ) identified electrophoretic types among only p. multocida isolates from australian poultry by mlee. in a previous study of mannheimia haemolytica (davies and donachie, ) , strains were sub-divided into three distinct groups based on their omp profiles and these were subsequently shown to represent phylogenetically distinct lineages by mlee (davies et al., ) . however, no such demarcation was apparent among the omp profiles of the avian p. multocida isolates. omp patterns have been shown to be closely associated with electrophoretic types and clones identified by mlee in other species (achtman et al., ; musser et al., musser et al., , achtman and pluschke, ; kapur et al., ; davies et al., ) . the exclusive association of isolates of the less common capsular types with specific omp-types provided evidence that omp-types mark individual clonal groups of p. multocida (achtman and pluschke, ) . for example, isolates of omp-type . were associated with capsular type f, isolates of omp-type were untypable, isolates of omp-type . were associated with capsular type b and isolates of omp-type . were associated with capsular type d (table ) . furthermore, almost all of the isolates representing each of these groups originated from a different regional laboratory. this is significant because a characteristic feature of clonal bacterial populations is that strains representing the same clone originate from widespread geographic origins (selander and musser, ) . the association of capsular types b and d and certain untypable isolates, with specific omptypes is also important because it demonstrates for the first time that strains of these uncommon capsular types, together with untypable isolates, probably represent specific clones of p. multocida. in contrast, dziva et al. ( ) were unable to demonstrate a relationship between rapd patterns and capsular serogroups in their study of zimbabwean isolates of p. multocida (dziva et al., ) . isolates of omp-types . and . were associated with capsular types a and f. this observation is probably due to the close relationships of these two capsular types (townsend et al., ) . in many pathogenic bacterial species, the majority of cases of infectious disease are often caused by a small proportion of the total number of extant clones (selander and musser, ) . in this respect, avian p. multocida strains differ from many other pathogens because the majority of cases of disease were associated with a relatively large number of omp-types/clones. a possible reason for this is that the isolates were recovered from a diverse range of lesions and tissues and were associated with different types of infection ranging from pneumonia and septicaemia to sinusitis, conjunctivitis and swollen head. high levels of diversity were also observed among eschericha coli strains isolated from chickens with swollen-head syndrome and from birds with colibacillosis (white et al., ) . it was suggested that the large number of clonal genotypes associated with these avian diseases was due either to the opportunistic nature of the infections or to the widespread occurrence of unknown virulence factors (white et al., ; whittam, ) . swollen-head syndrome associated with e. coli is thought to be the result of a secondary infection subsequent to an initial viral infection caused by paramyxovirus, coronavirus, or pneumovirus. the high level of diversity observed among avian p. multocida isolates, together with the wide range of clinical symptoms and tissues of origin, similarly suggests that a high proportion of the isolates might represent opportunistic pathogens of relatively low virulence. in particular, isolates associated with conjunctivitis, sinusitis and swollen head could potentially be secondary pathogens following initial viral infection. confirmation of this hypothesis will require the comparison of bacterial isolates from diseased birds with the normal avian flora. the ompa and omph proteins of avian isolates of p. multocida were shown to be heterogeneous since numerous molecular mass variants were identified (fig. ) . however, the omph protein ( . - . kda) is clearly more heterogeneous than the ompa protein ( . - . kda). comparative nucleotide sequence analysis of the omph proteins representing the somatic serotypes of p. multocida has shown that the molecular mass heterogeneity of this protein is due to variation in the number of amino acids ( - ) in the protein (luo et al., ) . however, most of this variation is confined to two discrete hypervariable regions (amino acids - and - ) which are thought to correspond to external surface-exposed loops (luo et al., ) . similar heterogeneity occurs in the corresponding p (omph) and p (ompa) proteins of haemophilus influenzae, and has also been shown to be due to differences in the size of hypervariable surface-exposed loop regions (forbes et al., ; sikkema and murphy, ; duim et al., ; webb and cripps, ) . these surface-exposed loops are thought to interact with the host immune system and, by undergoing antigenic variation, provide the bacterium with an important defence mechanism murphy, , ; neary et al., ) . furthering knowledge of the molecular basis of this diversity in p. multocida will lead to a better understanding of the role of these proteins in avian disease and contribute to the development of improved vaccines. in summary, this investigation of capsule and omp variation has confirmed the view that avian p. multocida isolates are very diverse. a possible explanation for the high level of strain diversity observed in the study is that many of the isolates were associated with chronic infections, were recovered from a wide range of lesions and tissues, and represent opportunistic pathogens. the association of certain capsular types with specific omp-types suggests that omp profiles mark individual clones of p. multocida. in particular, isolates of the uncommon capsular types b and d and certain untypable isolates, represent distinct clonal groups. the ompa and omph proteins exhibit extensive molecular mass heterogeneity that might provide a selective advantage to the pathogen by generating antigenic variation. clonal analysis of descent and virulence among selected escherichia coli six widespread bacterial clones among escherichia coli k isolates major heat-modifiable outer membrane protein in gram-negative bacteria: comparison with the ompa protein of escherichia coli immunologic response to turkey poults of various ages to an avirulent pasteurella multocida vaccine in the drinking water characterisation of pasteurella multocida isolated from fowl cholera outbreaks on turkey farms population structure and diversity of avian isolates of pasteurella multocida from australia molecular epidemiology of pasteurella multocida in turkeys identification of type d pasteurella multocida by counterimmunoelectrophoresis transmission of pasteurella multocida on california turkey premises in - intra-specific diversity and host specificity within pasteurella haemolytica based on variation of capsular polysaccharide, lipopolysaccharide and outer-membrane proteins outer membrane protein and lipopolysaccharide variation in pasteurella haemolytica a under different growth conditions evolutionary genetics of pasteurella haemolytica isolates recovered from cattle and sheep molecular variation in the major outer membrane protein p gene of nonencapsulated haemophilus influenzae during chronic infections random amplification of polymorphic dna and phenotypic typing of zimbabwean isolates of pasteurella multocida phenotypic characterisation of pasteurella multocida isolates from australian poultry variation in length and sequence of porin (ompp ) alleles of non-capsulate haemophilus influenzae molecular characterisation of avian pasteurella multocida isolates from australia and vietnam by rep-pcr and pfge bacterial outer membranes: evolving concepts serum resistance is correlated with encapsulation of avian strains of pasteurella multocida characteristics of pasteurella multocida isolated from waterfowl and associated avian species in california outer membrane protein patterns mark clones of escherichia coli o and o strains that cause avian septicemia pasteurella multocida produces a protein with homology to the p outer membrane protein of haemophilus influenzae cleavage of structural proteins during the assembly of the head of bacteriophage t cloning and characterization of the major outer membrane protein gene (omph) of pasteurella multocida x- sequence analysis of pasteurella multocida major outer membrane protein (omph) and application of synthetic peptides in vaccination of chickens against homologous strain challenge a modification of the lowry procedure to simplify protein determination in membrane and lipoprotein samples a population genetic framework for the study of invasive diseases caused by serotype b strains of haemophilus influenzae clonal population structure of encapsulated haemophilus influenzae reclassification of the genus pasteurella trevisan on the basis of deoxyribonucleic acid homology, with proposals for the new species pasteurella dagmatis, pasteurella canis, pasteurella stomatis, pasteurella anatis, and pasteurella langaa antibodies to loop of the p porin protein of nontypeable haemophilus influenzae are bactericidal against multiple strains genetic diversity of pasteurella multocida fowl cholera isolates as demonstrated by ribotyping and s rrna and partial atpd sequence comparisons the pathogenesis of pasteurella multocida serotype a: , infection in turkeys: a comparison of two vaccine strains and a field isolate capsular groups of pasteurella multocida isolated from avian hosts virulence of avian capsular serogroup b pasteurella multocida for turkey poults fowl cholera somatic serotypes of pasteurella multocida strains isolated from avian hosts ( - ) virulence and toxigenicity of capsular serogroup d pasteurella multocida strains isolated from avian hosts fowl cholera serogroup f, a new capsule serogroup of pasteurella multocida pasteurella multocida characterization of the major envelope protein from escherichia coli. regular arrangement on the peptidoglycan and unusual dodecyl sulphate binding vaccination of turkey breeder hens and toms for fowl cholera with cu strain population genetics of bacterial pathogenesis molecular analysis of the p porin protein of nontypeable haemophilus influenzae use of an rrna probe and restriction endonuclease analysis to fingerprint pasteurella multocida isolated from turkeys and wildlife characterization of a heat-modifiable outer membrane protein of haemophilus somnus genetic organization of pasteurella multocida cap loci and development of a multiplex capsular pcr typing system characterization of an outer membrane protein of pasteurella multocida belonging to the ompa family role of outer membrane protein h (omph)-and ompa-specific monoclonal antibodies from hybridoma tumors in protection of mice against pasteurella multocida the kda major outer-membrane protein of pasteurella multocida capsular serotype d secondary structure and molecular analysis of interstrain variability in the p outer-membrane protein of non-typable haemophilus influenzae isolated from diverse anatomical sites genetic relationships among strains of avian escherichia coli associated with swollen-head syndrome genetic population structure and pathogenicity in enteric bacteria comparison of dna fingerprinting and serotyping for identification of avian pasteurella multocida isolates pasteurella multocida isolated from wild birds of north america: a serotype and dna fingerprint study of isolates from to mapping of a strain-specific bactericidal epitope to the surface-exposed loop on the p porin protein of non-typeable haemophilus influenzae importance of an immunodominant surface-exposed loop on outer membrane protein p of nontypeable haemophilus influenzae molecular cloning of the pasteurella haemolytica poma gene and identification of bovine antibodies against poma surface domains this study was supported by a wellcome trust university award to r.l. davies ( /z/ /z). we are extremely grateful to staff of the veterinary laboratories agency (vla) for the provision of isolates, including those at bury st. edmunds for making available strains of their p. multocida collection. key: cord- -e rpryrh authors: tomasello, elena; pollet, emeline; vu manh, thien-phong; uzé, gilles; dalod, marc title: harnessing mechanistic knowledge on beneficial versus deleterious ifn-i effects to design innovative immunotherapies targeting cytokine activity to specific cell types date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: e rpryrh type i interferons (ifn-i) were identified over years ago as cytokines critical for host defense against viral infections. ifn-i promote anti-viral defense through two main mechanisms. first, ifn-i directly reinforce or induce de novo in potentially all cells the expression of effector molecules of intrinsic anti-viral immunity. second, ifn-i orchestrate innate and adaptive anti-viral immunity. however, ifn-i responses can be deleterious for the host in a number of circumstances, including secondary bacterial or fungal infections, several autoimmune diseases, and, paradoxically, certain chronic viral infections. we will review the proposed nature of protective versus deleterious ifn-i responses in selected diseases. emphasis will be put on the potentially deleterious functions of ifn-i in human immunodeficiency virus type (hiv- ) infection, and on the respective roles of ifn-i and ifn-iii in promoting resolution of hepatitis c virus (hcv) infection. we will then discuss how the balance between beneficial versus deleterious ifn-i responses is modulated by several key parameters including (i) the subtypes and dose of ifn-i produced, (ii) the cell types affected by ifn-i, and (iii) the source and timing of ifn-i production. finally, we will speculate how integration of this knowledge combined with advanced biochemical manipulation of the activity of the cytokines should allow designing innovative immunotherapeutic treatments in patients. specifically, we will discuss how induction or blockade of specific ifn-i responses in targeted cell types could promote the beneficial functions of ifn-i and/or dampen their deleterious effects, in a manner adapted to each disease. type i interferons (ifn-i) were the first cytokines discovered, over years ago, based on their potent anti-viral effects ( , ) . ifn-i play a crucial, non-redundant role in vertebrate anti-viral defenses ( ) ( ) ( ) . ifn-i also mediate protective effects in other physiopathological contexts, including cancer ( ) and multiple sclerosis (ms) ( ) . on the contrary, ifn-i responses can be deleterious in a number of circumstances, including bacterial or fungal infections ( - ), many autoimmune diseases ( ) , and, paradoxically, certain chronic viral infections ( ) ( ) ( ) . it is only recently that an integrated picture has emerged of the cellular and molecular mechanisms regulating the production of ifn-i and underlying their functions. much knowledge was gained recently on another class of potent innate anti-viral interferons, the lambda, or type iii ifns (ifn-iii). we will review knowledge on ifn-i/iii (ifns) and discuss how it could be harnessed to develop innovative therapeutic strategies aimed at surgically tuning ifn activity toward protective responses in a manner adapted to each disease. we will focus on ifn-α/β/λ because they are the best characterized ifns and already used therapeutically. recent reviews are covering information on other ifn-i subsets including ifn-ε, which is produced at mucosal sites and promotes local anti-viral defenses ( , ) . dendritic cells (dcs) are rare heterogeneous mononuclear phagocytes functionally characterized by their unique efficacy for antigen-specific activation of naïve t lymphocytes. dcs are sentinel cells of the immune system, able to sense and integrate a variety of danger input signals for delivery of output signals instructing the activation and functional polarization of effector immune cells. in mammals, five major dc subsets exist, which differ in their expression of innate immune recognition receptors (i r s) and in their functional specialization: monocyte-derived dcs (modcs), langerhans cells, cd b + dcs, xcr + dcs, and plasmacytoid dcs (pdcs) ( ) . a recurrent theme of this review will be the intricate relationships between ifns and dcs, since these cells can be a major source and/or target of these cytokines under various conditions. www.frontiersin.org the first section will synthesize current knowledge on ifn production and protective anti-viral functions. the i r s and downstream signaling pathways responsible for ifn-i production during viral infection will be listed. the roles of different cell types for this function will be discussed. the two main mechanisms through which ifn-i promote anti-viral defense will be reviewed, succinctly for direct anti-viral effects and in greater details for immunoregulatory functions. the second section will focus on the detrimental functions of ifn-i. selected diseases will be discussed to illustrate how different, and sometimes opposite, processes underlie deleterious ifn-i responses depending on the physiopathological contexts. ifn-i induction of unbridled inflammatory responses causing lethal tissue damage will be discussed as a major pathological mechanism during bacterial encounters secondary to influenza infection or in some autoimmune diseases. inappropriate functional polarization of immune responses by ifn-i will be discussed as one potential cause for enhanced susceptibility to bacterial or fungal infections. the complex and disputed role of ifn-i in chronic viral infections will be reviewed, with emphasis on the physiopathology of the infections by human immunodeficiency virus type (hiv- ) and human hepatitis c virus (hcv), with an outlook for the development of novel immunotherapeutic strategies to combine with anti-viral drugs. the third section will recapitulate how the balance between beneficial versus deleterious ifn-i responses is modulated by several key parameters including (i) the source and timing of ifn-i production, (ii) the cell types affected by ifn-i, and (iii) the signaling pathways activated by ifn-i. in the last section, we will speculate how integration of all the knowledge discussed before combined with advanced biochemical manipulation of the activity of the cytokines should allow designing innovative immunotherapeutic treatments, based on induction or blockade of specific ifn-i responses in targeted cell types. this"activity-by-targeting"concept is based on the design of novel "immuno-ifns" consisting in covalent association between a mutated ifn-i with decreased affinity for its receptor and an antibody with high avidity for a molecule specifically expressed on target cell types ( ) . this design ensures lack of activity of the immuno-ifns on all cell types but those targeted, contrary to previous strategies using ifns with close to maximal potency that were still able to mediate strong off-target effects despite their coupling to cell-type specific antibodies and/or their local delivery. type i interferons expression is not detectable under steady state conditions in vivo using classical methods such as gene expression analysis by rt-pcr or protein titration by elisa or bioassays. however, mice deficient for the expression of the alpha chain of the ifn-i receptor (ifnar ) harbor alteration in the ontogeny or functions of various cell types ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . hence, extremely small or localized but functionally relevant quantities of ifn-i must be produced under steady state conditions ( ) . indeed, the existence of steady state responses to ifn-i in various organs in vivo was demonstrated by using reporter mice expressing the firefly luciferase under the control of the promoter of ifnb ( ) or of mx ( ) , a canonical ifn-i-stimulated gene (isg). steady state ifn-i responses are promoted by gut commensals ( ) . early and transiently after many viral infections, large amounts of ifns can be detected, in blood and spleen in the case of systemic infections or locally in the case of confined infections. ifn induction during viral infections results from the detection of specific danger signals by specialized i r s. this includes the detection of pathogen-associated molecular patterns as well as the sensing of stress signals or damage-associated molecular patterns ( , ) . based on the nature and intracellular location of the danger signals that induce the production of the cytokines, the cellular sources of ifns during viral infection can be classified in two main groups. infected cells often contribute to ifn production as a response to their sensing of endogenous viral replication, or consecutive to the metabolic stress induced during massive translation of viral structural proteins, or as a result of plasma membrane perturbations upon viral entry. specific subsets of uninfected cells can also significantly contribute to ifn production upon engulfment of material containing viral-derived nucleotide sequences and sensing of these molecules in endosomes by specific i r s. all sensing pathways leading to ifn induction converge on the activation of interferon response factors or (irf / ), which are the master transcription factors inducing ifn genes. most cell types constitutively express irf but not irf or only at low levels. irf expression requires ifn-i stimulation. ifn-β can directly be induced by irf . all but one of the ifn-α subtypes require irf for their induction. hence, ifn-β secretion promotes its own production and that of ifn-α in an autocrine manner ( , ) . this positive feedback loop strongly amplifies ifn production during viral infections, promoting fast and widespread induction of cell-intrinsic anti-viral defenses in uninfected cells to prevent virus dissemination. other feedback loops tightly regulate ifn-i production positively or negatively. this section reviews different mechanisms controlling ifn production and how they could play different roles in host/virus interactions. different innate immune recognition receptors are involved in sensing various types of viral nucleic acids in distinct categories of cells during viral infections, which may promote different types of anti-viral defenses. for each selected sensor shown, the types of viral nucleic acids recognized and the downstream signaling cascade induced are represented in a simplified, schematic manner. the potential specific role of each cell type in anti-viral defenses is also indicated at the bottom of each panel. (a) potentially all types of infected cells can detect endogenous viral replication through cytosolic sensors triggering their local production of ifn-β/λ to control viral replication in an autocrine and paracrine fashion in infected tissues. (b) uninfected xcr + dcs selectively produce high levels of ifn-λ and ifn-β upon engulfment of materials containing dsrna and the consecutive triggering of tlr in endosomes. the receptor of ifn-λ is mostly expressed by epithelial cells. hence, xcr + dcs might be involved in inducing local ifn responses in virally infected epithelial tissues. since xcr + dcs are especially efficient at producing ifn-iii upon hcv stimulation, they might contribute to local or systemic ifn production during infection with this virus, to promote ifn-λ-mediated protection of hepatocytes. uninfected xcr + dcs and other uninfected cells may produce some ifn-β upon engulfment of materials containing ssrna and the consecutive triggering of tlr in endosomes. the contribution of this pathway to anti-viral defense is not well understood yet, in part because mouse tlr is deficient for this function. (c) uninfected pdcs selectively produce high levels of all subsets of ifns upon engulfment of materials containing ssrna or cpg dna and the consecutive triggering of tlr / in endosomes. however, pdcs seem to be activated for this function only in lymphoid tissues. hence, pdc might contribute to systemic ifn production during blood-borne viral infections or as a failsafe mechanism activated upon abnormal widespread dissemination of a viral infection once it has escaped local confinement at its portal of entry. cm, cell membrane; nm, nuclear membrane. particular nucleotide sequences or tertiary structures, their signaling pathways and their physiological significance have been recently reviewed ( , ) . cytosolic rna sensors encompass dexd/h helicases among which the retinoic-acid-inducible gene (rig)-i-like receptors (rlrs) have been the most studied, namely rig-i and melanoma differentiation associated gene (mda ). rig-i recognizes rna with a -ppp or -pp ( ) (uncapped) moiety, or double-stranded rna (dsrna), both structures being present in viral, but not in cytosolic eukaryotic, rna molecules. mda might specifically recognize long dsrna fragments. both rig-i and mda contain a dexd/h box-containing rna helicase domain, and caspase recruitment domains (card / ), which bind to mitochondrial anti-viral signaling protein (mavs). rna/rlr/sting molecular complexes initiate a signaling cascade leading to irf / -dependent induction of ifns (figure ). other dexd/h helicases can promote ifn-i production in dcs, www.frontiersin.org although their physiological roles for in vivo immune defenses against viral infections remain to be established ( ) . cytosolic dna sensors able to induce ifn-i (mostly ifn-β) and ifn-iii encompass molecules belonging to different protein families, including dexd/h helicases, the inflammasome component ifn-γ-inducible protein (ifi ) , the z-dna binding protein (zbp ), and the cyclic gmp-amp (cgamp) synthase (cgas) ( , ) . most of the cytosolic dna sensors activate sting and lead to irf / -and nfκb-dependent induction of ifn-β and ifn-iii. many cell types express zbp and are able to produce ifn-i upon triggering of this molecule, including macrophages, dcs, and fibroblasts following an hsv- infection ( , ) . upon dna binding, cgas catalyzes the production of cgamp. cgas is critical for the detection of lentiviruses including hiv- / ( , ) and can contribute to sensing of, and protection against, other rna viruses, including in vivo in mice ( ) . cgamp also acts as a secreted second messenger signal alerting uninfected cells to directly induce their expression of intrinsic immune anti-viral defenses. the cgas/sting/irf signaling cascade and the irf transcription factor are "master" inducers of cell-intrinsic immunity able to control the replication of most dna and some rna viruses at least in part independently of ifns ( ) . infected cells become a factory for production of viral particles. hijacking of the translation apparatus of the host cell for massive production of viral structural proteins leads to an overload of the capacity of the er for correct folding of newly synthesized proteins. er overload induces a homeostatic response of the cell, the unfolded protein response (upr). upr aims at restoring normal er functions by inhibiting translation. upr activation in infected cells contributes to prevent viral replication, including through inhibition of the production of viral proteins, promotion of ifn-i production, and induction of cell suicide ( ) . toll-like receptors (tlrs) are among the first and best characterized i r s. tlrs are transmembrane proteins with a leucine-rich repeat extracellular domain involved in ligand recognition and an intracellular toll/interleukin- receptor domain essential for signaling ( ) . among the nine tlrs conserved between mouse and human, tlr , tlr , tlr , and tlr are located in endosomes where they can detect the abnormal presence of nucleic acids such as occurs upon endocytosis of virions or of virally infected cell material. tlr recognizes dsrna, tlr / ssrna, and tlr dna sequences containing unmethylated cytidinephosphate-guanosine (cpg) motifs. tlr fine specificity and signaling pathways have been reviewed recently ( ) and are summarized in figure . we will discuss the expression patterns and functions of endosomal tlrs with regards to ifn production in uninfected specialized immune cell types, pdcs and xcr + dcs. plasmacytoid dcs uniquely produce very large amounts of ifns in response to in vitro stimulation with many viruses, without being infected ( ) . ifn-i mrnas represent up to % of all mrnas in pdcs at the peak of their activation ( ) . in vitro, upon exposure to influenza virus, herpes virus type , cytomegaloviruses, or vesicular stomatitis virus, individual pdcs produce - times more ifns than total pbmcs, monocytes, modcs, cdcs, neutrophils, and fibroblasts ( ) ( ) ( ) ( ) ( ) ( ) . however, in vitro, high molarity infection of cdcs with certain viruses unable to inhibit ifn-i production in their target cells can also induce massive ifn-β secretion ( ) . pdcs produce high levels of all subtypes of ifns, contrary to many other cell types including infected cells, which often preferentially produce ifn-β ( , ) . in vivo, pdc depletion during systemic viral infections leads to over % decrease of ifn-i production, while the total number of pdcs producing ifn-i (< , in one mouse) is much lower than the total number of infected cells ( ) ( ) ( ) ( ) ( ) ( ) . this shows that in vivo also individual activated pdcs produce much more ifn-i/iii than most other cell types, including virus-infected cells. the professional ifn-producing function of pdcs largely results from their high constitutive and selective expression of irf , tlr , and tlr (figure ) . these molecules are pre-associated in readyto-signal complexes located in specialized endosomes specific to pdcs ( , ) . pdcs must also be equipped for efficient sensing and up-take of virions and virus-infected cells. the corresponding cell surface i r s remain to be identified. selective expression of tlr in xcr + dc endows them with a unique ability to produce very high amounts of ifn-β and ifn-iii upon stimulation with dsrna or hcv irrespective of their own infection. xcr + dcs are very potent for antigenspecific activation of cd + t cells, in particular through crosspresentation of exogenous antigens that they have captured from other cells and processed for association with class i major complex histocompatibility (mhc-i) molecules ( ) . in mice, xcr + dcs are crucial for the initiation of protective adaptive immune responses against tumors and a variety of viruses ( ) . mouse and human xcr + dcs constitutively and selectively express high levels of tlr (figure ) . they produce large amounts of ifn-iii and ifn-β upon stimulation with a synthetic mimetic of dsrna, polyinosinic:polycytidylic acid (polyi:c) ( , ) . human xcr + dcs uniquely respond to stimulation with hcv by producing large amounts of ifn-iii in a tlr -dependent manner ( , ) , irrespective of their own infection. positive feedback loops. in addition to irf induction, other positive feedback mechanisms exist to amplify the production of ifns rapidly after initiation of a viral infection as illustrated by the following selected examples. ifns induce the expression of many cytosolic rna/dna sensors and of tlr . this broadens the spectrum of host's cell types able to detect endogenous viral replication for ifn induction. induction of oasl by ifns in human cells enforces rig-i signaling, counteracting viral immune frontiers in immunology | microbial immunology evasion genes interfering with this sensing pathway ( ) . the ifninducible ribonuclease l (rnasel) generates viral and cellular rna degradation products, which engage rlrs for amplification of ifn production ( , ) . the ifn-inducible protein kinase r (pkr) stabilizes ifn-i mrna ( ) . to prevent unbridled responses deleterious for the host, ifn activity must be tightly controlled including during viral infections. several negative feedback loops exist to terminate ifn production, after anti-viral defenses have been activated. the isg ubiquitin specific peptidase (usp ) binds to ifnar , preventing it from recruiting signal transducer and activator of transcription (stat ). ifns induce the expression of tam receptor tyrosine kinases in dcs, monocytes, and macrophages. tam receptors associate and signal in part through ifnar . they activate the suppressors of cytokine signaling- / (socs- / ). socs inhibit tlr and rlr signaling, thereby terminating ifn production ( ) . tam receptor ligands, gas and pros, bind phosphatidylserine on dying cells and are produced by activated dcs and monocytes/macrophages. thus, ifn induction of tam inhibitory receptors on uninfected phagocytic immune cells could limit their propensity to produce the cytokines upon engulfment of dying virally infected cells. ifns induce tetherin on most cell types. pdcs express a receptor for tetherin, leukocyte immunoglobulin-like receptor, subfamily a (with tm domain), member (lilra ). lilra triggering on pdcs inhibits their production of ifn-i. hence, through lilra engagement by tetherin, pdcs can monitor their efficacy at inducing an antiviral gene expression program in neighboring cells through ifns, and timely terminate their ifn production. how positive and negative feedback loops integrate in time and space to promote optimal kinetics and intensity of ifn production in order to efficiently control viral infection without causing severe immunopathology is not completely understood. positive feedback loops may occur very rapidly after initiation of viral infection to allow rapid secretion of high levels of the cytokines for fast and strong induction of anti-viral cell-intrinsic immunity. negative feedback loops occur likely later to terminate the response and thus avoid chronicity of cytokine production and its ensuing deleterious effects. pdcs do not constitute the major source of ifn production upon local infections by several viruses in the lung or in the female reproductive tract. pdcs are dispensable for resistance against these infections ( , , ) . during pulmonary infection by newcastle disease virus (ndv), ifn-i are produced locally in the lungs mainly by infected alveolar macrophages. lung pdcs do not express the cytokines ( ) . selective depletion of lung alveolar macrophages leads to systemic dissemination of ndv, and to a strong activation of pdcs for ifn-i production specifically in the spleen. even in the case of systemic viral infections such as caused by intravenous injection of ndv or intraperitoneal injection of mouse cytomegalovirus (mcmv), pdc ifn production is confined to the spleen. it is not detected in other organs even those with high viral replication ( , ) . hence, splenic pdcs are especially prone to high level ifn production upon systemic acute viral infections. pdcs located in non-lymphoid organs, in particular mucosal barrier tissues, appear to be inhibited for ifn production. thus, ifn production by infected cells serves as first line of defense to block virus replication at its portal of entry in the body. ifn production by uninfected pdcs might constitute a failsafe mechanism mainly activated in the spleen when viral infection gets systemic ( ) . under these conditions, to promote health over disease, the benefits for the host of producing high circulating levels of ifns in order to induce widespread cell-intrinsic anti-viral defenses might prevail over the deleterious effects that this could cause on certain cell types or tissues. indeed, pdcs are required for protection against hsv- and hsv- in mice only in systemic but not local infections ( ) . this observation is consistent with the crucial role of pdcs for protection of mice against systemic infection by mouse hepatitis virus (mhv), a fast replicating coronavirus ( ) . conflicting results have been obtained on the role of pdcs during intranasal influenza infection ( , ( ) ( ) ( ) . a possible explanation is that pdc ifn production contributes to resistance to highly pathogenic influenza strains that might systemically spread from the lung early after infection, even if at low levels. another intriguing observation is that ifns are critical for host resistance to mcmv and that pdcs are the major source of ifns in this infection model but are dispensable for virus control ( ) . studies are ongoing to understand this apparent paradox. patients bearing genetic mutations disrupting endosomal tlr signaling do not appear to suffer from life-threatening viral infections ( , ) , contrary to patients impaired in ifnar signaling ( , ) . a notable exception is the specific susceptibility to severe herpes virus encephalitis in individuals' deficient for tlr signaling ( , ) . however, contrary to extracellular tlr, endosomal tlr have evolved under strong purifying selection in human beings ( ) . hence, while pdcs and endosomal tlr might have been required for protection of our species against viral infections in the past, this appears not to be the case anymore perhaps due to improved social, hygiene, and health care in modern society ( ) . attesting to the importance of ifns for anti-viral defense in vertebrates, many mammalian viruses encode immune evasion genes specifically inhibiting the production of ifns in infected cells ( , ) . pdcs or xcr + dcs might be essential for ifndependent host protection against these viruses, because they are spared from the intracellular functions of viral immune evasion genes ( ) . to the best of our knowledge, mcmv does not encode for immune evasion genes inhibiting ifn production. however, mcmv manipulates ifn-i responses through specific inhibition of stat functions in infected cells. thus, pdcs might be dispensable for resistance against systemic mcmv infection due to sufficient levels of ifn production by infected cells locally in all infected tissues. hepatocyte responses to ifn-iii appear to play a www.frontiersin.org critical role in human resistance to hcv. in infected hepatocytes, hcv induces the expression of cellular micrornas binding to ifn-iii mrna and leading to its degradation. uninfected xcr + dcs produce high levels of ifn-iii in vitro upon hcv stimulation ( , ) . hence, during acute hcv infection in vivo, xcr + dc may be a strong and early source of ifn-iii not subjected to virus immune evasion strategies, therefore, contributing to protect naturally resistant individuals. in secondary lymphoid organs, a subset of macrophages is critical for the clearance of viruses from the lymph ( ) . these macrophages are located on viral entry routes, near to subcapsular sinuses where the afferent lymph drained from non-lymphoid tissues flows. contrary to other subsets of macrophages, subcapsular sinus macrophages are highly susceptible to viral infection, because they constitutively express only low levels of effector molecules of cell-intrinsic anti-viral immunity and because their responses to ifns are inhibited by their high constitutive expression of usp . subcapsular sinus macrophages rapidly become infected by viruses incoming from the lymph and produce large amounts of ifns. this altruistic suicide prevents virus dissemination to other adjacent cell types and promotes the induction of innate and adaptive anti-viral immunity ( ) . upon instruction by ifns, cells express a wide variety of viral restriction factors, whose combined action blocks pathogen invasion by interfering with the different stages of viral life cycle (figure a ). this has been extensively reviewed recently ( ) and will only be described succinctly here. virus fusion with host cell membrane can be blocked by cholesterol- hydrolase (ch h) that inhibits sterol biosynthesis. some viruses enter cells by escaping from endosomes/lysosomes, which can be blocked by interferon inducible transmembrane (ifitm) proteins. virus uncoating can be blocked by tripartite motif (trim) proteins, such as trim α, which bind to hiv- capsid thus promoting its degradation, and by myxoma resistance gtpases, mx , and mx , which efficiently trap viral structural proteins at an early stage following virus entry into the cell. mx inhibits a number of viruses, including influenza virus through sequestration of its nucleocapsid. mx associates with host cyclophilin a and hiv- capsid protein. virion assembly can be blocked at transcriptional, translational, and posttranslational levels. the adenosine deaminase acting on rna (adar ) and the apolipoprotein b mrna editing enzyme, catalytic polypeptide-like (apobec) deaminases induce viral rna destabilization and hypermutation ( , ) . the sterile alpha motif and histidine-aspartic domain (hd) containing protein (samhd ) blocks reverse transcription by hydrolyzing dntps ( ) . adar , apobec, and samhd functions have been mainly studied in infections by hiv- and other retroviruses. the , -oligoadenylate synthase (oas) proteins, the ifn-induced proteins with tetratricopeptide repeats (ifit), and pkr inhibit viral and host protein translation by using complementary mechanisms ( ) . the major post-translation modification induced by ifns is the binding of the ubiquitinlike modifier isg to several viral and host proteins, a process called isgylation. most of the known isgylated proteins are targeted for degradation, with few exceptions that are on the contrary stabilized like irf ( ) . finally, the egress and budding of virions of many enveloped viruses can be inhibited by tetherin or by viperin ( ) . many anti-viral isgs have been functionally characterized only recently, largely thanks to large-scale screening approaches. they display a variable degree of viral specificity ( , ) that might inversely relate to the extent of their side effects on host cells ( figure b ). anti-viral effectors acting on a broad spectrum of viruses often target key metabolic pathways that are also crucial for host cell functions. this is the case for the control of cholesterol metabolism by ch h ( ) or of protein translation by pkr, oas, or ifits ( ) . other anti-viral restriction factors such as mx may specifically target one molecule of a very restricted set of viruses with no apparent side effects on host cells. some anti-viral isgs target specific functions critical for only a restricted array of viruses and might similarly exert side effects only on a subset of host cell types. for example, samhd inhibits retrovirus replication through dntp depletion, which might more specifically affect proliferating host cells. hence, the infected host must balance the intensity, breadth, and location of isg induction to circumvent viral replication while preventing life-threatening damages to vital cell types or tissues. one of the mechanisms contributing to this balance is translational control of the expression of isgs, especially those with pro-apoptotic or anti-proliferative functions ( ) . while many anti-viral isgs are transcriptionally activated in most ifn-stimulated cells, their translation can be specifically blocked in uninfected cells by cellular microrna. this inhibition is relieved upon cell infection through negative regulation of the function of the rna-induced silencing complex. hence, ifn stimulation of uninfected cells prepares them for rapid and strong induction of cell-intrinsic anti-viral defenses upon viral infection while avoiding their unnecessary exposure to the toxic effects of certain isgs. further knowledge on the functions and the dynamic regulation of isgs is essential to develop novel therapeutic strategies against viral infections aiming at modulating ifn responses to promote their protective anti-viral cell-intrinsic functions over their deleterious toxic effects. a better understanding of the immunoregulatory effects of ifns will also help. type i interferon can modulate the functions of a broad spectrum of immune cells ( figure a ). we will review this knowledge, focusing on the functions of dcs, nk cells, t cells, and b cells, since they are involved in the control of most viral infections. we will discuss the hypothesis that dcs play a central role in ifn-i orchestration of innate and adaptive immunity for the induction of optimal anti-viral defenses (figure ) . during viral infections and cancer immunosurveillance, ifn-i constitute one of the most important input signal acting on dcs to promote their delivery of appropriate output signals to t cells, b cells, and nk cells for protective immunity ( figure a ). dcs deliver three types of signals to activate and functionally polarize t cells. signal is the triggering of the t cell receptor by viral peptide-mhc complexes. signal is the triggering of activating t cell co-stimulation receptors such as cd or cd by the cd / and cd co-stimulation molecules expressed on dcs. signal corresponds to cytokines, which can promote t cell proliferation and acquisition of specific effector functions. under steady state conditions, most dcs are in an immature state characterized by low level expression of mhc-ii (signal ) and co-stimulation molecules (signal ) and by the lack of production of t cell-activating www.frontiersin.org cytokines (signal ). upon activation, including early after viral infections in vivo, dcs up-regulate their expression of signal and activating signal and secrete t cell-activating cytokines. this process is called dc maturation. gene expression profiling of dcs stimulated by microbial stimuli identified a core set of genes upregulated in mature dcs irrespective of the stimulus they receive, irrespective of the subset they belong to, and conserved across evolution ( ) . most of these genes are induced during dc maturation in part through cell-intrinsic ifn-i signaling ( ) . consistently, cell-intrinsic ifnar signaling in dcs is required in many circumstances for the induction of protective immunity, including efficient cd t cell responses during viral infection or tumor development ( ) ( ) ( ) , th responses upon polyi:c injection independently of il- or ifn-γ effects ( , ) , as well as follicular helper t cell and humoral responses ( , ) . mechanistically, ifn-i promote dc immunogenicity for efficient t cell activation through a variety of effects ( figure b) . it drives dc up-regulation of signal in vivo during viral infections ( ) and boosts their capacity to cross-present antigens for increased delivery of signal to cd t cells ( ) ( ) ( ) . it shapes their delivery of activating signal , in particular inducing il- and promoting or inhibiting il- depending on experimental conditions ( , ) . finally, it is necessary to induce their metabolic shift from mitochondrial oxidative phosphorylation to aerobic glycolysis, which fuels the increased needs in energy and the expansion of the intracellular organelles required for the production and proper intracellular routing of the signal and proteins ( , ). selective inactivation of ifnar on cdcs compromises mouse resistance to mcmv and mhv infections ( , ) . in contrast, ifnar expression is not required on nk cells for protection against mcmv and on pdcs, t cells, and b cells for early control of mhv replication ( , ) . although cell-intrinsic ifn-i signaling in nk cells can promote their activation ( ) (figure a) , ifn-i-induced il- trans-presentation by dcs plays a more prominent role for this function in many conditions including in vivo during mcmv infection ( , ) ( figure c) . cell-intrinsic ifn-i signaling in cd t cells ( ), cd t cells ( , ) , and b cells ( ) can also contribute to their efficient activation and functional polarization (figure ). this depends on experimental settings. cd t cell-intrinsic ifn-i responses are crucial for mounting efficient cytotoxic cd t cell responses against lcmv but are less critical against vaccinia virus and vesicular stomatitis virus ( , , ) . mechanistically, cell-intrinsic ifn-i signaling in cd t cells can promote their survival during their antigen-induced proliferation ( ) . cell-intrinsic signaling in dcs and cd t cells may act in a synergistic manner. indeed, conditional inactivation of ifn-i responsiveness was required to occur simultaneously in both of these two cell types to dramatically affect cd t cell expansion upon vaccination with a modified ankara vaccinia virus ( ) . in summary, ifn-i generally play a crucial, beneficial, role in immune defenses against viral infections, both through the induction of cell-intrinsic anti-viral defenses and through the orchestration of innate and adaptive immunity. however, if these responses are not properly regulated, they can contribute to diseases as we will now discuss. a frequent side effect of ifn-i treatment against cancer or chronic viral infections is the induction of autoimmune reactions. consistently, isg expression is a hallmark of many spontaneous systemic or tissue-specific autoimmune diseases, including systemic lupus erythematosous (sle), sjogren's syndrome, psoriasis, and other skin disorders ( ) . the dysregulation of ifn-i responses observed in patients with these autoimmune diseases likely results from both genetic and environmental factors. genome-wide association studies show that polymorphisms in genes involved in ifn-i responses strongly correlate with increased susceptibility to many autoimmune diseases ( ) . diverse environmental factors can also contribute to the onset of autoimmune diseases. microbial infections often precede first clinical manifestations of autoimmune diseases. whether infections ( ) and/or alterations in the commensal microbiota of the affected barrier tissues ( , ) are the cause or rather the consequence of autoimmunity is still matter of debate. infection-or dysbiosis-induced tissue damages and unbridled ifn-i responses can contribute to initiate autoimmune reactions. gender is another prominent factor affecting susceptibility to autoimmune diseases. women are more prone to autoimmunity, which may result from endocrine regulation of ifn-i responses. pdc ifn-i production is enhanced in human and mouse females, due at least in part to cell-intrinsic enhancement of tlr / responses by the female hormone estradiol ( ). in autoimmune diseases, different mechanisms could operate to initiate the dysregulation of immune responses leading to a vicious circle of reciprocal activation between innate ifn-i responses and adaptive self-reactive lymphocyte responses (figure ) . adaptive immune cells are educated to spare "self." this occurs through negative selection of potentially autoimmune b and t cells during their development in the bone marrow or thymus, respectively, a process called central tolerance. self-reactive b or t cells that have escaped this pruning can be either deleted or functionally inactivated once they have egressed in secondary lymphoid organs or non-lymphoid tissues, a process called peripheral tolerance. in some individuals, polymorphisms in genes involved in the promotion of central or peripheral tolerance lead to a higher number, diversity, and/or responsiveness of self-reactive lymphocytes in the periphery, in particular of b cells secreting anti-dna or anti-rnp antibodies ( , ) . mammalian dna or rna are poor inducers of pdc ifn-i induction under normal conditions. however, pre-existing anti-dna or anti-rnp autoantibodies can break this innate tolerance of pdc. indeed, antibodies binding to self nucleic acids can protect them from degradation and compact them into nanoparticles that are very effective for the induction of ifn-i in pdc (figure ) . dna-containing immune complexes (ics) are frequently found in the serum of sle patients (sle-ics) and can activate pdc ifn-i production ( ) . in turn, pdc ifn-i activate cdcs, monocytes ( ) , and b cells, leading to a vicious circle of reciprocal activation between dcs and frontiers in immunology | microbial immunology figure | a simplified model of the deleterious role of ifn-i in several autoimmune diseases. when exposed to different kinds of injuries (microbial infection, commensal microbiota dysbiosis, chemical or physical insults), healthy tissues can undergo cell damage and death. these events induce the release of apoptotic bodies encompassing self rna or dna. neutrophil recruitment and activation in inflamed tissues can also constitute a potent source of self nucleic acids, through the release of neutrophil extracellular traps (net). self rna or dna can associate with cationic peptides (e.g., ll ) as shown in psoriatic patients or with inflammatory molecules (e.g., high mobility group box , hmgb ) to generate nanoparticles that are extremely efficient for ifn-i production by pdc and eventually other cell types. pdc can also be efficiently activated for ifn-i production by immune complexes (ics) generated by the association between self nucleic acids and auto-antibodies as frequently found in the serum of systemic lupus erythematosus patients. ifn-i promote the differentiation and/or the maturation of antigen-presenting cells, in particular different subsets of dc. activated dc can then present self-antigens for activation of auto-reactive t cd + cells, including follicular helper lymphocytes, which in turn activate auto-reactive b cells for auto-antibody secretion, leading to a vicious circle of reciprocal activation between innate and auto-reactive adaptive immune cells. idc, immature dc; mdc, mature dc; mo-dc, monocyte-derived dc. see main text for further details. self-reactive lymphocytes and to the exacerbation of autoimmune responses (figure ) . certain infections or dysbiosis of the commensal microbiota of the affected barrier tissues could promote chronic production of host amphiphatic peptides able to combine with eukaryotic dna or rna, likely released from dying cells, thus forming pdc-activating nanoparticles. indeed, in psoriatic skin, both a high expression of ll and a massive infiltration of pdcs is observed ( ) (figure ) . hence, to treat many autoimmune diseases, novel therapeutic strategies could be designed to target dysregulated pdc ifn-i production or b cell activation by ifn-i. one of the most common complications of primary infections by many respiratory viruses, in particular influenza virus, is a lifethreatening pneumonia due to secondary pulmonary infections by bacteria, such as streptococcus pneumoniae, staphylococcus aureus, or haemophilus influenza ( , ) . these pathologies affect especially infants, elderly, and immunocompromised patients. retrospective studies indicate that secondary bacterial pneumonia was highly recurrent in lung tissues isolated from patients who died during last century influenza pandemics, independently of antibiotic availability ( , ) . influenza virus induces high ifn-i responses in human beings and mice. in both hosts, secondary bacterial infections are lethal only when they occur in a limited time window following primary viral infection ( - days), around the peak of ifn-i responses, before complete virus clearance. mouse models of viral/bacterial coinfections are being used to dissect disease mechanisms ( ) . ifnar -deficient mice appear more resistant to secondary pulmonary bacterial infections, showing that ifn-i responsiveness contributes to disease ( ) . similarly, after lymphochoriomeningitis virus (lcmv) infection, wild-type but not ifnar -deficient mice are more susceptible to lpsinduced septic shock ( ) . several mechanisms may contribute to the detrimental role of ifn-i in secondary bacterial infections ( figure ) . early during viral infection, ifn-i decrease the host ability to control bacterial replication, by dominantly polarizing immune responses toward anti-viral functions, simultaneously inhibiting the development of the types of immune responses required for protection against most bacterial infections. ifn-i can inhibit the production of chemokines required for the recruitment to the respiratory tract of antibacterial effector innate immune cells, in particular neutrophils or monocytes/macrophages ( , ) (figure ) . depending on the experimental models used, ifn-i can on the contrary induce a ccr -dependant recruitment of classical monocytes ( ) . in infected tissue, ifn-i might skew the functional polarization of resident or infiltrating monocytic cells toward immunosuppression, because it does limit their antibacterial functions by inhibiting their il- production ( ) ( ) ( ) while it might promote their production of il- and nitric oxygen intermediates. the exact nature of infiltrating monocytic cells is not clear and could correspond to activated classical monocytes, modcs, monocyte-derived macrophages, or myeloidderived suppressor cells (mdscs). the boundaries between these putatively different cell types are currently ill-defined ( ) . these cells could fuel local replication of monocyte/macrophage-tropic bacteria ( ) , be immunosuppressive ( ) or contribute to local immunopathology ( ) . the role of ifn-i on monocytes/macrophages is complex and will require further investigations to determine when it is protective versus deleterious and what the underlying mechanisms are. depending on the context, ifn-i can either promote or inhibit the induction of th cytokines such as il- and ifn-γ, and myeloid cell responses to ifn-γ ( , ( ) ( ) ( ) . ifn-i can also polarize cd t cell responses toward th at the expense of th , while the th -type cytokines il- a and il- are required for host defense against pulmonary www.frontiersin.org bacteria by inducing the production of anti-microbial peptides and of tissue repair molecules ( figure ) ( ) ( ) ( ) . ifn-i may not only affect host resistance to bacterial infection, but also host tolerance, i.e., the ability of the host to tolerate a given burden of pathogen without undergoing excessive tissue damages ( , ) . hence, to counter ifn-i deleterious effects during secondary bacterial infections, it will be important to better delineate the respective contribution of lung tissue tolerance modulation and of immune-mediated resistance weakening. another well documented example of deleterious effects of ifn-i due to their inappropriate functional polarization of immune responses is the enhanced susceptibility to fungal infections of patients with genetically determined hyperactive ifn-i responses, as exemplified in the hereditary disease chronic mucocutaneous candidiadis (cmc) (figure ) ( ) . patients with cmc have a significant deficit in th cd t cells, at least in part as a consequence of altered responsiveness to il- or il- . several stat mutations were identified in patients with autosomal dominant cmc. gain-of-function stat mutations were found to hard wire cd t cell responses to cytokines toward stat signaling, compromising their stat -dependent ability to produce il- upon il- or il- stimulation. this was associated to induction of a global ifn-i transcriptomic signature in blood ( ) . deleterious ifn-i effects on immunity to candida might not only occur in cmc patients but also in other types of individuals upon secondary fungal infections occurring shortly after a primary viral infection, likewise to the situation discussed above for secondary bacterial infections. indeed, polyic induced ifn-i abrogate innate immunity to systemic candidiasis in mice ( ) , and ifnar-deficient mice can be more resistant to candida infection under certain experimental settings ( ) . however, the role of ifn-i in the modulation of the ability of immunocompetent hosts to control fungal infection is disputed ( , ) . the inhibition of th responses by ifn-i could be protective in at least one important human pathology, ms (figure ) . ms represents a striking exception to the previously discussed detrimental role of ifn-i in autoimmune diseases. indeed, a large proportion of ms patients have low serum ifn-i activity and low isg levels. these ms patients present a significant reduction of ms relapse upon ifn-β administration ( ) . the underlying mechanisms are not yet completely unraveled. however, in the experimental autoimmune encephalitis mouse model of ms, th responses bear a major contribution to nervous system damages and are inhibited by the il- and il- induced upon ifn-i administration ( ) . in summary, ifn-i responses can be deleterious in autoimmunity by promoting a vicious circle of reciprocal activation between innate immune cells and auto-reactive cd t or b lymphocytes. ifn-i responses can also be deleterious upon secondary bacterial or fungal infections in the lung or the kidneys occurring shortly after a primary viral infection, by compromising the recruitment of anti-microbial innate effector cells and/or by preventing the proper functional polarization of immune responses. we will now discuss how ifn-i responses can also compromise host immune defenses against certain viruses and promote chronic infections. different lcmv strains such as armstrong and clone- (cl ), respectively, lead to acute versus chronic infections in mice. a hallmark of chronic lcmv infection is the loss of the proliferative potential and effector functions of anti-viral cd t cells, a process called exhaustion. exhausted cd t cells are characterized by a high expression of the inhibitory receptors pd- , ctla , and lag- ( ) . in vivo blockade of these inhibitory receptors can reverse t cell exhaustion and allow resolution of the chronic infection ( ) . ifn-i and isgs are induced early after infection with all strains of lcmv, albeit to lower levels with those leading to chronic infection. this early ifn-i production is critical to limit viral replication ( ). in models of acute infection, ifn-i responses rapidly return to normal, undetectable, levels, before viral replication is completely controlled. in contrast, isg induction is maintained in chronic infection, including the expression of pd- ligands on apcs and of the immunosuppressive il- cytokine, consistent with a prolonged expression of ifn-i albeit at low levels ( , ) . in vivo neutralization of ifn-i by antibody administration promoted resolution of chronic lcmv cl infection, allowing the frontiers in immunology | microbial immunology restoration of functional anti-viral cd t cell responses at least in part through cd t cell-and ifn-γ-dependent mechanisms ( , ) . during persistent lcmv cl infection, chronic low level ifn-i production polarizes cd t cell responses toward t follicular helper (tfh) rather than th functions. thus, chronic ifn-i responses promote enhanced anti-viral b cell responses but facilitate cd t cell exhaustion due to deficient cd t cell help, therefore contributing to host failure to prevent chronic infection ( ) . strikingly, establishment of chronic infection by lcmv cl could also be prevented by early administration of two shots of a high dose of exogenous ifn-i, at days and post-lcmv inoculation. this treatment allowed viral clearance by rescuing anti-viral cd t cell from exhaustion ( ) . altogether, these studies show that the timing and duration of ifn-i production during viral infections is critical in determining how this response will impact the balance between the virus and the host. an early and robust but transient production of ifn-i promotes strong induction of cell-intrinsic viral restriction mechanisms as well as adequate polarization of adaptive anti-viral immune responses, which combined effects lead to viral clearance. in contrast, if the production of ifn-i is too low and/or too late, both viral replication and low ifn-i responses become chronic, their combined action leading to induction of immunosuppressive effects and to inadequate functional polarization of cd t cells. this results in cd t cell exhaustion and maintenance of chronic infection. chronic viral replication and cd t cell exhaustion is also a hallmark of hiv- infection. we will now discuss the complex and disputed role of ifn-i in this disease. both in hiv- infection and in its most relevant animal model, infection of non-human primates with simian immunodeficiency virus (siv), disease progression after the acute phase of the infection is associated with high and chronic expression of isgs while ifn-i production is inconsistently detected ( ) ( ) ( ) . in contrast, the individuals that do not progress toward disease despite persistent high viral loads show much lower immune activation, in particular low isg expression, after the acute phase of the infection ( ) ( ) ( ) ( ) . hence, chronic low levels of ifn-i are associated to disease progression independently of the level of viral replication. therefore, an outstanding question still open for a better understanding of the physiopathology of hiv- infection is whether chronic ifn-i responses are merely a marker of progression, or whether they are implicated in driving disease development. in addition to mechanisms similar to those uncovered in the mouse model of chronic lcmv infection, during hiv- infection other effects of ifn-i could promote a vicious circle of reciprocal activation between chronic viral replication and sustained, deleterious immune responses (figure ). very early after hiv- infection, in most individuals, ifn-i production might be too weak or too late to induce a combination of cell-intrinsic defense mechanisms and of immune responses efficient enough to prevent later establishment of chronic infection. on the contrary, as demonstrated in the case of the mouse model of lcmv infection, ifn-i responses could favor cd t cell exhaustion, either by direct cell-intrinsic effects on cd t cells (figure , ) or by contributing to deprive them from cd t cell help (figure , ) . several effects of ifn-i might compromise anti-hiv- th responses or more generally contribute to the global depletion of cd t cells. these mechanisms include functional polarization of anti-hiv- cd t cells toward tfh rather than th responses, cxcl production leading to enhance recruitment of memory cd t cells to the sites of viral replication where they fuel chronic viral replication with new hiv- target cells (figure , to ) , direct pro-apoptotic and anti-proliferative effects on cd t cells (figure , ), as well as trail induction on pdcs licensing them for killing cd t cells irrespective of their infection (figure , ) ( , ) . altogether, these mechanisms entertain chronic viral replication and continuous depletion of cd t cells, leading to the dramatically enhanced susceptibility to opportunistic infections (figure , ) characteristic of the acquired immunodeficiency syndrome (aids) (figure , ) . other lines of evidences have been reported to support a deleterious role of pdc activation during hiv- infection. women undergo faster hiv- disease progression than men with similar viral loads, which may result in part from the highest ifn-i production of women's pdcs including in response to hiv- stimulation ( ) . pdc recruitment and activation in the vaginal mucosa of female macaques early after local siv inoculation contribute to attract and activate cd t cells, which can then be infected and promote virus dissemination from its portal of entry ( ) . however, in vivo blockade of pdc ifn-α production by administration of tlr / -antagonistic oligonucleotides early after siv infection of macaques did not decrease t lymphocyte activation, which suggests that additional sources of ifn-i likely contribute to the immune dysfunction observed in siv/hiv- infections. targeting dysregulated ifn-i responses during hiv- infection might represent an interesting adjuvant therapeutic strategy to highly active antiretroviral treatments. administration of ifn-i in the non-pathogenic siv infection model of sooty mangabeys was not sufficient to switch it into a pathogenic model. no cd t cell depletion ensued, no hyperactivation of immune responses were observed. viral loads were even significantly decreased. however, this could be consistent with the positive impact of early and high dose ifn-i administration in chronic lcmv infection ( ) . indeed, during the review process of this manuscript, it was reported that, early during primary siv infection in the pathogenic rhesus macaque model, a high dose injection of ifn-i was protective while neutralization of endogenous ifn-i was deleterious. in contrast, in the same animal model, prolonged ifn-i administration accelerated disease development in the chronic stage of the infection ( ) . in mice with a humanized immune system, pdc depletion strongly decreased isg induction and enhanced viral replication both in the acute and chronic phases of hiv- infection. however, pdc depletion during chronic infection decreased infection-induced t cell apoptosis and increased t cell numbers in lymphoid organs ( ) . these results further emphasize the dual role of ifn-i and pdcs in the physiopathology of hiv- infection. a strong and transient production of ifn-i early after infection benefits the host by lowering the set-point of viral replication during chronic infection. sustained production of low levels of ifn-i during chronic infection contributes to immune dysregulation and cd t cell depletion. further studies will be necessary to examine whether complementing standard-of-use antiretroviral drugs with pdc www.frontiersin.org depletion, ifn-i neutralization, or selective inhibition of t cell responses to ifn-i could yield additional benefits to chemotherapy in non-human primates during chronic siv infection. ifn-i administration has been used for many years to treat another human chronic viral disease, hcv infection. roughly, half of the patients do not show sustained virological responses (svr). the treatment causes severe side effects in many individuals. new chemotherapeutic drugs very potent at blocking hcv replication in vivo have recently become available. hence, whether ifn-i administration still constitutes a viable treatment against chronic hcv infection is being questioned ( , ) . we will now discuss this issue. chronic hcv infection is the main cause of liver cirrhosis and hepatocellular carcinoma. there is currently no vaccine against hcv. the most common therapy for chronic hcv patients is the administration of recombinant pegylated ifn-α (peg-ifn-α) combined with the anti-viral drug ribavirin. however, because of ifnar pleiotropic expression, ifn-α administration induces severe side effects including flu-like syndrome, fever, fatigue, myalgia, and nervous depression ( figure a ) ( ) . moreover, only about half of treated patients harbor svr ( ) . prior-totreatment high hepatic isg expression is a negative predictor of svr upon peg-ifn-α therapy. high isg expression in untreated patients likely results from chronic but low ifn-i production triggered by persistent hcv replication. indeed, hepatocytes from non-responder patients were found to be infected at a greater frequency and to exhibit dampened antiviral and cell death responses ( ) . what the cellular sources of ifn-i production are and why they persist only in non-responder patients still remain to be established. in chronic hcv infection, cytotoxic effector lymphocytes may contribute to the development of hepatocarcinoma by causing low level but sustained hepatocyte death and renewal. in contrast, local production of ifn-γ in the liver by nk and t lymphocytes could promote resistance to disease through non-cytolytic control of viral replication. as discussed previously for lcmv and hiv- , low chronic production of endogenous ifn-i in hcv patients could compromise both innate and adaptive anti-viral immune responses. chronic exposure to ifn-i could dampen the ability of frontiers in immunology | microbial immunology utr of ifnl mrna to promote their degradation. the favorable ifnl allele associated with responsiveness to peg-ifn-α treatment may allow endogenous expression of sufficient levels of ifnl for efficient induction of cell-intrinsic anti-viral defenses in hepatocytes. this process is, however, hampered by the limited expression of the receptor for this cytokine (ifnλr ) in these patients. peg-ifn-α treatment might promote resolution of the infection by inducing ifnλr in these patients, potentiating their response to their endogenous production of ifnl . in the patients that do not respond to peg-ifn-α treatment, endogenous levels of ifnl are insufficient for efficient induction of cell-intrinsic anti-viral defenses in hepatocytes, due to the degradation of the corresponding mrna in infected hepatocytes. in these patient's hepatocytes, however, ifnλr is already expressed to high levels prior to treatment due to their high endogenous ifn-i responses. administration of exogenous ifn-λ might cure these patients. see main text for further details. nk and cd t cells to produce ifn-γ ( , ) and promote cd t cell exhaustion ( ) . it could also induce an antagonist form of cxcl , a chemokine required for recruitment to the liver of anti-viral nk and cd t cell effectors ( ) . it may also polarize monocytes toward immunosuppressive functions ( ) . therefore, better understanding ifn-i effects in hcv infection is critical to improve care of both responders and non-responder patients to peg-ifn-α. for responder patients, the issue is to modify the treatment to favor beneficial antiviral and immunoactivating effects over side effects strongly affecting patient's quality of life (figure a ). this might be achieved by specific delivery of ifn-i to targeted cell types as discussed later. for non-responder www.frontiersin.org patients, the issue is to understand the mechanisms underlying treatment failure to determine whether alternative therapies could be designed (figure a) . genome-wide association studies identified various single nucleotide polymorphisms (snp) in the gene encoding il- b/ifn-λ , one of the ifn-iii, as well in its and non-coding regions ( ) ( ) ( ) ( ) . one snp, called rs , is located kb upstream of the ifnl gene. patients harboring the cc genotype have a favorable prognosis to ifn-i treatment. patients with the tt genotype are at high risk of treatment failure ( , ) . in europeans, the favorable cc genotype is the major, most common, ifnl allele. the unfavorable tt snp is the minor allele. the frequency of these alleles is reversed in africans. the favorable allele allows escape of ifnl mrna from degradation by cellular microrna induced upon hcv infection ( ) . until recently, ifnl genotypes and hepatic isg expression were considered as independent predictors of response to peg-ifn-α treatment in hcv patients ( ) . here, we propose a potential explanation, which integrates both factors in a relatively simple model ( figure b ). our main hypothesis is that efficient control of hcv infection depends on hepatocyte response to ifn-λ rather than ifn-α. this is supported by reports that ifn-λ induces a stronger and more sustained isg expression in hepatocyte cell lines in vitro ( ) , and that polyi:c-induced control of hcv replication in humanized liver in chimeric mice is correlated to the induction of ifn-λ but not ifn-i in human hepatocytes ( ) . responder patients harboring the favorable ifnl allele preventing the degradation of the corresponding rna in infected cells might express significant levels of endogenous ifn-λ , although this is disputed. however, they express only low levels of ifn-λr , which limits ifnλ efficiency ( figure b ) ( ) . how these patients benefit from peg-ifn-α treatment could be that it induces ifn-λr expression on hepatocytes thus boosting endogenous ifn-λ effects ( ) . in contrast, high isg-expressing non-responder patients harboring the unfavorable ifnl allele might not express enough ifn-λ for virus control. however, they do express ifn-λr as a result of their endogenous production of ifn-i. hence, peg-ifn-α might be ineffective in these patients because they already express ifn-λr but fail to produce endogenous ifn-λ due to the degradation of its mrna in infected hepatocytes (figure b) . these patients may be good candidates for peg-ifn-λ therapy, currently undergoing clinical development. since the expression of ifn-λr is mainly restricted to epithelial cells, melanocytes, and hepatocytes, some of the side effects related to ifn-i treatment might be strongly attenuated in peg-ifn-λ therapy. however, as ifn-i are key to induce anti-viral immune responses, it will be critical to determine whether, beside viral clearance, peg-ifn-λ therapy can also induce long-term immune protection against hcv. ifn-i transduce intracellular signals through a single receptor, ifnar, but via a multitude of downstream signaling pathways. the janus activated kinase (jak)/stat pathway was the first to be identified ( ) . ifnar is composed of two distinct subunits, ifnar and ifnar , which are constitutively associated with members of the jak family, tyrosine kinase (tyk ) and jak , respectively ( ) . the binding of ifn-i to their receptor leads to the phosphorylation of jak and tyk , which in turn induce the phosphorylation and activation of the stat proteins ( ) . different stat complexes can form upon triggering of ifnar (figure ) . a transcriptional complex that forms in most conditions of ifn-i stimulation and induces the expression of many molecules of cell-intrinsic anti-viral immunity is interferonstimulated gene factor (isgf ), a heterotrimer composed of pstat , pstat , and irf ( ) (figure ) . following its translocation into the nucleus, isgf binds to isre regulatory sequences in target genes. many molecules playing a key role in the function of innate or adaptive immune leukocytes are also induced by isgf , including cd , cd , or il- in dc, and granzyme b in nk cells. isgf is generally composed of stat phosphorylated on tyr and ser and of stat phosphorylated on tyr . however, alternative isgf complexes have been described in various contexts which could participate to the diversity of ifn-i effects ( ) . the pstat homodimer also plays a prominent role in cell-intrinsic ifn-i-dependent gene induction. it binds ifnγactivated sequences (gas) and controls the expression of many pro-inflammatory molecules ( ) . pstat homodimers can form upon stimulation with either ifn-i or ifn-γ. many gasregulated genes can be induced by either cytokines. depending on cell types, jak signaling downstream of ifnar can lead to the activation of virtually all stat proteins and to their combinatorial association into a variety of complexes with different affinities for specific gas elements ( ) ( ) ( ) (figure ) . this diversity contributes to ifn-i induction of different transcriptional programs in distinct cell types ( ) . stat complex formation depends in part on the relative abundance of stat molecules in the cell ( ) . while stat , stat , stat , and stat can be activated in most cell populations, stat and stat are mainly activated in lymphocytes ( ) . for example, quiescent nk cells express more stat than stat , leading to constitutive association of ifnar to stat in these cells. hence, quiescent nk cells mount pstat homodimer-dependent responses to ifn-i stimulation, including ifn-γ production and t-bet-driven proliferation (figure ) ( , ) . changes in stat levels can also occur upon the differentiation/activation of a given cell type and lead to a shift in its functional response to the cytokines ( ) . upon activation, nk cells decrease their expression of stat and increase that of stat , shifting their ifn-i response from stat -dependent in a quiescent state to stat dependent in pre-activated cells. this translates into opposite ifn-i effects on ifn-γ production and proliferation for quiescent versus pre-activated nk cells ( ) . however, this outcome can be modulated by simultaneous exposure to other cytokines such as il- or il- / . a reverse stat -to-stat shift occurs in dc during their maturation, shifting their functional responses from inhibition to activation of il- production in response to combined stimulations with ifn-i and cd l ( ) . this frontiers in immunology | microbial immunology ifn-i binding to ifnar triggers the phosphorylation of tyk and jak , which in turn phosphorylate a variety of stat proteins. activated stats are able to form complexes, as homo-or hetero-dimers. the heterodimer stat -stat binds to a third partner, ifn-regulatory factor (irf ), in order to form the isgf complex. this complex translocates into the nucleus and binds to specific regulatory sequences, ifn-stimulated response elements (isre), to activate the expression of many interferon-stimulated genes (isgs). in particular, isgf induces most, if not all, of the isgs encoding effector molecules of cell-intrinsic anti-viral defenses such as oas or mx . alternative jak/stat pathways include the formation of stat or stat homodimers, which may drive different functional responses to ifn-i. stat homodimers bind to ifnγ-activated sequences (gas) in the promoter of certain isgs, which may promote inflammatory, anti-proliferative, and pro-apoptotic responses. stat homodimers also bind to gas but promote ifn-γ production and pro-proliferative responses. enables mature dc to efficiently activate cd t cells. other yet unknown mechanisms control the formation of different stat complexes in distinct cell types. the nature and dynamics of the signaling pathways triggered by ifn-α or -β were evaluated in bulk cultures of human blood leukocytes using flow cytometry ( ) or high throughput mass cytometry ( ) . a diversity of phosphorylation patterns of stat / / was observed upon ifn-i stimulation. ifn-α activation induced phosphorylation of stat , stat , and stat in most cell types, peaking at min ( ) . ifn-β-induced stat phosphorylation was found to be poor in b cells as compared to monocytes and t cells ( ) . however, the underlying mechanism remains to be identified since b cells did not express lower amount of ifnar or stat or enhanced levels of the inhibitory socs molecule. the high stat activation in monocytes led to their induction of ifn-i-dependent pro-apoptotic genes while this was not the case in b cells. these results strikingly differ from those obtained in the other study upon ifn-α stimulation, where stat phosphorylation was on the contrary lower in cd + monocytes and was prolonged in b cells and nk cells ( ) . the differences between these two studies might have resulted from the use of different subsets and doses of ifn-i. in any case, both studies consistently reported that cd t cells showed the highest activation of stat . all cd t cells but not all cd t cells activated stat and for a longer time ( ) . ifn-β activation of stat was delayed in cd t cells and b cells as compared to cd t cells and monocytes ( ) . different stat complexes may lead to distinct transcriptional programs linked to different biological functions (figure ) . more systematic studies are needed to understand this complexity. besides changing stat levels between cell types or www.frontiersin.org activation states, the processes controlling differential formation of stat complexes downstream of ifnar triggering remain to be identified. in addition to jak/stat signaling, other pathways can be activated downstream of ifnar, including those involving the phosphatidylinositol -kinase (pi k), mitogen-activated protein kinases (mapk), and the crk adaptor molecules ( , ) . this leads to the activation of other transcription factors such as irf, nf-κb, or pu. , which contribute to orchestrate cell responses to the cytokines by regulating both distinct and overlapping sets of genes as compared to stat ( , ) . in summary, ifnar signals through a remarkable diversity of pathways, including but not limited to diverse combinations and kinetics of stat phosphorylations. this explains at least in part the diversity of ifn-i effects, including their induction of opposite responses depending on the physiopathological contexts and/or the nature of the principal responding cell types ( , ) . ifn-iii induce the same signaling pathways as ifn-i, although they engage a different heterodimeric receptor, composed of the il- ra and il- rb chains and preferentially expressed on epithelial cells including hepatocytes. in mice and human beings, numerous ifn-i subtypes exist. functional and population genetic analyses showed that these ifn-i subtypes significantly differ in their functions ( ) ( ) ( ) ( ) ( ) ( ) . hence, one of most extraordinary feature of ifn-i biology is how ifn-i subtypes can elicit so many pleiotropic and diverse functions by interacting with the same receptor complex ( ) . both ifnar and ifnar are required for the initiation of ifn-i-dependent signals, as mice deficient in either one are highly susceptible to viral infections ( , ) . the assembling of the ifnreceptor ternary complex is a two-step process. first, a binary complex is formed by the binding of one side of the ifn molecule to ifnar . then, a single binary complex interacts with ifnar via the other side of the ifn molecule. the stability of the ternary complex will be determined in part by the association and dissociation kinetics between the cytokine and the two receptor chains, as well as by ifnar expression levels since the cell surface concentrations of the receptor subunits are relatively low. hence, both the affinity of ifn-i subsets for ifnar and the amounts of ifn-i, ifnar , and ifnar will regulate their biological effects ( figure a ) ( , ) . cell membrane density of ifnar and ifnar is also involved in differential ifn-β-versus ifn-α-induced functional activities, such as anti-proliferative function ( ) . a variety of cell-intrinsic parameters can also impact the lifetime of the ifn-receptor ternary complex, such as the rate of endocytosis/degradation/recycling of signaling complexes, and negative isg regulators such as usp that decrease the affinity of ifn-ifnar binding ( , ) . based on a definition of a prototypic cytokine-receptor binding module and by analogy with the epo receptor system, ifn-i subtypes were originally postulated to form ternary complexes of differing architectures, resulting in distinct geometry and assembling of intracellular signaling components ( ) . experimental evidence rejected this hypothesis. rather, the differential activities of ifn-i subtypes are determined by the stability of the ligand/receptor ternary complex ( , ) . differential affinities of the ifn-i subtypes for ifnar and ifnar extracellular domains generate subtype-specific signaling cascades and biological outcomes ( figure a ) ( , ) . crystal structure of ternary ifn-i/ifnar /ifnar complex illuminated the biochemical complexity of ifn-i interaction with their cognate receptors ( ) . the main conformational features of ifn-i/ifnar /ifnar ternary complexes are conserved among the different ifn-i, but are quite different from the other cytokine receptors ( , ) . in the formation of the binary ifn-i/ifnar complex, ifn-i ligand discrimination resides on differential energetics during the interaction of anchor points with ifnar , shared by all ifn-i, as well as on key amino acid substitution among ifn-i subtypes ( ) . ifnar then performs major conformational changes to interact with ifn-i associated in the binary complex, thus displaying an optimized functional plasticity ( ) . these differences in the chemistry of ifn-i subtype interaction with ifnar and ifnar thus explain the different affinities of ifn-α versus ifn-β within ternary complex and their differential activities ( ) . the functions regulated by ifn-i strongly depend on the main responding cell types ( figure b ). this has been studied in vitro by examining the functional consequences of the stimulation of different cell types with ifn-i, and in vivo by determining the contribution of cell-intrinsic ifn-i responses of different cell types to resistance or susceptibility to various diseases. an emerging concept is the central role of dc responses to ifn-i for induction of protective immunity against viral infections or tumors (figure ) . the development of mutant mice allowing conditional genetic inactivation of ifnar in a cell-type specific manner using the cre-lox system ( ) has been instrumental in accelerating our understanding of how different cell types respond to ifn-i in vivo and what their respective contribution is to protective or deleterious ifn-i responses. this has been investigated most extensively in viral infections ( , , , , ) but also in cancer ( , ) , bacterial infections ( ), autoimmunity ( , ) , sepsis ( ), or inflammatory diseases ( ) . efforts are being pursued to better understand which cell types respond to ifn-i in a manner promoting protective versus deleterious effects in different physiopathological settings. that knowledge will considerably help to develop novel strategies to modulate ifn-i functions for promoting health over disease. the development of mutant mice allowing conditional genetic inactivation of stat , stat , and stat ( - ) will help better understanding how different signaling pathways in different cell types determine the outcome of ifn-i response in vivo in various conditions. this knowledge might lead to the development of strategies aiming at targeting a given cell type with a specific subset of ifn-i, or in the presence of antagonists of certain signaling pathways, to surgically tune ifn-i responses in vivo toward the most desirable outcome. frontiers in immunology | microbial immunology for example, the affinity of ifn-β for ifnar is -times higher than that of ifn-α , and ifnβ is much more potent in inhibiting cellular proliferation or (continued ) ifn-i can also determine distinct functional outcomes. for example, during viral infections, early and transient high levels of ifn-i promote protective dc and t cell responses, while delayed, chronic and low level ifn-i production compromises host immune defenses and promotes chronic viral infections. within a given cell type, the outcome of ifn-i stimulation also depends on time of exposure to these cytokines relative to other modulatory signals (timing relative to other stimuli). for example, in naïve cd t cells, tcr signaling prior to ifn-i stimulation leads to increased expression of stat and promotes ifn-γ production and proliferation, while ifn-i stimulation prior to tcr triggering leads to stat -dependent anti-proliferative and pro-apoptotic effects. the formation of specific stat complexes is a highly dynamic process. it depends not only on the cell type but also on its specific state at the time it sees ifn-i. hence, major parameters controlling the effects of ifn-i in a given cell type also include its microenvironment ( figure c ) and the timing of its exposure to the cytokines both in terms of duration of the stimulation and of previous activation history ( figure d) . the tam receptor ligand gas is expressed within tumor cells in various solid cancers ( , ) . elevated gas expression is of bad prognosis in different cancers ( , ) . in a mouse model of ovarian cancer, early during tumorigenesis tumor-infiltrating dcs were found to be immunogenic and promote antitumor immunity, but they were later altered in the course of tumor development to acquire immunosuppressive properties beneficial to the tumor ( ) . one may thus hypothesize that expression of tam soluble ligands in certain tumors and of tam receptors on tumor-infiltrating dcs might contribute to dampen dc response to ifn-i and therefore facilitate their polarization by the tumor microenvironment into immunosuppressive cells (figure c) . acute versus chronic exposure to ifn-i can lead to strikingly opposite effects on a given cell type ( , , ) . in addition to duration, the time when a cell is exposed to ifn-i can also dramatically impact its functional response, depending on its previous activation history ( figure d) . in vitro stimulation of dcs with ifn-β can lead to opposite outcomes depending whether it occurs simultaneously to, or after, tnfα-induced maturation. ifn-β polarizes dcs toward th induction in the former case, and toward il- -secreting t cells in the latter case. these opposite effects result at least in part from the differential expression of il- / by dcs ( ) . similarly, ifn-i effect on the functional polarization of cd t cells is strongly modulated by the other cytokines present in the lymphocyte microenvironment at the same time ( ) . ifn-i can also mediate opposite effects on cd t cells depending whether it occurs before or after cognate engagement of the t cell receptor. indeed, while cd t cells have the potential to respond to ifn-i by inducing both stat -and stat -dependent genes, this depends upon their activation history. naïve cd t cells respond mostly to ifn-i through stat signaling, leading to the inhibition of their proliferation and eventually to the induction of their apoptosis. however, cognate triggering of the t cell receptor causes a decrease in stat and an increase in stat expression in cd t cells. this leads to a shift of their ifn-i response from stat -to-stat signaling, resulting in the promotion of their proliferation and ifn-γ production. during lcmv infection, this mechanism promotes stat -dependant expansion of anti-viral cd t cells, but stat -dependant inhibition of naïve cd t cell proliferation ( ) . since the late s the clinical potential of ifn-i for the treatment of patients suffering of viral infection or cancer diseases has been widely acknowledged ( ) . today, this expectation is tempered because ifn-i treatment can induce severe side effects and sufficient doses cannot be administered in patients. therefore, there is a strong need to create tuned ifn molecules devoid of side effects. based on our current understanding of ifn-i responses as reviewed above, many parameters could be tuned individually or in a combined manner to modulate ifn-i activity to promote their beneficial effects over the deleterious ones in a number of diseases. these parameters include modifying the affinity of ifn-i for its receptor, playing with the local quantity/concentration of ifn-i and with the duration of its delivery, and modulating the nature of the cells that are responding to ifn-i. we will discuss here novel strategies being developed to deliver ifn-i to, or block ifn-i responsiveness of, a specific target cell type in vivo (figure ). if ifn-i-induced side effects are a consequence of the pleiotropic nature of ifn-i, and if the bioactivities mediating deleterious effects have some degree of independence from those mediating beneficial effects, one could mutate the ifn-i molecules in order to skew their activity toward a desired bioactivity. indeed, introducing key mutation in ifn-α allowed increasing its affinity to ifnar by a factor of . accordingly, this ifn-α mutant is times more potent in inhibiting cell proliferation, but as potent as wt ifn-α in inducing an anti-viral state ( ) ( ) ( ) . hence, it is possible to tune ifn activity by modifying its binding to ifnar. however, translating such an approach for the design of molecules for clinical application is severely hampered by the poor understanding we have on the ifn-i bioactivities mediating the side effects. furthermore, we are far from having established the list of bioactivities that could be differentially modulated by changing the stability of the ifn-i/ifnar complex. we know more about the frontiers in immunology | microbial immunology cell types that mediate beneficial versus deleterious ifn responses in various diseases. hence, we will now discuss strategies aimed at focusing ifn activity to specific cell types to promote health over disease. several strategies have been developed to specifically target ifns on tumor cells, tumor-infiltrated immune cells or infected tissues. these strategies include intra-lesional injection ( , ) , adenoviral-mediated gene transfer ( ) ( ) ( ) , engineered tumorinfiltrating monocytes ( ) , and fusion of ifns with a cleavable protecting shell ( ) . another strategy to increase cytokine accumulation within the tumor or infected tissue is antibody-mediated targeting of cytokine delivery, where a cytokine moiety is fused to an antibody directed against a specific cell surface marker (figure ) . the fusion molecule retains both antigen-binding and ifn-i bioactivities, and is enriched at the targeted site upon in vivo injection ( ) ( ) ( ) ( ) . when targeted to human cd , ifn-i inhibited the proliferation of lymphoma cells engrafted in immunodeficient mice ( ) . an ifn-i targeted to a tumor antigen can also amplify the therapeutic effect of the antibody by acting on tumor-infiltrated dcs, thus increasing antigen cross-presentation and antitumor cytotoxic t cell responses ( ) . on non-targeted cells, the antibody conjugation negatively impacts ifn-i potency, but only modestly ( , , ) (figure a) . fusion molecules generally retain full ifn-i biological activity on the cells expressing the antibody target ( figure b) . hence, this difference only leads to a modest ratio between the ifn-i specific activity measured on target and non-target cells (figure b) . such a targeting efficiency is definitely too low to reduce the toxic effect of ifn-i administration, because it will not specifically focus ifn-i activities on "beneficial cells" without stimulating "deleterious cells." the engineering of immuno-ifn-i must be improved to reach the very high targeting efficacy required to significantly diminish the treatment side effects. we recently reported an innovative strategy reaching this goal ( ) . it is based on the postulate that the antibody moiety of an immuno-ifn-i stabilizes the ifn-i/receptor-complex by avidity. it also takes into account the fact that the biological potency of an ifn-i is proportional to the stability of the ifn-i/receptor complex up to a certain threshold beyond which increasing the stability does not increase its potency ( , ) . ifn-α and ifn-β are used in most immuno-ifn-i studies. they have evolved to retain close to maximal potency. hence, their targeting by an antibody that only provides a modest gain in terms of biological potency. however, it is expected that decreasing the affinity of the ifn-i for its receptor, by introducing a mutation, would increase the targeting effect of the antibody (figures c,d) . this is indeed the case. using an ifn-i with a single point mutation that dramatically decreases its affinity for ifnar ( figure c ) allows engineering immuno-ifns that are up to -fold more potent on cells expressing the antibody target ( figure d) . the three log targeting efficiency of these novel types of immuno-ifns is found for various activities measured in vitro or in vivo when delivered in mice. if the toxic side effect experienced by the patients treated with ifn-i is due to systemic ifn-i activity, this targeting technology may find considerable clinical applications since such engineered immuno-ifns are virtually inactive while "en route" and are activated only after binding of the fused antibody to the desired target. it remains to define the useful targets according to pathologies, for example, tumor cells themselves and professional cross-presenting xcr + dcs for cancer ( , , ) , or hepatocytes for chronic hcv infection. to treat autoimmune diseases, novel therapeutics targeting ifn-i have been developed, including two ifn-α-neutralizing monoclonal antibodies currently in clinical trials (sifalimumab and rontalizumab) ( , ) . however, long-term systemic neutralization of ifn-i activity may increase susceptibility to viral infection and tumor development. alternative strategies are needed to specifically inhibit ifn-i deleterious effects in these diseases without globally compromising ifn-i anti-viral and www.frontiersin.org anti-tumoral functions. the sequential nature of the assembling of the ifn-i/receptor complex opens the possibility to design ifn-i antagonists specifically targeting the cell subsets responsible for ifn-i deleterious effects. an ifn-α carrying a single amino acid substitution that blocks the ifn-i/ifnar interaction engages ifnar in a complex, which cannot bind ifnar ( ) . since the binary ifn-i/ifnar complex is devoid of any ifn-i activity, such mutant behaves as a potent ifn-i antagonist. when linked to an antibody specific for a cell surface marker, the antagonistic activity of the mutant ifn-i should be significantly reinforced specifically on the cells expressing the target. hence, it should be possible to design and construct targeted antagonists that inhibit responsiveness to endogenous ifn-i specifically on the cell subsets on which the cytokines act to promote autoimmunity or severe side effects, leaving the other cells fully responsive. for example, in chronic hcv patients treated with peg-ifn-α, one of the most deleterious side effects is nervous depression, which might be prevented by co-administration of an ifn-i antagonist specifically targeting neurons or other cells of the central nervous system. in the last decade, several major technological breakthroughs and the generation of novel animal models have remarkably advanced our understanding of the mode of action of ifns. in vitro high throughput screening allowed systematically studying the functions of isgs by ectopic expression or knock-down. advance biophysical investigation of the interactions between ifn-i and the ifn-i receptor allowed to rigorously investigate the mechanistic basis for the differential bioactivities of ifn-i subtypes. the analyses of the responses of different cell types to ifns or to viral infection, in vitro but also in vivo in various pathologies, demonstrated that ifn-i often mediate beneficial versus deleterious roles by acting on different cell types. from integrative analysis of these data, a picture is now emerging suggesting that it will be possible to segregate protective from deleterious ifn-i effects, based (i) on their differential induction depending on ifn-i subsets or on the magnitude/timing of ifn-i production, (ii) on their conditioning in different tissues, (iii) or on their occurrence in different cell types. hence, innovative immunotherapeutic treatments are being designed to tune ifn-i activity toward desired effects in order to promote health over disease in a manner adapted to each physiopathological condition. in particular, a proof-of-concept has been made in vitro that it will be possible to target ifn-i activity on given cell types or tissues to administer to patients sufficiently high doses of the cytokine at the site of interest while limiting unwanted effects in other tissues or cell types. the next steps will be to demonstrate efficacy of this strategy in vivo in preclinical animal models. importantly, to foster the development of these innovative immunotherapies, major efforts are still warranted to continue delineating which cell types are mainly responsible for the protective versus deleterious effects of ifn-i in different diseases. combining high throughput technologies and systems biology approaches will also advance our understanding of the molecular mechanisms dynamically controlling ifn-i responses in health and diseases, which should reveal potentially novel therapeutic targets. virus interference. i. the interferon virus interference. ii. some properties of interferon functional role of type i and type ii interferons in antiviral defense inborn errors of interferon (ifn)-mediated immunity in humans: insights into the respective roles of ifn-alpha/beta, ifn-gamma, and ifn-lambda in host defense suppressor of cytokine signaling regulates the immune response to infection by a unique inhibition of type i interferon activity new insights into cancer immunoediting and its three component phases 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virulent influenza a virus plasmacytoid dendritic cells are dispensable during primary influenza virus infection clearance of influenza virus from the lung depends on migratory langerin+cd b− but not plasmacytoid dendritic cells human tlr- -, - -, and - -mediated induction of ifn-alpha/beta and -lambda is irak- dependent and redundant for protective immunity to viruses pyogenic bacterial infections in humans with myd deficiency impaired response to interferon-alpha/beta and lethal viral disease in human stat deficiency herpes simplex virus encephalitis in a patient with complete tlr deficiency: tlr is otherwise redundant in protective immunity herpes simplex encephalitis in children with autosomal recessive and dominant trif deficiency evolutionary dynamics of human toll-like receptors and their different contributions to host defense type interferons and the virus-host relationship: a lesson in detente subcapsular sinus macrophages prevent cns invasion on peripheral infection with a neurotropic virus enforced viral replication activates adaptive immunity and is essential for the control of a cytopathic virus interferon-stimulated genes: a complex web of host defenses protein kinase pkr and rna adenosine deaminase adar : new roles for old players as modulators of the interferon response dna deamination mediates innate immunity to retroviral infection samhd host restriction factor: a link with innate immune sensing of retrovirus infection a diverse range of gene products are effectors of the type i interferon antiviral response the transcription factor stat- couples macrophage synthesis of -hydroxycholesterol to the interferon antiviral response reciprocal inhibition between intracellular antiviral signaling and the rnai machinery in mammalian cells plasmacytoid, conventional, and monocyte-derived dendritic cells undergo a profound and convergent genetic reprogramming during their maturation crosspriming of cd + t cells stimulated by virus-induced type i interferon type i interferon is selectively required by dendritic cells for immune rejection of tumors host type i ifn signals are required for antitumor cd + t cell responses through cd {alpha}+ dendritic cells dendritic cells require a systemic type i interferon response to mature and induce cd + th immunity with poly ic as adjuvant direct type i ifn but not mda /tlr activation of dendritic cells is required for maturation and metabolic shift to glycolysis after poly ic stimulation type i interferons potently enhance humoral immunity and can promote isotype switching by stimulating dendritic cells in vivo johansson-lindbom b. type i interferon signaling in dendritic cells stimulates the development of lymph-noderesident t follicular helper cells differential responses of immune cells to type i interferon contribute to host resistance to viral infection a type i interferon autocrine-paracrine loop is involved in toll-like receptorinduced interleukin- p secretion by dendritic cells tlrdriven early glycolytic reprogramming via the kinases tbk -ikkvarepsilon supports the anabolic demands of dendritic cell activation type i ifn-mediated protection of macrophages and dendritic cells secures control of murine coronavirus infection direct action of type i ifn on nk cells is required for their activation in response to vaccinia viral infection in vivo dendritic cells prime natural killer cells by trans-presenting interleukin cutting edge: the direct action of type i ifn on cd t cells is critical for sustaining clonal expansion in response to a viral but not a bacterial infection type i interferons act directly on cd t cells to allow clonal expansion and memory formation in response to viral infection direct stimulation of t cells by type i ifn enhances the cd + t cell response during cross-priming cutting edge: enhancement of antibody responses through direct stimulation of b and t cells by type i ifn cd t cells specific for lymphocytic choriomeningitis virus require type i ifn receptor for clonal expansion innate inflammatory signals induced by various pathogens differentially dictate the ifn-i dependence of cd t cells for clonal expansion and memory formation concomitant type i ifn receptor-triggering of t cells and of dc is required to promote maximal modified vaccinia virus ankara-induced t-cell expansion antiviral immune responses: triggers of or triggered by autoimmunity? is chronic plaque psoriasis triggered by microbiota in the skin? genetic dysbiosis: the role of microbial insults in chronic inflammatory diseases the tlr-mediated response of plasmacytoid dendritic cells is positively regulated by estradiol in vivo through cell-intrinsic estrogen receptor alpha signaling accumulation of peripheral autoreactive b cells in the absence of functional human regulatory t cells cvid-associated taci mutations affect autoreactive b cell selection and activation human lupus autoantibody-dna complexes activate dcs through cooperation of cd and tlr ifn priming is necessary but not sufficient to turn on a migratory dendritic cell program in lupus monocytes plasmacytoid dendritic cells sense self-dna coupled with antimicrobial peptide the pathology of influenza virus infections how do viral infections predispose patients to bacterial infections? predominant role of bacterial pneumonia as a cause of death in pandemic influenza: implications for pandemic influenza preparedness pathology of human influenza revisited immune dysfunction and bacterial coinfections following influenza type i interferon: friend or foe? a role for ifn-alpha beta in virus infection-induced sensitization to endotoxin type i ifns mediate development of postinfluenza bacterial pneumonia in mice synergistic stimulation of type i interferons during influenza virus coinfection promotes streptococcus pneumoniae colonization in mice intranasal poly-ic treatment exacerbates tuberculosis in mice through the pulmonary recruitment of a pathogen-permissive monocyte/macrophage population innate and adaptive interferons suppress il- alpha and il- beta production by distinct pulmonary myeloid subsets during mycobacterium tuberculosis infection host-directed therapy of tuberculosis based on interleukin- and type i interferon crosstalk inflammation. -hydroxycholesterol suppresses interleukin- -driven inflammation downstream of type i interferon dendritic cells, monocytes and macrophages: a unified nomenclature based on ontogeny cytomegalovirus impairs antiviral cd + t cell immunity by recruiting inflammatory monocytes tnf/inos-producing dendritic cells are the necessary evil of lethal influenza virus infection influenza a inhibits th -mediated host defense against bacterial pneumonia in mice influenza a virus exacerbates staphylococcus aureus pneumonia in mice by attenuating antimicrobial peptide production role of tissue protection in lethal respiratory viral-bacterial coinfection disease tolerance as a defense strategy gainof-function human stat mutations impair il- immunity and underlie chronic mucocutaneous candidiasis type i interferon inhibits interleukin- production and inflammasome activation type i interferons promote fatal immunopathology by regulating inflammatory monocytes and neutrophils during candida infections ifnalpha/beta signaling is required for polarization of cytokine responses toward a protective type pattern during experimental cryptococcosis interferonbeta production via dectin- -syk-irf signaling in dendritic cells is crucial for immunity to c. albicans aberrant type i interferon regulation in autoimmunity: opposite directions in ms and sle, shaped by evolution and body ecology janus-like effects of type i interferon in autoimmune diseases coregulation of cd + t cell exhaustion by multiple inhibitory receptors during chronic viral infection type i interferon suppresses de novo virus-specific cd th immunity during an established persistent viral infection timing and magnitude of type i interferon responses by distinct sensors impact cd t cell exhaustion and chronic viral infection distinct transcriptional profiles in ex vivo cd + and cd + t cells are established early in human immunodeficiency virus type infection and are characterized by a chronic interferon response as well as extensive transcriptional changes in cd + t cells chronic cd + t-cell activation and depletion in human immunodeficiency virus type infection: type i interferon-mediated disruption of t-cell dynamics host genes associated with hiv- replication in lymphatic tissue global genomic analysis reveals rapid control of a robust innate response in siv-infected sooty mangabeys nonpathogenic siv infection of african green monkeys induces a strong but rapidly controlled type i ifn response downregulation of robust acute type i interferon responses distinguishes nonpathogenic simian immunodeficiency virus (siv) infection of natural hosts from pathogenic siv infection of rhesus macaques comparative transcriptomics of extreme phenotypes of human hiv- infection and siv infection in sooty mangabey and rhesus macaque hiv turns plasmacytoid dendritic cells (pdc) into trail-expressing killer pdc and down-regulates hiv coreceptors by toll-like receptor -induced ifn-alpha plasmacytoid dendritic cells express trail and induce cd + t-cell apoptosis in hiv- viremic patients sex differences in the toll-like receptor-mediated response of plasmacytoid dendritic cells to hiv- glycerol monolaurate prevents mucosal siv transmission type i interferon responses in rhesus macaques prevent siv infection and slow disease progression plasmacytoid dendritic cells suppress hiv- replication but contribute to hiv- induced immunopathogenesis in humanized mice chronic hepatitis c: future treatment host-targeting agents in the treatment of hepatitis c: a beginning and an end? the interferons: years after their discovery, there is much more to learn immune responses to hcv and other hepatitis viruses interferon lambda alleles predict innate antiviral immune responses and hepatitis c virus permissiveness altered interferon-alpha-signaling in natural killer cells from patients with chronic hepatitis c virus infection natural killer cells are polarized toward cytotoxicity in chronic hepatitis c in an interferon-alfa-dependent manner coexpression of pd- , b , cd and klrg on exhausted hcv-specific cd + t cells is linked to antigen recognition and t cell differentiation evidence for an antagonist form of the chemokine cxcl in patients chronically infected with hcv monocyte activation by interferon alpha is associated with failure to achieve a sustained virologic response after treatment for hepatitis c virus infection genetic variation in il b and spontaneous clearance of hepatitis c virus genetic variation in il b predicts hepatitis c treatment-induced viral clearance a variant upstream of ifnl (il b) creating a new interferon gene ifnl is associated with impaired clearance of hepatitis c virus the favorable ifnl genotype escapes mrna decay mediated by aurich elements and hepatitis c virus-induced micrornas distinct and overlapping genomic profiles and antiviral effects of interferon-lambda and -alpha on hcv-infected and noninfected hepatoma cells targeted induction of interferon-lambda in humanized chimeric mouse liver abrogates hepatotropic virus infection ifn-lambda receptor expression is induced in chronic hepatitis c and correlates with the ifn-lambda genotype and with nonresponsiveness to ifn-alpha therapies how cells respond to interferons jak-stat pathways and transcriptional activation in response to ifns and other extracellular signaling proteins regulation of type i interferon responses stat and irf : beyond isgf . jakstat ( ) single-cell mass cytometry of differential immune and drug responses across a human hematopoietic continuum multiplexed mass cytometry profiling of cellular states perturbed by small-molecule regulators major differences in the responses of primary human leukocyte subsets to ifn-beta critical role for stat activation by type interferons in the interferongamma response to viral infection immunomodulatory functions of type i interferons high basal stat balanced by stat induction to control type interferon effects in natural killer cells type interferon induction of natural killer cell gamma interferon production for defense during lymphocytic choriomeningitis virus infection changing partners at the dance: variations in stat concentrations for shaping cytokine function and immune responses to viral infections dendritic-cell maturation alters intracellular signaling networks, enabling differential effects of ifn-alpha/beta on antigen cross-presentation mechanisms of type-i-and type-ii-interferon-mediated signalling type i interferon [corrected] gene induction by the interferon regulatory factor family of transcription factors complex modulation of cell type-specific signaling in response to type i interferons alternate interferon signaling pathways ifn-beta induces serine phosphorylation of stat- in ewing's sarcoma cells and mediates apoptosis via induction of irf- and activation of caspase- usp -based negative feedback control is induced by type i and type iii interferons and specifically inactivates interferon alpha response interferon-alpha and -beta differentially regulate osteoclastogenesis: role of differential induction of chemokine cxcl expression differential activity of type i interferon subtypes for dendritic cell differentiation evolutionary genetic dissection of human interferons the receptor of the type i interferon family interferons as biomarkers and effectors: lessons learned from animal models protection against progressive leishmaniasis by ifn-beta multifaceted activities of type i interferon are revealed by a receptor antagonist receptor density is key to the alpha /beta interferon differential activities structural and dynamic determinants of type i interferon receptor assembly and their functional interpretation the type i interferon receptor: structure, function, and evolution of a family business differential receptor subunit affinities of type i interferons govern differential signal activation structural linkage between ligand discrimination and receptor activation by type i interferons distinct and nonredundant in vivo functions of ifnar on myeloid cells limit autoimmunity in the central nervous system type i interferons protect t cells against nk cell attack mediated by the activating receptor ncr lymphadenopathy in a novel mouse model of bartonella-induced cat scratch disease results from lymphocyte immigration and proliferation and is regulated by interferon-alpha/ beta cytosolic rig-i-like helicases act as negative regulators of sterile inflammation in the cns expression of type i interferon by splenic macrophages suppresses adaptive immunity during sepsis myeloid type i interferon signaling promotes atherosclerosis by stimulating macrophage recruitment to lesions stat activation is responsible for il- -dependent t cell proliferation through preventing apoptosis: generation and characterization of t cell-specific stat -deficient mice generation of mice with a conditional stat null allele conditional stat ablation reveals the importance of interferon signaling for immunity to listeria monocytogenes infection loss of stat from mouse mammary epithelium results in an increased neu-induced tumor burden inactivation of stat in mouse mammary epithelium during pregnancy reveals distinct functions in cell proliferation, survival, and differentiation meta-analysis of microarray data identifies gas expression as an independent predictor of poor survival in ovarian cancer axl and growth arrest-specific gene are frequently overexpressed in human gliomas and predict poor prognosis in patients with glioblastoma multiforme gas expression identifies high-risk adult aml patients: potential implications for therapy ovarian cancer progression is controlled by phenotypic changes in dendritic cells ifnalpha activates dormant haematopoietic stem cells in vivo timing of ifn-beta exposure during human dendritic cell maturation and naive th cell stimulation has contrasting effects on th subset generation: a role for ifn-beta-mediated regulation of il- family cytokines and il- in naive th cell differentiation combinatorial flexibility of cytokine function during human t helper cell differentiation regulating type ifn effects in cd t cells during viral infections: changing stat and stat expression for function interferons at age : past, current and future impact on biomedicine inquiring into the differential action of interferons (ifns): an ifn-alpha mutant with enhanced affinity to ifnar is functionally similar to ifn-beta an interferon alpha mutant optimized by phage display for ifnar binding confers specifically enhanced antitumor activities the stability of the ternary interferon-receptor complex rather than the affinity to the individual subunits dictates differential biological activities intra-lesional low-dose interferon alpha a therapy for primary cutaneous marginal zone b-cell lymphoma intratumoral injection of interferon-alpha and systemic delivery of agonist anti-cd monoclonal antibodies synergize for immunotherapy delivery of interferon alpha using a novel cox -controlled adenovirus for pancreatic cancer therapy the efficacy of radiotherapy relies upon induction of type i interferondependent innate and adaptive immunity a trial of intrapleural adenoviral-mediated interferon-alpha b gene transfer for malignant pleural mesothelioma tumortargeted interferon-alpha delivery by tie -expressing monocytes inhibits tumor growth and metastasis targeting cytokines to inflammation sites livertargeting of interferon-alpha with tissue-specific domain antibodies antibody-based targeting of interferon-alpha to the tumor neovasculature: a critical evaluation targeting ifn-alpha to b cell lymphoma by a tumor-specific antibody elicits potent antitumor activities targeting the tumor microenvironment with interferon-beta bridges innate and adaptive immune responses targeted delivery of interferon-alpha via fusion to anti-cd results in potent antitumor activity against b-cell lymphoma targeted delivery of interferon-alpha to hepatitis b virus-infected cells using t-cell receptor-like antibodies variations in the unstructured c-terminal tail of interferons contribute to differential receptor binding and biological activity safety and pharmacodynamics of rontalizumab in patients with systemic lupus erythematosus: results of a phase i, placebo-controlled, double-blind, dose-escalation study safety profile and clinical activity of sifalimumab, a fully human anti-interferon alpha monoclonal antibody, in systemic lupus erythematosus: a phase i, multicentre, double-blind randomised study mutation of the ifnar- receptor binding site of human ifn-alpha generates type i ifn competitive antagonists the studies performed in the laboratories are supported by funding from inserm, cnrs, aix-marseille university, the labex mabimprove, and the european community's seventh framework programme fp / - (grant agreement for gilles uzé, european research council starting grant agreement number for marc dalod including salary support to emeline pollet and thien-phong vu manh). we thank past and present laboratory members for their contribution to studies on dcs or ifns. we apologize for not quoting certain studies because of space limitations. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. key: cord- - kozsv z authors: dewidar, bedair; kahl, sabine; pafili, kalliopi; roden, michael title: metabolic liver disease in diabetes – from mechanisms to clinical trials date: - - journal: metabolism doi: . /j.metabol. . sha: doc_id: cord_uid: kozsv z abstract non-alcoholic fatty liver disease (nafld) comprises fatty liver (steatosis), non-alcoholic steatohepatitis (nash) and fibrosis/cirrhosis and may lead to end-stage liver failure or hepatocellular carcinoma. nafld is tightly associated with the most frequent metabolic disorders, such as obesity, metabolic syndrome, and type diabetes mellitus (t dm). both multisystem diseases share several common mechanisms. alterations of tissue communications include excessive lipid and later cytokine release by dysfunctional adipose tissue, intestinal dysbiosis and ectopic fat deposition in skeletal muscle. on the hepatocellular level, this leads to insulin resistance due to abnormal lipid handling and mitochondrial function. over time, cellular oxidative stress and activation of inflammatory pathways, again supported by multiorgan crosstalk, determine nafld progression. recent studies show that particularly the severe insulin resistant diabetes (sird) subgroup (cluster) associates with nafld and its accelerated progression and increases the risk of diabetes-related cardiovascular and kidney diseases, underpinning the critical role of insulin resistance. consequently, lifestyle modification and certain drug classes used to treat t dm have demonstrated effectiveness for treating nafld, but also some novel therapeutic concepts may be beneficial for both nafld and t dm. this review addresses the bidirectional relationship between mechanisms underlying t dm and nafld, the relevance of novel biomarkers for improving the diagnostic modalities and the identification of subgroups at specific risk of disease progression. also, the role of metabolism-related drugs in nafld is discussed in light of the recent clinical trials. finally, this review highlights some challenges to be addressed by future studies on nafld in the context of t dm. nonalcoholic fatty liver diseases (nafld) is currently defined by lipid deposition that exceeds more than % of hepatocytes, as assessed from liver biopsy, and/or by more than . % hepatocellular fat content per liver weight, as assessed from magnetic resonance (mr) methods, in the absence of significant alcohol consumption and other causes of fatty liver [ , ] . nafld, which affects about % of the population [ ] , comprises a broad range of abnormalities ranging from simple fatty liver (steatosis) to non-alcoholic steatohepatitis (nash), characterized by inflammation, necrosis, and hepatocellular ballooning, and progression to liver fibrosis, cirrhosis, and hepatocellular carcinoma (hcc) [ ] . some gene variants promote risk of nafld by altering lipid droplet formation and de novo lipogenesis (dnl), such as variants of patatin-like phospholipase domain-containing protein (pnpla ) and glucokinase regulatory protein [ ] , or by decreasing very-low-density lipoproteins (vldl) export as shown for a missense mutation (e k) in transmembrane superfamily member (tm sf ) [ ] . nevertheless, nafld is tightly associated with common acquired metabolic diseases such as obesity and type diabetes (t dm). the mutual relationship between both diseases is illustrated by several epidemiological data. the prevalence of steatosis and nash has been estimated to be and %, respectively, in t dm [ ] . the age-adjusted relative risk of nafld is about . fold higher in t dm compared to healthy humans [ ] . t dm is also an emerging risk factor for nash progression to advanced fibrosis, cirrhosis and hcc [ , ] . diabetes was even a better predictor for hcc development in people with nash and cirrhosis compared to other metabolic risk factors such as hyperlipidemia, body mass index (bmi) and hypertension [ ] . recently, a consensus panel has proposed to rename nafld a metabolic-dysfunction-associated fatty liver disease (mafld) based on the presence of overweight/obesity, t dm and evidence of so-called "metabolic dysregulation" [ ] . future will tell, if this will help to better understand the multiple relationships between nafld and t dm. in this context, nafld per se associates with more than double risk of incident diabetes pointing to specific liver-related mechanisms [ , ] . moreover, multicenter studies in skandinavia and germany have recently found that diabetes can be stratified into subtypes j o u r n a l p r e -p r o o f the circulation. on the contrary, a recent study demonstrated that obesity-induced insulin resistance preceded inflammation in adipose tissues of mice [ ] . indeed, adipose tissue inflammation might be a protective feedback mechanism as its local inhibition in mice induced ectopic lipid accumulation in liver, glucose intolerance, and systemic inflammation [ ] . besides released inflammatory cytokines and ffa, adipose tissue could still communicate with the liver and muscle through secretion of different adipokines such as adiponectin and leptin. persons with nash have lower serum adiponectin compared to those with nafld with or without normal liver enzymes [ ] . by contrast, the circulating levels of leptin were higher in people with nafld and t dm, probably due to increased leptin resistance, and were associated with disease severity [ ] . increased ffa influx to skeletal muscle promotes accumulation of intramyocellular lipid (imcl). reduced mitochondrial oxidation contributes as well to imcl as shown in aging and insulin-resistant humans [ ] . consequently, skeletal muscle exhibits insulin resistance, which often precedes the onset of t dm and insulin resistance in the liver [ ] . lipid intermediate metabolites, in particular sn , diacylglycerols (dag), link imcl to skeletal muscle insulin resistance through activation of protein kinase c-theta (pkcθ) resulting in its translocation from cytoplasm to the plasma membrane [ ] . muscles of insulin resistant humans with obesity and t dm showed increased dag content and pkcθ activity as compared to healthy humans [ ] . mutation studies highlighted serine amino acid residue (ser ) of irs to be a substrate for activated pkcθ [ ] . as a consequence of skeletal muscle insulin resistance, postprandial energy storage shifts from glycogen synthesis in the muscle into triacylglycerol (tag) in the liver, promoting nafld development [ ] . gut microbiome is increasingly recognized as a modulator of liver pathogenesis through what is called the "gut-liver axis" [ ] . distinctive alterations of gut microbiome were reported in humans having nash and t dm [ ] . the intestinal microbiome alters host metabolism by modulating the production of short-chain fatty acids (scfa) e.g. butyrate, acetate, and propionate, which have beneficial effects on insulin sensitivity, lipid and glucose metabolism [ ] . also, intestinal dysbiosis could associate with increased intestinal permeability permitting translocation of bacterial lipopolysaccharide (lps) into the systemic circulation, j o u r n a l p r e -p r o o f which could induce fat deposition in the liver, nash progression, weight gain, and diabetes [ ] . moreover, intestinal microbiome could suppress the expression of fasting-induced adipocyte factor (fiaf) in intestinal epithelium, which functions as an inhibitor of circulating lipoprotein lipase, resulting in increased tag storage in the peripheral tissues [ ] . also, ethanol-producing microbiome could increase blood alcohol concentration in nash, which is metabolized in the liver generating high levels of reactive oxygen species (ros). the last mechanism could explain the histological similarity between nash and alcoholic steatohepatitis [ ] . furthermore, microbiota metabolize liver-derived primary bile acids into secondary bile acids. the latter are reabsorbed into bloodstream and may act as signaling molecules via a variety of receptors including farnesoid x receptor (fxr), which regulates the transcription of different metabolic genes involved in bile acid synthesis, transport, lipogenesis, and glucose homeostasis [ ] . insulin resistance in both skeletal muscle and adipose tissues initiates liver steatosis by providing precursors and substrates for dnl and mitochondrial β-oxidation e.g. glucose, ffa and glycerol [ ] . although reesterification of ffa derived from diet and adipose tissue is the dominant contributor to tag pool in the liver ( %), it did not increase in people with nafld. on the other side, dnl-derived ffa contribute by about %, which is severalfold higher as compared to individuals without nafld ( %) [ ] . later, insulin resistance of the liver develops, resulting in increased gluconeogenesis and elevation of endogenous glucose production (egp) from the liver, which contributes to fasting hyperglycemia in individuals with t dm [ ] . insulin signaling inhibits typically hepatic gluconeogenesis through akt-induced phosphorylation of forkhead box (foxo ) and induces lipogenesis through activation of sterol regulatory element-binding proteins (srebp c) and mammalian target of rapamycin complex (mtorc ) pathways. during hepatic insulin resistance, insulin does not suppress gluconeogenesis efficiently, while dnl is preserved or even increased. to explain this discrepancy, pathway-selective hepatic insulin resistance was postulated, which means that only one arm of insulin signaling is defective i.e. akt/foxo leading to reduced insulin-mediated suppression of gluconeogenesis, whereas insulin-activated srebp- c/mtorc pathway remains intact and activates lipogenesis [ ] . actually, dnl was reduced after induction of hepatic insulin resistance by feeding mice with j o u r n a l p r e -p r o o f a high-fat diet (hfd) [ ] , which challenges the concept of selective hepatic insulin resistance and suggest the existence of alternate mechanisms that contribute to increased dnl and gluconeogenesis in insulin-resistant liver, for example, hyperinsulinemia, insulinindependent re-esterification of adipose tissue-derived ffa, and increased acetyl coa generation from β-oxidation of ffa, which allosterically activates pyruvate carboxylase enzyme, leading to enhanced gluconeogenesis [ ] . in addition, increased blood glucose level can activate carbohydrate response element-binding protein (chrebp) signaling pathway, thereby stimulating expression of several glycolytic genes, which provide additional metabolic precursors for dnl [ ] . in line, chrebp overexpression induced stearoyl-coa desaturase (scd ) expression, an enzyme responsible for the biosynthesis of monounsaturated fatty acids (mufa), resulting in increased liver fat content [ ] . pkc epsilon (pkcε) is crucial in mediating hepatic insulin resistance and once activated by dag, it translocates to the cell membrane, and phosphorylates specific threonine residue (thr in human and thr in mouse) on insulin receptor leading to destabilization of the active configuration of insulin receptor kinase and inhibition of its tyrosine kinase activity [ ] (figure ). in general, both hyperglycemia and toxic lipids such as ceramides, dag, ffa, and cholesterol can induce deleterious effects on liver cells (glucolipotoxicity), which might initiate nafld progression from simple steatosis to nash and fibrosis via various mechanisms, including cell death, oxidative stress, endoplasmic reticulum (er) stress and mitochondrial disorders [ ] . alterations in the activity and abundance of oxidative phosphorylation (oxphos) proteins and antioxidant enzymes were described in various animal models of nafld [ ] . impaired hepatic mitochondrial function was evident as well in t dm and nash [ , ] . in a mouse model of choline-deficient diet-induced nafld, mitochondrial oxphos was increased at weeks but lost at a later time point [ ] . also, the higher maximal respiration rate of liver mitochondria was severalfold higher in insulin-resistant obese individuals with fatty liver as compared to lean individuals [ ] . these studies highlight the flexibility of mitochondria to adapt to increased metabolic inputs to keep energy homeostasis and to protect against the harmful effects of increased ffa and tag in the liver. in nash, mitochondrial flexibility was lost, which was associated with increased ros production and exhaustion of protective antioxidant enzymes [ ] (figure ). whether loss of mitochondrial flexibility is a cause or consequence for nafld progression remains obscure. depletion of atp due to j o u r n a l p r e -p r o o f mitochondrial disorders, together with hyperglycemia and lipid overload could be inducers for another signaling pathway, termed unfolded protein response "upr", which is adaptive response to resolve unfolded or misfolded proteins and to restore er homeostasis [ , ] . prolonged upr stress can activate jnk and nf-kb signaling pathways, which are involved in insulin resistance, liver steatosis, and inflammation [ ] . furthermore, er stress could increase insulin resistance through induction of lipin- expression, which is a phosphatase enzyme that catalyzes biosynthesis of dag leading to activation of dag/pkcε axis [ ] . interestingly, upr could also increase liver steatosis through activation of srebp- c signaling pathway [ ] . elevated ros and upr are well-identified pathways that could induce hepatocytes death in nash. hepatocytes apoptosis and necroptosis are the main forms of cell death in human steatohepatitis and diet-induced mouse models of nash [ ] . the key fibrogenic liver cells, hepatic stellate cells (hscs), usually exist in a quiescent state and get activated by engulfment of apoptotic bodies, the inflammatory milieu, or damage-associated molecular patterns (damps) released from stressed and dying hepatocytes [ ] . interestingly, ffa-mediated lipotoxic effects stimulate hepatocytes to release extracellular vesicles (evs) with distinctive micrornas (mirna) profile that increase the expression of fibrogenic genes in the surrounding hscs [ ] (figure ). free cholesterol could directly sensitize hscs to the action of tumor growth factor (tgf)-β, a potent fibrogenic cytokine [ ] . treatment of human and rat immortalized hscs cell lines with saturated fatty acid increased the expression of various profibrogenic genes [ ] . macrophages aggregate as well around dead hepatocytes forming a crown-like structure, a phenomenon that exists only in persons with nash but not in those with simple steatosis [ ] . recruitment of more inflammatory cells from systemic circulation is facilitated by "find me" signals that are released from dead cells [ ] . inhibition of inflammatory monocytes recruitment via inhibition of c-c chemokine receptors type and type (ccr /ccr ) suppressed fibrogenesis and steatohepatitis in murine nash [ ] . macrophages can modulate also hepatic insulin resistance and favor tag accumulation in hepatocytes through secretion of il- β that downregulates peroxisome proliferator-activated receptor (ppar) α, a key transcriptional pathway involved in fatty acid oxidation [ ] . liver sinusoidal cells (lsecs) are fenestrated cells that exist in close vicinity to hepatocytes, j o u r n a l p r e -p r o o f macrophages and hscs, which, under physiological conditions, regulate lipid transport, maintain the quiescence of kupffer cells, resident liver macrophages, and hscs [ ] . at early stage of nafld, lsecs lose their fenestrae, a process termed capillarization, which could favor liver steatosis and initiate hscs activation [ ] (figure ). during nash, lsecs display a pro-inflammatory phenotype that promotes steatohepatitis [ ] . autophagy is a self-degradative process which is used by the cells to remove misfolded proteins and damaged organelles [ ] . singh et al. showed that the cells can use autophagic process as lipolytic mechanism to mobilize lipids from intracellular lipid store, which is termed in this case "macrolipophagy" [ ] . various in vitro and in vivo studies showed that autophagy was decreased in fatty hepatocytes [ ] . impairment of autophagy by palmitic acid in macrophages induces inflammatory il- β production [ ] . on the other side, selective loss of autophagic activity reduced liver fibrogenesis in cultured hscs [ ] and protected against diet-induced insulin resistance [ ] . the results highlight that the net effect of autophagy on nafld might depend on the tissue or type of the cells that show autophagic dysfunction and the stage of nafld. the liver biopsy is considered the gold standard for nafld diagnosis, especially for distinguishing steatosis from inflammation and fibrosis [ ] . nevertheless, this technique has several limitations not only resulting from the invasive procedure and rare post-interventional complications, but also due to the small tissue volume, which might not be representative for the whole liver and may not take into account inhomogeneous distribution of nafldassociated alterations. novel imaging modalities such as ultrasound-or mr-based techniques are of increasing value, as recently reviewed [ ] , but are still not generally available, so that there is an urgent need for noninvasive screening tools. non-invasive detection of nash and fibrosis remains challenging today. biomarker-based panels such as aspartate aminotransferase (ast)/platelets count ratio index, fibrosis- (fib- ) index, fibrotest, fibrospect ii, and nafld fibrosis score (nfs) offer variable diagnostic efficacy for assessing different stages of liver fibrosis [ , ] . although combination of these panels could enhance their predictive values [ ] , they still suffer from limited accuracy even compared to the alanine aminotransferase (alt) and ast, particularly in t dm [ ] . in this regard, transient elastography looks like a promising alternative in diabetes clinics for detection of liver fibrosis in nafld [ ] . numerous biomarkers have been developed to specifically track features of nash progression and fibrosis. cytokeratin (ck) , an intermediate filament protein that is cleaved during cell death to ck m and ck m , has been intensively investigated as a surrogate of nash-associated liver cell damage, but a recent meta-analysis of studies reported a maximum sensitivity of . for nash diagnosis [ ] and suboptimal diagnostic value was shown in t dm [ ] . the ecm turnover marker, type iii procollagen, can offer a diagnostic efficacy of . and . to detect moderate and advanced liver fibrosis in t dm, respectively [ ] . in , the european associations for the study of the liver, obesity and diabetes (easl-easo-easd) jointly released recommendations for diagnosis and monitoring of disease severity in persons with suspected nafld and metabolic risk factors [ ] . people with metabolic risk factors, such as t dm, should undergo assessment by abdominal ultrasound, serum transaminases and fibrosis markers (e. g. fib- , nfs). elevated transaminases or steatosis plus abnormal fibrosis test shall require referral to a specialist. this strategy has raised the question of a possible overreferral when adhering to these guidelines [ ] . however, recent analyses show that a refined strategy of specific combining indices such as fli and fib- could reduce the number of people with t dm for further diagnostic work up to a reasonable size [ ] . this retrospective analysis also found that certain non-invasive biomarkers are consistently associated with different patterns of diabetes-related complications. analyses of the plasma metabolomic of insulin-resistant individuals with nafld suggested that bile salts, e. g. glycocholate, taurocholate, and glycochenodeoxycholate allow to detect nash in one study [ ] , while another study reported an association with insulin resistance j o u r n a l p r e -p r o o f but not with liver necroinflammation [ ] . also amino acids, specifically a glutamate-serineglycine index, was associated with hepatic insulin resistance and transaminases and discriminated individuals with fibrosis grade - from those with grade - independently of bmi [ ] . the diagnostic performance of a serum-based lipidomic analysis was substantially lower for nafld detection in t dm compared to healthy individuals [ ] . nevertheless, certain sphingolipid species were recently found to be increased in insulin-resistant nash and to correlate with hepatic oxidative stress and inflammation [ ] . one circulating small noncoding rna, mirna- , was found to be more than . fold in steatosis and further doubled in nash [ ] . this mirna was also higher in t dm with nafld than in those without nafld and provided better prediction of nafld when combined with ldl and waist-to-hip ratio [ ] . moreover, extracellular vesicles (ev), which among other cargo also transport mirna, may be promising biomarkers for nafld as shown by higher serum levels in people with nafld, obesity and diabetes [ ] . recently, metagenomics data derived from gut microbiota alterations allowed to detect advanced fibrosis in nafld patients, of whom % had t dm, with a robust diagnostic accuracy (auroc: . ) [ ] . the current guidelines recommend only lifestyle modification and -for certain groupsbariatric or metabolic surgery to treat nafld [ , ] . although the numerous activities in this field, no pharmacological treatment is currently approved or expecting approval for the use in nafld with or without concomitant t dm (table and ), except for obeticholic acid (oca). marketing authorization application for oca has been submitted to the u.s. food and drug administration (fda) and european medicines agency (ema), awaiting fda decision by june [ ] . against this background, current guidelines and recommendations primarily advise lifestyle modification (healthy nutrition and exercise) and weight loss in overweight/obese persons. the easl-easo-easd guidelines recommend to consider pharmacological treatment in people with nash when combined with fibrosis and in those with less severe disease, but high risk conditions for disease progression such as t dm [ ] . the american association for the study of liver diseases (aasld) recommends to limit pharmacological treatment to those with biopsy-proven nash and fibrosis [ ] . j o u r n a l p r e -p r o o f weight management and physical exercise are key to the treatment of both nafld and t dm. a proof-of-concept study showed that a hypocaloric diet with weight loss of ~ kg within weeks not only normalized fasting egp and thereby hyperglycemia, but decreased hepatic tag content down to normal concentrations in obese t dm humans with nafld [ ] . subsequent studies in larger cohorts extended these results by demonstrating that a very low-caloric diet with weight loss of about % rapidly normalized liver fat content in in dividuals with t dm, which persisted for one year if weight loss was maintained [ ] . interestingly, mediterranean, low-saturated fat and high plant-based protein diets also improve steatosis in nafld combined with t dm despite minor or no relevant weight loss [ , ] . in addition, physical activity and exercise training interventions can also decrease liver fat content, which may not be exclusively depending on concomitant weight loss [ ] . although the beneficial effects of structured behavioral treatment to improve histological endpoints, likely extend beyond reduction of hepatic fat content to ameliorating the grade and stage of inflammation and fibrosis [ ] , only a minority of the people manages to adhere to dietary weight loss and exercise programs, which raises the issue of other therapeutic approaches [ ] . weight-loss inducing drugs could be an attractive option for persons with nafld with a bmi > kg/m or > kg/m in the presence of at least one metabolic comorbidity, as an adjunct treatment to lifestyle modifications [ , ] . currently, the most-popular weight-loss inducing medications associated with at least % body weight decrease in one year as compared to placebo are orlistat, the fixed combination of phentermine and topiramate or naltrexone and bupropion, and the glucagon-like peptide- (glp- ) receptor agonist (glp- ra), liraglutide [ ] . of these drugs, orlistat treatment failed to improve liver histology when compared to placebo [ ] , while no reports are available for topiramate, naltrexone, bupropion and phentermine regarding hepatic endpoints in humans with nafld [ ] . only bariatric or metabolic surgery is a therapeutic option to induce sustained weight loss partricularly in people with combined nafld and t dm. in obese humans, bariatric surgery resulted in % resolution of nash within one year [ ] and in % complete remission of t dm [ ] . surgical weight loss improves glucose metabolism and insulin sensitivity by different mechanisms such as increased glp- secretion [ ] and epigenetic modification [ , ] . several antihyperglycemic drug classes were or are currently being investigated in clinical trials and preclinical models to evaluate their efficacy for people with nafld and with or without t dm. metformin reduces body weight, hepatic gluconeogenesis -by yet unclear mechanisms [ ] , and the risk of macrovascular complications, which is still controversially discussed due to lack of optimally designed clinical trials [ ] . despite its action on hepatic metabolism, former small-scale studies reported conflicting results [ , ] (table ) and recent metaanalysis failed to demonstrate any effect of metformin on liver histology [ ] . nevertheless, epidemiological, observational and preclinical studies suggest that metformin may reduce the risk of hcc and also in t dm, possibly by promoting apoptosis, but controlled clinical trials are not available [ ] . dpp degrades incretins such as glp- so that dpp- i treatment increases the postprandial levels of endogenous glp- , which leads to lower glucose peaks after meals [ , ] . in individuals with nafld with prediabetes or recent onset diabetes, sitagliptin did not improve liver steatosis or liver fibrosis compared to placebo as assessed by mr-based techniques [ ] . in line, another recent trial reported no benefits for sitagliptin versus placebo on liver fibrosis or steatohepatitis in t dm [ ] (table ) . in contrast, a moderate reduction in liver fat content was observed with vildagliptin in individuals with t dm [ ] . j o u r n a l p r e -p r o o f the actions of endogenous glp- are mimicked by glp- receptor agonists (glp- ra), which effectively reduce blood glucose levels and also reduce the risk for cvd in t dm [ ] . in individuals with nafld and t dm, liraglutide in combination with metformin reduced liver, subcutaneous, and visceral fat [ ] . also, liraglutide improved hepatic steatosis measured by mr-based methods as well as resolved biopsy-proven nash without worsening of fibrosis [ , ] (table ) . however, a recent subanalysis did not detect an effect of liraglutide on liver fat content quantified by h-mrs [ ] (table ) . respective trials with semaglutide and dulaglutide are still waiting for results (nct , nct ). in an animal model of t dm, exenatide also counteracted hcc development by suppression of stat -regulated genes [ ] . glucose-dependent insulinotropic polypeptide (gip) represents the second important incretin with proposed beneficial effects on peripheral energy metabolism [ ] . tirzepatide, a novel dual agonist for gip and glp- receptors with probably greater efficacy than glp- ra [ ] , decreased transaminases and surrogate markers of liver injury paralleled by increased circulating adiponectin levels in people with t dm [ ] . a clinical trial on nash resolution is currently ongoing in persons with nash with or without t dm (nct ). sglt i inhibit reabsorption of glucose in the proximal renal tubule resulting in glucosuria and calory loss, thus effectively reducing blood glucose level and body weight in t dm. moreover, sglt is show beneficial effects on cardiovascular risk and progression of nephropathy [ , ] . most, but not all recent randomized controlled trials (rcts) showed that sglt i treatment resulted in reduction in liver fat content compared to placebo [ ] [ ] [ ] [ ] [ ] (table ) . in an open-label pilot study in liver-biopsy proven cohort of nash with t dm, treatment with empagliflozin for months reduced liver steatosis, ballooning and fibrosis and induced nash resolution in approximately half of those persons [ ] . animal models further suggested anti-inflammatory, anti-oxidant and pro-apoptotic actions of sglt i with cardio-protective effects as well as inhibition of nash progression and tumor growth of hcc [ , ] . the dual sglt / i, licogliflozin, is expected to block both intestinal and renal glucose (re)absorption [ ] and currently investigated to evaluate its efficacy on resolution of nash as monotherapy and as combination therapy with the fxr agonist, tropifexor, in people with nash and fibrosis (nct ). first results hint at improvement of transaminases and reduction of steatosis by licogliflozin in nash [ ] . ppars is a family of nuclear receptors composed of multiple isoforms with wide tissue distribution [ ] . the ppar-γ agonist pioglitazone had convincing efficacy on nash resolution in individuals with and without t dm, but with conflicting results on fibrosis [ ] [ ] [ ] (table ) . of note, pioglitazone has been withdrawn in many european countries because of its disadvantageous safety profile [ ] . however, pioglitazone exerts beneficial effects on cardiovascular outcomes in persons with t dm and a history of cvd [ ] . it has been proposed that non-ppar-γ dependent mechanisms, as the inhibition of the mitochondrial pyruvate carrier (mpc), might mediate the beneficial effects of pioglitazone on nafld [ ] . however, a novel drug with ppar-γ sparing effects and mpc binding activity did not improve histological components of nash in individuals with t dm compared to placebo [ ] . elafibranor is a dual agonist for ppar-α and ppar-δ without ppar-γ stimulation [ ] . based on a post-hoc analysis, nash was resolved without worsening of fibrosis in a higher percentage of people with and without t dm in the elafibranor group as compared to the placebo [ ] and a follow-up study on these findings has been initiated ( table ) . recent announcements on an interim analysis state that elafibranor treatment failed to resolve nash and improve fibrosis when compared to placebo [ ] . saroglitazar is another dual ppar-α/γ agonist with higher affinity for ppar-α. in a mouse model of nash, saroglitazar reduced liver steatosis, inflammation and prevented fibrosis development and a respective clinical trial is ongoing [ ] ( table ) . lanifibranor is a pan-ppar agonist for ppar-α, β, and γ receptors focused on treatment of liver fibrosis and nash (nct ). in an animal model of liver fibrosis, lanifibranor decreased hepatic collagen deposition [ ] . several other strategies for pharmacological targeting of nash have emerged over the last few years; however, these strategies are not specifically designed for t dm, but for the whole nafld collective. they comprise antiinflammatory drugs, but also modulators of other pathways, which are briefly summarized in the following sections. j o u r n a l p r e -p r o o f fxr can be activated by bile acids in a negative feedback mechanism to suppress bile acid synthesis [ ] . oca is a potent semisynthetic and selective fxr agonist approved for treatment of primary biliary cholangitis [ ] . oca treatment resulted in histologic improvement of nash in people with and without t dm compared to placebo [ , ] ; however, concerns were raised about oca-induced changes in plasma lipoprotein profile [ ] . tropifexor, another potent fxr agonist [ ] , is currently being tested in combinatorial approaches of nash treatment (cenicriviroc and licogliflozin; nct ). oltipraz is a synthetic dithiolethione with antisteatotic effects by inhibiting the activity of lxr-α. it activates adenosine monophosphate activated protein kinase (ampk) and inactivates s k , affecting lxr-α thus reducing lipogenesis and increasing lipid oxidation [ ] . a recent -week phase clinical trial found decreased steatosis measured by h-mrs with oltipraz compared to placebo treatment [ ] and a respective phase clinical trial is ongoing ( table ). chemokine receptors type (ccr ) and type (ccr ) are expressed on various inflammatory and fibrogenic cells [ ] . cenicriviroc is a ccr /crr dual antagonist that reduced insulin resistance, liver inflammation and fibrosis in diet-induced models of nash [ ] . in recent rcts in a nash cohort with fibrosis, cenicriviroc treatment did not improve nas but may reduce liver fibrosis [ , ] . currently, there is an ongoing clinical trial to evaluate the effects of cenicriviroc on fibrosis in nash ( table ) . both people with obesity, metabolic syndrome or t dm as well as those with nafld are at increased risk for dyslipidemia. statins, inhibitors of -hydroxy- -methyl-glutaryl-coenzyme a (hmg-coa) reductase, are generally safe and have unmet efficacy to decreased serum ldl and prevent cardiovascular outcomes, despite slightly increasing the risk of t dm [ ] . use of statins associated with a % lower relative risk of hepatic decompensation and mortality in cirrhosis and a trend towards lower fibrosis progression in non-cirrhotic liver diseases [ ] . however, the data on liver histology ist limited [ ] so that statins are not currently recommended for the management of nafld [ ] . the inhibitor of intestinal j o u r n a l p r e -p r o o f cholesterol absorption, ezetimibe, decreased the histological nas, but not consistently liver fat content in an analysis of the few available studies [ ] . fenofibrate, a ppar-α agonist, does not affect or even increase liver fat content or volume [ , ] . nevertheless, the combination of statins with certain anti-nash drugs, such as oca, could be beneficial to counteract drug-related increases in ldl during long-term treatment. inhibition of dnl in nafld may be achieved by inhibition of acc as this enzyme catalyzes the conversion of acetyl coa into malonyl coa, which acts as a substrate for fatty acid synthesis and inhibitor for fatty acid β-oxidation [ ] . in a recent clinical trial in individuals with nash with and without t dm, treatment with the dual acc and acc inhibitor gs- decreased liver steatosis without improvement of fibrosis [ ] . the major concern of this strategy is that decreased dnl in nafld might channel lipids towards other harmful directions, e.g. increased blood lipids [ ] . scd is a key enzyme for hepatic lipogenesis that catalyzes the conversion of saturated fatty acids to mufa and its downregulation protected mice from high carbohydrate-induced liver steatosis [ ] . a clinical trial with aramchol, an inhibitor of scd , showed a reduction in liver fat content, resolution of nash and improvement of liver fibrosis in persons with nafld with prediabetes or t dm [ ] . these results paved the way for a respective phase clinical trial ( table ) . mitochondrial uncoupling describes any process that uncouples the electron transport from atp synthesis in mitochondria [ ] . , -dinitrophenol (dnp) was widely used for the treatment of obesity before its discontinuation due to life-threatening serious adverse events [ ] . to overcome the side effects of dnp, improved formulas of dnp were recently developed to decrease the toxic to effective dose ratio (dnp-methyl ether (dnpme) and controlled-release mitochondrial protonophore (crmp)) [ , ] . oral crmp targets mainly the liver due to the first-pass effect and decreased hepatic insulin resistance, hepatic steatosis and liver fibrosis in a methionine-choline deficient rat model of nash [ ] . crmp was also effective in nonhuman primates with diet-induced nafld for reduction of hepatic steatosis and egp [ ] . thyroid hormone deficiency has been associated with nafld development [ ] . thyroid hormone analogues decreased liver fat, liver transaminases, and inflammatory and fibrosis markers in animal models of nash [ ] . resmetirom is a liver-targeted highly selective thr-β agonist which showed efficacy in reducing liver fat content in nash with and without t dm [ ] . currently, there is ongoing clinical trial for evaluation of long-term outcomes of resmetirom and its efficacy on nash resolution ( table ). vitamin e is a potent antioxidant [ ] . in nash without t dm, vitamin e improved nas without worsening of fibrosis [ ] . despite concerns regarding adverse effects of long term vitamin e usage, treatment with vitamin e for ≥ years reduced the risk of liver failure in a nash cohort with advanced fibrosis and with or without t dm [ ] . therapeutic manipulation of intestinal microbiome is still in its infancy. rodent data showed efficacy for fecal microbiota transplantation in improving nafld in a diet-induced nash model [ ] . also, modulation of the intestinal microbiota by antibiotic treatment reduced liver transaminases in nafld [ ] . the use of prebiotics and probiotics in obese nafld/nash is currently not supported by high-quality clinical studies with mr-or biopsy-based endpoints [ ]. the evidence for shared pathophysiological mechanisms between t dm and nafld will help to develop strategies for detecting and treating both diseases and preventing their leading complication, cvd. altered lipid and energy metabolism, insulin resistance, low-grade inflammation and intestinal dysbiosis represent key targets. in addition to weight loss by lifestyle modification or bariatric surgery, glp- ra and sglt i are promising antihyperglycemic concepts with beneficial effects on nafld and cvd. in addition, many j o u r n a l p r e -p r o o f metabolism-based drugs are currently studied comprising ppar agonists, endocrine dual coagonists to modulators of hepatic metabolism or microbiota. nevertheless, several roadblocks need to be overcome to reduce the burden of nafld in t dm. first, there is still lack of preclinical animal models that encapsulate essential features of human nash and diabetes. second, available biomarkers lack diagnostic efficacy to identify nafld progression. innovative strategies such as cluster analysis already enabled detection of a diabetes subtype (sird) with high risk of nafld [ ] . combination with computational integration of multiomics data shall identify specific disease signatures and pave the way to precision medicine and targeted management of t dm and nafld. finally, studies on so-called endpoints are scarce, which may be due to the need of long-term studies to evaluate liver-related mortality, to the current neglect to accept cvd morbidity and mortality as nafld outcome and to ongoing discussions on the relevance of surrogate markers. the research of the authors is supported in part by grants from the german federal ministry phase iii registered interventional trials on "clinicaltrials.gov" for nafld as accessed on th april . bl, baseline; ccr / , c-c chemokine receptors type and type ; fxr, farnesoid x receptor; hba c , glycated haemoglobin; lxr, liver x receptor; mpc, mitochondrial pyruvate carrier; na, data not available; nafld, non-alcoholic fatty liver disease; nfs, nafld fibrosis score; ppar, 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of liraglutide therapy on liver fat content in patients with inadequately controlled type diabetes: the lira-nafld study placebo-controlled randomised trial with liraglutide on magnetic resonance endpoints in individuals with type diabetes: a pre-specified secondary study on ectopic fat accumulation the anti-diabetic drug exenatide, a glucagon-like peptide- receptor agonist, counteracts hepatocarcinogenesis through camp-pka-egfr-stat axis ly , a novel dual gip and glp- receptor agonist for the treatment of type diabetes mellitus: from discovery to clinical proof of concept unimolecular dual incretins maximize metabolic benefits in rodents, monkeys, and humans effects of novel dual gip and glp- receptor agonist tirzepatide on biomarkers of nonalcoholic steatohepatitis in patients with type diabetes sglt inhibitors for primary and secondary prevention of cardiovascular and renal outcomes in type diabetes: a systematic review and meta-analysis of cardiovascular outcome trials 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steatohepatitis-related hepatocarcinogenesis: association with sglt expression in hepatocellular carcinoma empagliflozin ameliorates obesity-related cardiac dysfunction by regulating sestrin -mediated ampk-mtor signaling and redox homeostasis in high-fat induced obese mice sglt inhibition: pros and cons lik (licogliflozin), an sglt / inhibitor, robustly decreases alt and improves markers of hepatic and metabolic health in patients with nonalcoholic fatty liver disease: interim analysis of a -week, randomized, placebo-controlled, phase a study n pparγ signaling and metabolism: the good, the bad and the future long-term pioglitazone treatment for patients with nonalcoholic steatohepatitis and prediabetes or type diabetes mellitus: a randomized trial pioglitazone, vitamin e, or placebo for nonalcoholic steatohepatitis randomized, placebocontrolled trial of pioglitazone in nondiabetic subjects with nonalcoholic steatohepatitis pioglitazone for the primary and secondary prevention of cardiovascular and renal outcomes in patients with or at high risk of type diabetes mellitus: a meta-analysis targeting metabolism, insulin resistance, and diabetes to treat nonalcoholic steatohepatitis insulin sensitizer msdc- k in non-alcoholic steatohepatitis: a randomized, double-blind, placebo-controlled phase iib study the opportunities and challenges of peroxisome proliferator-activated receptors ligands in clinical drug discovery and development elafibranor, an agonist of the peroxisome proliferator-activated receptor-α and -δ, induces resolution of nonalcoholic steatohepatitis without fibrosis worsening announces-results-from-interim-analysis-of-resolve-it-phase- -trial-of-elafibranor-in-adultswith-nash-and-fibrosis.html dual pparα/γ agonist saroglitazar improves liver histopathology and biochemistry in experimental nash models synthesis , and evaluation of a novel series of indole sulfonamide peroxisome proliferator activated receptor (ppar) α/γ/δ triple activators: discovery 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steatohepatitis with fibrosis cenicriviroc treatment for adults with nonalcoholic steatohepatitis and fibrosis: final analysis of the phase b centaur study statin use and risk of cirrhosis and related complications in patients with chronic liver diseases: a systematic review and meta-analysis prescription of statins in suspected non-alcoholic fatty liver dise ase and high cardiovascular risk, a population-based study ezetimibe decreased nonalcoholic fatty liver disease activity score but not hepatic steatosis effect of fenofibrate and niacin on intrahepatic triglyceride content, ve ry low-density lipoprotein kinetics, and insulin action in obese subjects with nonalcoholic fatty liver disease effects of free omega- carboxylic acids and fenofibrate on liver fat content in patients with hypertriglyceridemia and non-alcoholic fatty liver disease: a double-blind, randomized, placebo-controlled study acetyl coa carboxylase inhibition reduces hepatic steatosis but elevates plasma triglycerides in mice and humans: a bedside to bench investigation acetyl -coa carboxylase inhibitor gs- for weeks reduces hepatic de novo lipogenesis and steatosis in patients with nonalcoholic steatohepatitis hepatic stearoyl-coa desaturase- deficiency protects mice from carbohydrate-induced adiposity and hepatic steatosis one-year results of the global phase b randomized placebo-controlled arrest trial of aramchol, a stearoyl coa desaturasemodulator in nash patients n mitochondrial uncoupling and lifespan emerging pharmacological targets for the treatment of nonalcoholic fatty liver disease, insulin resistance, and type diabetes reversal of hypertriglyceridemia, fatty liver disease and insulin resistance by a liver-targeted mitochondrial uncoupler controlled-release mitochondrial protonophore reverses diabetes and steatohepatitis in rats controlledrelease mitochondrial protonophore (crmp) reverses dyslipidemia and hepatic steatosis in dysmetabolic nonhuman primates nonalcoholic fatty liver disease and hypercholesterolemia: roles of thyroid hormones, metabolites, and agonists mgl- ) for the treatment of non-alcoholic steatohepatitis: a multicentre, randomised, double-blind, placebo-controlled, phase trial nonalcoholic fatty liver disease and diabetes: part ii: treatment vitamin e improves transplant-free survival and hepatic decompensation among patients with nonalcoholic steatohepatitis and advanced fibrosis total fecal microbiota transplantation alleviates high-fat diet-induced steatohepatitis in mice via beneficial regulation of gut microbiota efficacy of rifaximin on circulating endotoxins and cytokines in patients with nonalcoholic fatty liver disease key: cord- - bj gr authors: zimmet, p. title: the burden of type diabetes: are we doing enough? date: - - journal: diabetes & metabolism doi: . /s - ( ) - sha: doc_id: cord_uid: bj gr summary increasing levels of obesity, arising from energy-rich diets and sedentary lifestyles, are driving a global pandemic of type diabetes. the prevalence of type diabetes worldwide is set to increase from its present level of million, to million by the end of the decade and to as many as million by . shocking as they are, these figures represent only clinically diagnosed diabetes, and many more cases of diabetes remain undiagnosed and untreated. in addition, up to one-quarter of western populations have impaired glucose tolerance or the dysmetabolic syndrome, which are considered to represent pre-diabetic states. type diabetes is appearing increasingly in children and adolescents, and the frequency of diagnosis of paediatric type diabetes is outstripping that of type diabetes in some areas. the long-term complications associated with type diabetes carries a crushing burden of morbidity and mortality, and most type diabetic patients die prematurely from a cardiovascular event. diabetic patients are more than twice as costly to manage as non-diabetic patients, due mainly to the high costs associated with management of diabetic complications. indeed, diabetes care already accounts for about - % of the total national health care budgets of western european countries. controlling the type diabetes epidemic will require changes to the structure of healthcare delivery. well-resourced interventions will be required, with effective co-ordination between all levels of government, health care agencies, multidisciplinary health care teams, professional organisations, and patient advocacy groups. above all, intervention is needed today. diabetes metab, , , s - s • www.e med.com/dm p zimmet t oday we are seeing a paradox in global public health. in recent decades, we have seen the reemergence of communicable diseases such as tuberculosis, and a new group including hiv/aids, ebola virus and, most recently, the devastating epidemic of severe acute respiratory syndrome (sars). in addition, there is still a global crisis resulting from widespread malnutrition. however, against this background, we are seeing a dramatic rise in prevalence of chronic non-communicable diseases such as type diabetes, cardiovascular diseases, hypertension and obesity in developed and developing nations. there were already signals that diabetes was to become the epidemic of the st century in the early s. at this time, bennett and his colleagues discovered an extraordinarily high prevalence of type diabetes in the pima indians of the usa [ ] , and we reported on the equally high rates of diabetes in the micronesian population of nauru and other pacific island communities [ , ] . over subsequent decades, numerous reports have highlighted the high prevalence of type diabetes in a number of other populations including native americans, afro-americans, and mexican americans in the usa [ ] [ ] [ ] [ ] , native canadians [ ] , australian aborigines and torres strait islanders [ ] , and polynesians in new zealand [ , ] . the prevalence of type diabetes is rising relentlessly around the world (fig ) . current estimates suggest that, globally, the number of persons with diabetes will rise from million in the year , to million by the year , and to million by [ , ] . this rise is predicted to occur in virtually every nation, with the greatest increases expected in developing countries. type diabetes will account for nine patients in every ten of these diagnoses. this explosive increase in the prevalence of type diabetes, and the consequences of its complications and associated disorders, represents the greatest health care challenge facing the world today [ ] . the highest rates of type diabetes occur in native americans and pacific islanders, followed by hispanics or mexican americans, people originating from the indian subcontinent, south east asians and african americans [ , ] . in addition, a relatively high prevalence has been reported from some of the middle east arab states [ , ] , and from disadvantaged minorities in the developed countries, including australia's indigenous population [ , ] . type diabetes affects up to % of adults in native american and pacific island populations [ , , , ] and the years to saw an increase from . % to . % in the prevalence of diabetes among people age - years in the usa [ ] . in china, a prevalence figure of . %, from a survey of , subjects aged - from all parts of the country, was about three-fold higher than prevalence estimates from a decade before [ ] . the type diabetes prevalence in an urban south indian population among individuals aged over years rose from . % in to . % in [ ] , while in denmark, a % rise in diabetes prevalence has been reported over a year period [ ] . in our recent national study in australia, . % of adults were found to have diabetes compared to an estimated . % in [ ] . our studies in mauritius have provided a future guide to the magnitude of the global diabetes epidemic [ ] . the population of this nation are asian indian, creole (black) and chinese, and these three ethnic groups account for over % increasing prevalence of type diabetes by region [ ] . of the world's population. mauritians have a high diabetes prevalence [ ] and a % secular increase occurred between and in mauritian asian indians and creoles [ , ] . the mauritian chinese share this high prevalence [ ] . data showing that the prevalence of type diabetes doubled between and in singaporean chinese, together with the high prevalence in taiwan [ , ] , provide an alarming indicator of the magnitude of the anticipated epidemic in the people's republic of china (prc) [ ] . in the prc, the prevalence of type diabetes was, until recently, less than % [ ] , but recent studies show a three-fold increase in prevalence in certain areas of china within the last two decades [ ] . if china has just % of the prevalence of diabetes in taiwan, the number of individuals with diabetes will increase dramatically from million in to well over million by . diabetes prevalence is currently estimated at . % in the developed world, and forecast to rise to . % by [ ] . developing countries are starting from a lower baseline, at . %, but this is set to increase by more than a third, to . %, in . the lower overall prevalence in developing countries conceals considerable heterogeneity between individual nations. in latin american countries, the crude prevalence of type diabetes for ranges from . % in chile to . % in argentina [ ] and in africa from . % in tanzania to . % in the northern sudan [ ] . in asia, the overall prevalence is low in some countries, such as bangladesh (roughly - %), but higher in others, such as pakistan ( - %) [ , ] . the pacific islands bear an especially heavy burden of diabetes, as described above, with an estimated prevalence of % in kiribati, % in the cook islands, % in fiji, and % in nauru [ ] . increased rates of obesity due to low levels of physical activity and high-energy diets are driving this global epidemic [ ] . for example, between and , the average body weight of men and women in the usa increased by . kg and . kg, respectively, while the prevalence of diabetes increased from . % to . % [ ] . obesity, particularly abdominal obesity, promotes the development of insulin resistance and the dysmetabolic syndrome (also known as the "metabolic syndrome", or "syndrome x") and, ultimately, type diabetes [ ] [ ] [ ] [ ] . approximately one quarter of the population of the developed world are already believed to have the dysmetabolic syndrome [ , ] . impaired glucose tolerance or impaired fasting glucose develop in response to worsening insulin resistance before the clinical diagnosis of type diabetes is made, and signify an increased risk of developing type diabetes [ ] . between % and % of western populations may already have igt [ , ] . for example, the australian diabetes, obesity and lifestyle study (ausdiab) recently surveyed the glycaemic status of a population of , adults [ ] . the overall preva-lence of diabetes was . %, but the combined prevalence of impaired fasting glucose or impaired glucose tolerance was more than twice as high, at . %. these glucose-intolerant, but non-diabetic, individuals represent a reservoir of potential new diabetes cases. approximately - % of individuals with impaired glucose tolerance go on to develop type diabetes each year [ ] . demographic, social and cultural factors profoundly influence the prevalence of diabetes in a developing nation, as shown by recent studies in india. the overall prevalence of diabetes in that country was estimated at . % in the year [ ] . however, a survey in madras, an urban area of southern india, showed that the prevalence of diabetes had risen by % between - and - , and by a further % in the year [ , ] . increasing urbanisation and sedentary work was significantly associated with this increased prevalence [ , ] . indeed, % of urban residents, and only % of rural residents were found to have type diabetes in this survey. south asians who migrate to developed nations face a similar increase in the risk of developing diabetes [ , ] . a greater tendency to insulin resistance is already evident in children of south asian descent in the uk, which is associated with a steeply rising incidence of type diabetes [ , ] . the impact of urbanisation on diabetes prevalence in india is consistent with experience from other cultures, such as australian aborigines, pacific islanders and native americans [ , ] . global estimates predict that the ratio of people with diabetes in urban and rural areas in the developing world will rise from . -fold, at present, to more than -fold in [ ] . other socio-economic factors, particularly poverty, also increase the risk of type diabetes [ ] . patients with a diagnosis of diabetes represent the tip of the iceberg, and a large number of patients with undiagnosed diabetes may also be at risk of adverse clinical outcomes. in the ausdiab study, there was one undiagnosed case for every known case of diabetes [ ] . an observational study carried out in an apparently healthy, elderly ( - years) population in the us revealed that % of the , participants had undiagnosed diabetes. higher estimates have been observed in patients with a previous mi ( %; [ ] ) or awaiting coronary angioplasty ( . %; [ ] ). a study in the uk, which surveyed subjects without known diabetes in an impoverished urban area, illustrates how genetic and socioeconomic factors combine to amplify the problem of undiagnosed diabetes [ ] . the age-adjusted prevalence of diabetes in this population was high in subjects of south asian or african-caribbean descent ( % and %, respectively), as would be expected from earlier studies. however, the age-adjusted prevalence of type diabetes was unexpectedly high ( %) in subjects of european descent. similarly, data from the third us national health and nutrition examination survey suggested that the adult prevalence of undiagnosed type diabetes nationwide was . %, though this concealed wide vari- s diabetes metab, , , s - s • www.e med.com/dm p zimmet ations between men and women and between ethnic groups [ ] . for example, the prevalence of undiagnosed diabetes among middle-aged ( - years) mexican-american men and women was . % and . %, respectively, compared with . % and . %, respectively, for the general population within this age range. there is compelling evidence that undiagnosed diabetes is associated with cardiovascular risk factors characteristic of insulin resistance or the dysmetabolic syndrome, including hypertension and abdominal obesity [ ] [ ] [ ] . the additional increases in metabolic dysfunction associated with westernisation and urbanisation of the developing world, as described above, can only exacerbate this problem in the future. type diabetes mellitus in children, teenagers and adolescents is a serious new aspect of the type diabetes epidemic and is an emerging public health problem of significant proportions [ ] . while, globally, type diabetes is still numerically the major form in children, it is likely that type diabetes is set to be the predominant form within years in many ethnic groups and potentially in europid groups. type diabetes has already been reported in children from japan, the united states, pacific islands, hong kong, australia and the united kingdom [ ] . the prevalence of type diabetes in the general population of the usa has been estimated at . / - year olds [ ] . dabelea et al. have studied pima indian children since the late s, and have demonstrated rising rates of glucose intolerance with time and age, as well as a female preponderance [ ] . from - to - , the prevalence of type diabetes has markedly increased from . % in males and . % in females to . % in males and . % in females. diagnoses of type diabetes now outnumber diagnoses of type diabetes by : in children in parts of japan and china, compared with a ratio of about : of total diagnoses of diabetes in urban usa centres [ , ] . obesity and insulin resistance are driving this explosion of paediatric diabetes, as in adults [ ] . a diagnosis of diabetes has a profound impact on life expectancy, and a patient diagnosed with type diabetes in middle age ( - years) stands to lose as much as years of life expectancy [ ] . given the close association between type diabetes and the cardiovascular risk factors constituting the dysmetabolic syndrome, it is not surprising that most type diabetic patients ultimately die from a cardiovascular cause [ ] . indeed, type diabetes confers the same degree of risk of premature death as a previous myocardial infarction in a non-diabetic subject [ ] . a substantial proportion of type diabetic patients already have diabetic complications at the time of diagnosis of diabetes. for example, retinopathy, peripheral neuropathy and proteinuria were present in %, % and %, respectively, of the newly-diagnosed patients in the uk prospective diabetes study (ukpds) at baseline [ ] . the cost of diabetes in europe -type (code- ) study has investigated the prevalence of diabetic complications in a randomly selected cohort of type diabetic patients. less than half ( %) of the german cohort of , patients (mean age years) did not have diabetic complications, while % had at least two, and % had at least three complications [ ] . cardiovascular complications were present in % of patients, cerebrovascular complications in %, and neuropathy or diabetic foot syndrome, retinopathy and nephropathy were present in %, % and % of patients, respectively. the code- data are consistent with previous estimates of the prevalence of coronary heart disease and other complications in type diabetic patients in various countries (fig ) . the prognosis of type diabetes in patients who already have diabetic complications is extremely poor. a retrospective review of patients referred to a combined diabetesrenal clinic showed that their median survival was only months [ ] . similarly, odds ratios for all-cause and cardiovascular mortality in finnish type diabetes patients with diabetic retinopathy were . and . , respectively, compared with non-diabetic control subjects [ ] . diabetes is expensive to manage, and the per capita costs of managing a diabetic patient are - -fold higher than for a non-diabetic patient [ ] . however, the additional cost burden associated with type diabetes begins long before diabetes is diagnosed. analysis of data from a managed care organisation in the usa showed that the cumulative costs associated with inpatient, outpatient and pharmacy provision for patients with type diabetes were higher, on average, than those incurred by control subjects matched for gender and age for each of the years preceding diagnosis of diabetes [ ] . by the time of diagnosis, the total cost of treatment was $ , higher for a diabetic patient, compared with a control subject (fig ) . costs for the years following diagnosis were higher in diabetic subjects, as would be expected, with a total difference in average total costs of $ , between a type diabetic patient and a matched control subject (fig ) [ ] . most of the cost of managing type diabetes is associated with the management of diabetes-related complications, especially where hospital treatment is required. the ukpds performed separate health economic evaluations, based on the main cohort, who were managed with conventional dietbased treatment or intensive glycaemic management with a sulphonylurea or insulin [ ] , and on overweight patients, who were managed with diet or with intensive metforminbased treatment [ ] . the total costs incurred during years of follow-up are shown in table i . managing complications accounted for about two-thirds of the total costs in the diettreated group of the main cohort, and about three quarters of the costs in diet-treated overweight patients. complications accounted for more than half of the total costs during follow-up even after intensive glycaemic management with a sulphonylurea or insulin (main cohort) or metformin (overweight cohort), which significantly reduced the incidence of microvascular and macrovascular complications, respectively [ , ] . comparing the health economic costs between countries is difficult, because of variations in the costs of services and standard clinical practice, and variations in how data are presented. however, in general terms, the cost of managing complications drives the overall treatment costs in most countries. fig compares the average annual costs of managing a type diabetic patient across europe: in most countries, costs arising from hospital treatment (mainly associated with the management of complications) were markedly greater than out-of- columns show the average of estimates of the prevalence of individual complications for adults with type diabetes presented by amos et al. [ ] . numbers beneath columns show the number of estimates used in each case. chd: coronary heart disease; pvd: peripheral vascular disease. cumulative costs of managing a type diabetic patient and an age-and gender-matched control subject in a managed care organisation over a period of years before (a and b) or after (c and d) the diagnosis of diabetes [ , ] . hospital (ambulatory) costs [ ] . data from the usa, from a model based on the interventions used in the ukpds, show that the annual costs of managing complications in patients receiving diet-based therapy ($ , ) accounted for % of total direct costs of care ($ , ) [ ] . a retrospective analysis of , patients within a managed care organisation in the usa compared the costs of diabetes management in type diabetic patients with no evidence of significant cardiovascular or renal disease ("no complications"), in patients under treatment or investigation for risk factors for cardiovascular or renal conditions ("pre-event"), and in patients with a history of cardiovascular events or clinically significant renal dysfunction ("post-event") [ ] . direct costs were lowest in the "no complications" group (fig ) , and increased in the "preevent" group for men and women. however, the greatest differences in costs were between the "pre-event" and "postevent" groups, with a greater than doubling of costs for patients who developed cardiovascular complications. data are from separate analyses, from the perspective of a healthcare purchaser. all costs are undiscounted direct costs and are in uk £ ( values). additional costs for specialist care were for eye or renal care in lean patients and eye care in overweight patients. the analysis in lean patients presented individual costs from the clinical trial setting and adjusted the total for the routine community care setting; the analysis in obese patients presented all costs after adjustment for the routine care setting, hence na (= not available) for trial costs for overweight patients. the percentage of costs due to complications was calculated using trial costs for lean patients and standard practice costs for overweight patients, for the same reason. total costs (in uk £) associated with type diabetes and its management during years of follow-up in the uk prospective diabetes study [ , ] . direct medical costs per type diabetic patient in some european countries in [ ] . additional treatment costs arising from the development of cardiovascular or renal diabetic complications [ ] . costs are in prices from the perspective of a health care purchaser. patients were stratified as follows. no complication: patients in the analysis either had no history of cardiovascular or renal disease or treatment; pre-event: patients were under treatment with an antihypertensive drug, had made at least two visits to a cardiologist, or had had at least one positive test for microalbuminuria; post-event: patients had suffered at least one major cardiovascular event (e.g. myocardial infarction, stroke, revascularisation, or hospitalisation for congestive heart failure) or had documented clinical renal dysfunction indicated by high serum creatinine measurements. the high prevalence of type diabetes means that the overall costs of managing the disease, including costs arising from managing diabetic complications, are high enough to place a substantial burden on national economies. the total direct healthcare expenditure on the management of diabetes in the usa in was $ billion. of this amount, only $ billion was required for glycaemic management, with $ billion required for the management of complications and other problems related to diabetes [ ] . the total costs of managing type diabetes in europe already range between roughly % and % of national healthcare budgets in europe (fig ) . the burden on some other economies is even higher. for example, diabetes care in mexico accounts for % of the total healthcare budget, which equates to . % of gross domestic product [ ] . moreover, the burden grows heavier as the global epidemic of type diabetes evolves. projections based on the uk diabetic population suggest that the total costs of managing type diabetes will increase from £ . billion in to £ . billion by , an increase of % [ ] (fig ) . national governments have either failed to recognise the future socioeconomic burden of type diabetes or are ignoring it. the global epidemic of type diabetes is already threatening healthcare budgets, and the burden will continue to increase. we should not forget that these prevalence figures relate to people with a clinical diagnosis of type dia-betes. many more people have undiagnosed diabetes, and there is a vast reservoir of people with igt, most of whom will develop diabetes eventually, and who are already at substantial risk of developing cardiovascular disease [ ] . increasing rates of obesity will place ever more people at risk of developing the dysmetabolic syndrome, type diabetes, and cardiovascular disease. the increasing prevalence of type diabetes in children and adolescents underlines the urgency of taking action. the diabetes prevention program has shown us that preventing type diabetes with lifestyle interventions or with pharmacologic therapy is cost-effective in either the european or american settings [ , ] . however, extending these benefits beyond the tightly-controlled structure of a randomised clinical trial will require an unprecedented degree of public health intervention. educating the public on the dangers of obesity and glucose intolerance will certainly be important. some steps in this direction have been made. for example, efforts are underway to improve the lifestyles of school-age children in japan [ ] , and health workers in philadelphia in the usa patrol the streets armed with weighing machines to spread the message of the dangers of obesity [ ] . in finland, a simple questionnaire is being used as a screening tool to identify citizens at risk of type diabetes, who may benefit from further follow-up and intervention [ ] . mounting a successful challenge to the diabetes epidemic, however, will require major alterations to the structure of society. for example, the intervention in finland is being mounted in collaboration with a broad range of governmental agencies and healthcare advocacy groups, as part of a broader strategy dating back several decades [ ] . support from the highest level of governments is also needed, as resources to mount diabetes prevention efforts must be made available, and changes to taxation and reimbursement structures relating to resources for people to improve their lifestyles would also certainly help in the longer term. the rising tide of type diabetes and its complications will place an increasingly heavy burden of morbidity and mortality on patients and their families for decades to come. moreover, the expenditure required to manage these patients will stretch the healthcare systems even of 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descriptive epidemiology of non-insulin dependent diabetes mellitus detected by urine glucose screening in school children in japan us public health and the st century: diabetes mellitus the diabetes risk score: a practical tool to predict type diabetes dehko: development programme for the prevention and care of diabetes in finland key: cord- -ba wq t authors: sias, catia; salichos, leonidas; lapa, daniele; del nonno, franca; baiocchini, andrea; capobianchi, maria rosaria; garbuglia, anna rosa title: alpha, beta, gamma human papillomaviruses (hpv) detection with a different sets of primers in oropharyngeal swabs, anal and cervical samples date: - - journal: virol j doi: . /s - - -x sha: doc_id: cord_uid: ba wq t background: recent studies have shown a -fold increase of oropharyngeal cancer in the presence of hpv, while α-hpv detection seems to be rare in oral cavity in comparison to anal or cervical district, many novel β and γ types have been isolated in this anatomical site suggesting a wide tropism range. currently, there are no guidelines recommending hpv oral cavity screening as a mandatory test, and it remains unknown which hpv types should be included in hpv screening programs. our goal was to assess hpv prevalence in oropharyngeal, anal, and cervical swabs using different sets of primers,which are able to amplify α, β, γ hpv types. methods: we analysed the presence of hpv dna in oropharyngeal (n = ), anal (n = ), cervical specimens (n = ) from hiv positive and negative patients using fap / and my / primers. all untyped strains were genetically characterized through pcr amplification and direct sequencing of partial l region, and the resulting sequences were classified through phylogenetic analysis. results: hpv prevalence was . % in oropharyngeal swab samples, including infections with multiple hpv types ( . %). hpv prevalence in this anatomical site was significantly associated with serostatus: . %in hiv positive and . % in hiv negative patients (p < . ). unclassified types were detected in specimens. in our analysis, we did not observe any difference in hpv (α, β, γ) prevalence between men and women. overall, β species were the most frequently detected . %. when using anal swabs, for hiv positive patients, β genus prevalence was % and γ genus was . % including unclassified types. in cervical samples from hiv positive women ( hpv negative and positive by my / pcr), only one sample was positivite for β( ) species ( . %) using fap primers. six of the untyped strains clustered with sequences from species , , , , of γ genus. four sequences remained unclassified. finally, β and γ hpv prevalence was significantly lower than their respective hpv prevalence as identified by the luminex system in all anatomical sites that were analyzed in previous studies. conclusion: this study provides new information about viral isolates present in oropharyngeal site and it will contribute to improve the monitoring of hpv infection. electronic supplementary material: the online version of this article ( . /s - - -x) contains supplementary material, which is available to authorized users. human papillomavirus (hpv) persistent infections are considered the primary cause of ano-genital cancer, where greater than % of cervical cancer and more than % of anal cancer contain hpv dna [ , ] . throughout the past years, several studies have suggested the link between hpv infection and other epithelial cancers, including cutaneous and oropharyngeal cancers. oropharyngeal cancers are often referred to as "head and neck cancer" (hnc), and mainly include squamous-cell carcinoma which occurs in the oral cavity, base of tongue, tonsils, adenoids pharynx and larynx. recent studies on hpv infection in oral exfoliated cells have shown that there is a -fold higher risk for oropharyngeal cancer in the presence of virus [ ] [ ] [ ] . the highest prevalence (up to %) for these hpv associated cancers has been found in the tonsillar cancers sub-set [ ] [ ] [ ] . since hpv presented a global prevalence in oropharyngeal squamous cell carcinoma (opscc) and oral squamous cell carcinoma (oscc) of . and . % respectively [ ] , iarc categorized this genotype as a risk factor with for the pathogenesis of both cancers [ ] . additionally, other hpv types have also been linked with these cancers, including hpv , , and [ ] including β and ɣ types [ ] . furthermore, while α-hpv detection seems to be rare in oral cavity in comparison to anal or cervical districts [ ] , many novel β and γ types have been isolated in oral cavity [ ] suggesting a wide tropism range of these genera [ ] . at the same time, different studies on hpv prevalence provided contradicting result for β and ɣ types not only in oropharyngeal site, but also in cervical and anal anatomical sites [ ] [ ] [ ] [ ] [ ] [ ] . to date, there are no guidelines recommending hpv oral cavity screening as a mandatory test, and no hpv dna test has been approved for hpv detection in oral cavity. in addition, it remains unknown which hpv types should be included in oral cavity screening. agalliou [ ] highlighted the link between β, γ hpv types and oral cancers, assessing the presence of hpv types which are not generally detected with commercial assays in cervical cancer screening. even though different methods have been used to detect novel hpv types, the association between new β and γ hpv types and oral cavity cancer has not yet been established. in this study, we analyzed the presence of hpv dna in oral, anal, and cervical specimens collected from hiv positive and hiv negative individuals, living in the same geographic area (regione lazio) by using my / [ , ] fap / primers [ ] . these primers, both targeting highly conserved region within l orf, are considered broad range pv primers and allow the detection of the great majority of already known and officially recognized hpv types. my / had been used in α hpv detection in cervical sites, where they showed good sensitivity in alpha hpv types amplification [ ] , while fap primers officially recognized hpv from α-pv, β-pv, and ɣ-pv genera, and might detect potentially new types [ ] [ ] [ ] [ ] . this property is particularly relevant, since ɣ and β papillomaviruses have been already identified in several anatomic sites [ , ] . granted that that hpv prevalence is significantly higher among hiv infected people and at multiple anatomic sites [ ] [ ] [ ] [ ] we included both hiv positive and hiv negative people in our study in order to also verify whether β and ɣ hpv positivity is influenced by host immune status. this is a retrospective study carried out at laboratory of virology inmi l spallanzani on residual oropharyngeal samples collected for respiratory virus detection, anal and cervical swabs collected for hpv testing in diagnostic routine. the hospital ethics committee approved the protocol. date of birth, date of swabs sampling, hiv serostatus were recorded from institutional database. in hiv positive patients, the most recent cd t cell count (± month from the date of sample collection) and hiv rna viral load (± month from the date of sample) with a detection limit of cp/ml (abbott molecular inc., des plaines, il, usa) were used to correlate clinical features and hpv positivity. oropharyngeal swabs -oropharyngeal samples were collected as following: the nylon-flocked tip was rotated - -times against right and left buccal mucosa, palatine, tonsils, upper and lower pharynx area. the swab was then plunged and stirred in . ml dmem medium with streptomycin and ampicillin. specimens were refrigerated within - h after collection until processing. anal swabs -we retrospectively analysed anal swabs previously tested by my / primers and typed by clart hpv clinical array or sanger sequencing (see hpv detection and typing section, below). one hundred samples belonged to hiv positive women ( hpv positive by my / pcr and negative by my / pcr), anal swabs were collected from men who have sex with men (msm). all msm specimen resulted positive in my / pcr. cervical swabs -a total of cervical swabs ( hpv negative and hpv positive, assigned by my / pcr) were considered in this study. all samples belonged to hiv positive women. general characteristics of hiv infected patients are presented in additional file : table s . oropharyngeal swabs were removed from the medium which we divided in two parts: half part was used for detecting respiratory viruses panel (influenza-a, b, rsv, rhinovirus, coronaviruses, metapneumovirus, adenovirus) and the other half was employed in testing hpv dna, having been stored at − °c until use. before nucleic acid extraction, all specimens were pre-treated. briefly, μl of clinical material was digested with μl proteinase k solution (qiagen, hilden germany) and lysed with al lysis buffer (qiagen, hilden, germany) at °c for min. nucleic acid extraction was done with a magnetic bead-based automated platform (qiasymphony, hilden, germany) in accordance with the manufacturer's instructions. nucleic acids were eluted in μl of ave elution buffer (qiagen, hilden, germany). nucleic acid from anal and cervical swabs were extracted as described elsewhere [ , ] . samples that were β-globin negative were excluded from the study [ , ] . ten μl of eluted nucleic acids were employed for evaluating the presence of hpv types by using my ( 'cgtccmarrggawactgatc ') and my ( 'gcmcagggwcataayaatgg ') [ , ] pcr and faststart dna polymerase (roche diagnostics gmbh, mannheim, germany). the pcr assay conditions were: °c for min, then cycles (denaturation °c/ s, annealing °c/ s, and extension °c/ min). one last step for extension was employed at °c for min. fifteen μl of the pcr products were mixed with loading solution in . % agarose gel electrophoresis stained by ethidium bromide and run for min at v. hpv genotyping of positive samples was conducted using genomica clinical hpv array (genomica, madrid, spain). clart hpv clinical array hpv is able to detect: , , , , , , , , , , , , , , , , , , , all adequate samples were retested using fap ( 'taacwgtiggicayccwtatt ') and fap ( 'ccwatatcwvhcatitciccatc ') primers [ ] able to detect α, β and γ hpv types. faststart dna polymerase were used in this assay condition: min at °c, cycles (denaturation °c/ s, annealing °c / s, and extension °c min) followed by a final extension step at °c for min. pcr products were also run on agarose gel. positive samples with fap set of primers and those positive with my / pcr protocol, but negative in genomica typing assay were purified and sequenced on the automated abi prism instrument, by using a bigdye terminator cycle sequencing kit (applied biosystems, warrington, uk). hpv sequences were aligned using mafft [ ] using the l-ins-i method. after visual inspection using seaview [ ] we removed sample sequence q and q were excluded from the alignment because they represented mixed infection and the sanger sequence interpretation was not optimal. reference sequences hm _hpv and gu _hpv were also excluded from the alignment. then, we further implemented gblocks [ ] to remove ambiguous positions using the least stringent options. to infer a maximum likelihood tree, we implemented raxml [ ] using a gtrcat model, with bootstrap replicates. tree visualization was achieved using figtree [ ] . finally, pairwise similarity matrices were constructed using blast. oropharyngeal swabs-a total of oropharyngeal swabs with β-globin positive signal were considered in this study. hpv prevalence was . % ( / ), including infections with multiple hpv types ( / , . %) ( table ). my / pcr gave positive results in samples. however, a blast search against genbank indicated that amplified fragments were identical to human sequences ( / , %), thus true hpv positive rate was estimated at . %. out of , three samples were identified as α and ten as β types. alpha-types were detected in two hiv positive patients ( / , . %), and in hiv negative patient. hpv α-types, and , were detected in hiv positive subjects ( / , . %) after being typed by clart hpv clinical array, while hpv was amplified in hiv negative subject ( / , . %); it was typed by sanger sequencing. all β hpv types belonged to β species (table ) , and hpv was the most frequent type ( / ), both in hiv positive and hiv negative patients; samples were identified as positive by fap pcr. no specific amplicons were observed. seven fap-detected hpv types were found in samples that also gave positive results with my /my pcr (table ) . types β were the most represented (n = ), while one sample was identified as γ type (hpv , γ ). additionally, one mixed infection (q ) and untyped hpv strains were also detected. among β species, the most frequent types were hpv (n = , . %) and hpv (n = , . %). three multiple infections harboured β and β types, whereas β types and untyped strains were observed in other multiple infections. no multiple infection harboured α with β or γ species (table ) . overall, β species were most prevalent (n = / , . %). hpv distribution differed significantly when the number of hpv β, γ-types were considered in hiv positive and hiv negative subjects: ( / , . %) vs ( / , . %) respectively (chi square test, p < . ). hiv positive people with hpv in oropharyngeal swabs showed a mean cd t cell count of . ± . . among hiv positive patients with detectable hpv, / had no detectable hiv rna in plasma and the other patients (n = ) showed a mean rna copies/ml . ± . . hpv prevalence rates were compared for statistical significance, using a chi squared test, in men and women both hiv positive and hiv negative. however, no difference was observed (chi square test, p > . ). anal swabs-eighty-six anal swabs from msm resulted positive by my / pcr, and anal swabs from women ( hpv positive by my / pcr and negative by my / pcr) were retested with fap primers. among msm anal swabs, samples showed single hpv infection by clart array testing, and patients harboured multiple hpv infection with at least one high risk (hr) type. in . % of samples we observed a clinically evident anal pathology, including subjects with anal intraepithelial neoplasia (ain) ii, patients with ain i. among hpv positive women, harboured a single infection (ain ii, n = ; ain i, n = ); atypical squamous cells of undetermined significance (asc-us), n = ; normal, n = ) and samples showed hpv multiple infection, all with at least hr type ( %) (ain ,n = ; ain i, n = ; normal cytology, n = ). hpv type was undetermined in one sample. among single infection with ain i, one specimen was infected with a low risk (lr)type (hpv ). nineteen anal swab specimens were identified as hpv positive by fap primers detection (female anal swabs, n = ; male anal swabs, n = ) ( table ). overall α-type strains were detected in samples with fap primers which were not previously detected by my / primers. all but one α-type strains were also detected with α mucosal hpv multiple infection typed with clart hpv clinical array which included at least one hr type. hpv β types (hpv and hpv ) were found in two specimens co-infected with α hpv types; specimens harboured γ hpv types (hpv and hpv ), both associated with type α hpv multiple infections. all fap untyped strains (n = ) were found in anal specimens infected by α types with at least one high hr type. considering cytological aspects, / samples, which resulted positive with fap primer pcr, had ain (i, ii) lesions, while / samples showed normal cytology (table ) . among anal swabs from females (n = ), only samples ( . %), previously tested my / hpv positive, resulted hpv positive with fap primer (hpv , α species; hpv , α species; hpv , α species). no untyped hpv strains were observed among female samples. overall, β genus prevalence was % and γ genus was . % including untyped isolates. mean of cd t cell count was . ± . cells/μl. overall / patients ( . %) were under antiretroviral therapy (art). all patients without art had a cd t cell count > / μl. current art use was not associated with risk of β and ɣ infection (chi square test, p > . ). cervical swabs-twelve specimens showed a single hpv infection, and had multiple infections by clart hpv array; / samples harboured at least hr genotype. cytology findings were available for specimens already resulted positive by clart hpv clinical array: had normal cytology, were asc-us or low grade squamous intraepithelial lesion (lsil), harboured hpv single infections, showed hpv multiple infections, and were high grade squamous intraepithelial lesion (hsil). all my / hpv negative women (n = ) showed normal cytological findings. eleven/ cervical samples resulted fap positive ( . %). however, only hpv types were not previously detected by my / primers. five samples harboured α types (hpv , n = α ; hpv , n = , α ; hpv α , n = ), and only one specimen gave positivity for β (hpv ) ( . %). the mean cd t cell count was ± cells/μl. ninety-six percent of women were receiving art per who guideline of starting art at cd t cell count < cell/mm . the woman harboring β hpv strain was under art. she had a cd t cell count of cells/μl and hiv rna viral load was not detected. overall cervical and anal β and γ type positivity was not related to immune status as measured by cd t cell count (see table ) and art treatment. furthermore, the immune-re constitution could potentially prevent the persistence of β and ɣ types in anal and cervical sites or alternatively, β and ɣ types might show to have a less tropism to these anatomical sites. for the phylogenetic analysis of our untyped hpv samples we used partial cds sampled sequences from major capsid protein l fap pcr products and reference sequences. our reference sequences represent classified and untyped hpv sequences (see table ). according to our phylogenetic analysis, of our sampled sequences (q , q , q , q , q and q ) clustered with previously classified reference sequences from species , , , and -γ genus. sequences q , q , q and q remained unclassified while showing great similarity with unclassified genomic regions af (fams ), kp , and af . respectively (see fig. and table ). in hnc, particularly oropharyngeal cancer, the true prevalence and involvement of hpv in the carcinogenetic process is still unknown. in fact, many studies report low prevalence of hpv obtained with systems used for the determination of hpv in cervical cancer screening (specific for α types), while other authors found higher prevalence when they used a platform able to detect hpv types belonging to genera other than α genus [ ] both in oral cavity and in oral cancer [ , ] . agalliu recently found a correlation between the infection of some β, ɣ hpv types and oscc [ ] . furthermore, a recent meta-analysis provided additional evidence of the involvement of β hpv types in the development of scc in immunocompetent individuals [ ] . additionally, several mechanistic studies have consistently showed that e and e from several β hpv types are able to target cellular proteins, such as p and prb, and to deregulate fundamental events involved in cellular transformation [ ] . thus β and ɣ hpv types detection could be included in a hpv screening test for oral cancer prevention, while a correct and suitable detection of hpv infection is becoming a priority. currently, there are few studies that compare the hpv prevalence obtained using the protocols, which are normally for cervical cancer screening with those obtained using methods that are able to detect hpv types belonging to genera other than α or new ones. in this study, in order to assess the impact of pcr system in hpv prevalence determination, we carried out the detection of hpv with my / primer set, used in several genotyping molecular assays (for example linear array hpv genotyping test, roche molecular system, ca usa; clart hpv clinical arrays, genomica, madrid, spain,) and fap primers able to detect α, β, γ hpv types in several specimens. in oropharyngeal swabs samples, my / primer set was able to detect . % ( / ) of hpv positive samples, while fap set of primers detected the presence of hpv in / samples ( . %). beta hpv was the main genus detected with a prevalence of . % in hpv positive samples. this prevalence was in agreement with bottalico et al. that used fap primer and my / for detecting hpv in oral swabs [ ] . hpv was the most frequent β type and hpv the main type among β species as described by bottalico [ ] . hpv , which was reported as the main β type in forslund study [ ] , was absent in our samples. hr, namely hpv , was observed only in one specimen from an hiv positive subject, whereas hr hpv were frequently observed in bottalico study group [ ] . the low frequency of hr could be imputed to immune status of the patients. our patients were under haart therapy and most of them had a cd t cell count > /μl. no severe compromise of the immune-system could have limited a persistence of hr in this anatomical site. unfortunately, no information was given in the bottalico paper on patient cd t cell count, and this fact did not allow a correct comparison of these results. according to previous data reported by forslund et al. [ ] , no difference in hpv prevalence was observed among men and women suggesting that host specific factors could contribute to hpv persistence and neoplasia development. gamma genus represented . % of hpv positive samples. to note, / γ strains were untyped. phylogenetic analysis revealed that samples (q and q ) belonged to γ species, while q to γ species and q to γ species. interestingly, the oral untyped genotypes fell in different genera and cluster with the anal untyped strains, suggesting a specific tropism of these gamma types for oral mucosa. gamma and were the most representative γ species described by bottalico in oral rinse and agalliu in hnc, suggesting their potential involvement in oscc development. unlike forslund and hampras [ , ] , we did not identified any β , β , β hpv types. this may be due to the types of biological samples that were used or to differences in the efficiency of extraction methods that were applied, which could have influenced the outcome of pcr [ ] . finally, differences in results might also be explained by the geographical distribution of hpv types. in general, differences in hpv β and γ types and their prevalence were observed by the luminex platform. according to him study [ ] , among β genotypes, hpv and hpv were the prevalent genotypes. while, among β strains, hpv was the most represented,and it was never detected in our samples. interestingly hr hpv was also the main β type observed also in moscicki study, whereas hpv represented the main β type [ ] . a different pattern of β types could be related to a different cellular input as described in a previous study [ ] , where several β hpv types showed essentially increasing prevalence with increasing dna input. beta hpv types , , , , , , and showed increased prevalence only in higher dna input groups [ ] . this may impose compromises in comparisons between studies; for example, our hpv negative result could be explained by an insufficient dna input in the pcr assay. further research is needed to establish whether there is an influence between cell number input and hpv β detection in oropharyngeal anatomical site and which cut-off would be suggested to avoid false negative results. possible type-specific differences in sensitivity to cellular input have to be evaluated in wider studies. if they are confirmed, they may be explained by the different sensitivities in detection systems and/or in the fig. a maximum likelihood phylogenetic analysis was inferred on unknown collected samples (bold) and reference sequences using the gtrcat model. out of unknown samples, sequences were clustered within previously classified hpv species lineages. bootstrap supports lower than % were excluded from the tree. genbank accession number: mh -mh different viral load spectra. we detected α strains in anal site fap primers that had not been typed by genomica, and β types, hpv and hpv , that had not been reported among oropharyngeal swabs. Βeta types frequency was sensibly lower to that found by luminex system reported by torres et al. [ ] which found . % of β types in anal swabs from hiv positive msm and . % among hiv negative msm. in this study, hpv and hpv were the prevalent types among β species, while hpv and hpv were the most frequently observed β types. among γ genus, the γ species was the most prevalent in both msm hiv positive and -negative groups, while we observed only γ untyped strains. donà reported higher prevalence of β ( . %) and γ types ( . %) in a group of msm similar to ours using the luminex system [ ] . some hypothesis could put forth to explain these data: i) the luminex system could be much more sensitive than the fap system, or ii) that cross-reactivity could occur in β and γ types detection when α multiple infections are present. in literature, cross-reactivity was also observed among α genotypes. preisler observed cross-reactivity both in lr and hr genotypes using hc , cobas, and aptima assays, despite what manufacturer claims: about % of hpvdna results in primary screening accounting for cross-reactivity, regardless of the assay [ ] . to obtain improved analytical and clinical performance, cross-reactivity studies should be focused, since this aspect could influence the effectiveness in a future head neck hpv cancer screening. in cervical swabs the fap primers mainly showed the presence of α genotypes (hpv , α ; hpv , α ) which were not detected by clart hpv clinical array/ my - primers and β type (hpv ). this low prevalence of β types confirms the data reported by bottalico et al. [ ] , which found a prevalence of % of the β types and % of the γ types in the cervical samples, emphasizing a weak β and γ type tropism towards the female genital mucosa. however, the genotypes hpv and hpv , detected by bottalico in cervical specimens, were never observed among our cervical samples, suggesting a different geographic distribution of hpv β and γ type similarly to that described for α genus. conversely, these findings were in disagreement with those obtained by luminex system, as described in previous study [ ] . a quantitative detection of the viral load in hair follicles demonstrated that the β genotype copy number was considerably lower than that reported for mucosal high-risk types from α genus in cervical tissue [ ] . thus, only comparable viral load detection studies with different methods and the potential for multiple infections detection could explain these differences in prevalence of β types, which are reported in literature. overall, our results confirmed a prevalence of > % for hpv strains in the oropharyngeal anatomical district. the genus β and γ were predominant when the analysis was carried out with fap primers. the discrepancy on the prevalence and hpv types reported in other studies [ ] [ ] [ ] ] seems to be due to the detection system: highest prevalence was always obtained with the luminex system. different sensitivity and specificity of hpv detection methods were also a problem in the determination of α hpv types, as described in a systematic review where approximately - % of all positive results showed discordance [ ] . a global proficiency program like labnet for α types in cervical hpv infection surveillance programs should be planned also for α, β and γ hpv type detection in oropharyngeal samples [ ] . standardizing methods for oral sample collection and hpv detection would ensure comparability between different detection methods in oral cavity samples [ , ] . in addition, considering that γ genus has been growing rapidly (currently γ types have been identified) surpassing α and β genera, and that % of the new types deposited in the hpv center within belonged to γ genus [ - ], our data reinforces the relevance of using primer sets able to detect a wide spectrum of hpv strains including β and γ types as well as new genotypes for hpv detection in the oropharyngeal anatomical site. this seems to be a crucial point since the meta genomic approach applied in some analysis [ ] should be used with caution, taking in account the possibility of having an overestimation of hpv types [ ] and requiring a confirmation of positivity by the sanger method. additional file worldwide burden of cancer attributable to hpv by site, country and hpv type casecontrol study of human papillomavirus and oropharyngeal cancer associations of oral α-, β-, and γ-human papillomavirus types with risk of incident head and neck cancer detection and quantitation of human papillomavirus (hpv) dna in the sera of patients with hpv-associated head and neck squamous cell carcinoma hpv infections and tonsillar carcinoma human papillomavirus (hpv) in head and neck cancer. an association of hpv with squamous cell carcinoma of waldeyer's tonsillar ring incidence of human papillomavirus (hpv) positive tonsillar carcinoma in stockholm, sweden: an epidemic of viral-induced carcinoma? hpv dna, e /e mrna, and p ink a detection in head and neck cancers: a systematic review and meta-analysis preventable exposures associated with human cancers prevalence of microsatellite instability, inactivation of mismatch repair genes, p mutation, and human papillomavirus infection in korean oral cancer patients oral human papillomavirus in healthy individuals: a systematic review of the literature characterization of three novel human papillomavirus types isolated from oral rinse samples of healthy individuals the oral cavity contains abundant known and novel human papillomaviruses from the betapapillomavirus and gammapapillomavirus genera betapapillomaviruses in the anal canal of hiv positive and hiv negative men who have sex with men beta and gamma human papillomaviruses in the anal canal of hiv-infected and uninfected men who have sex with men prevalence of beta and gamma human papillomaviruses in the anal canal of men who have sex with men is influenced by hiv status cervical infection with cutaneous beta and mucosal alpha papillomaviruses prevalence and epidemiologic profile of oral infection with alpha, beta, and gamma papillomaviruses in an asian chinese population the use of polymerase chain reaction amplification for the detection of genital human papillomaviruses identification and assessment of known and novel human papillomaviruses by polymerase chain reaction amplification, restriction fragment length polymorphisms, nucleotide sequence, and phylogenetic algorithms a broad range of human papillomavirus types detected with a general pcr method suitable for analysis of cutaneous tumours and normal skin molecular methods for identification and characterization of novel papillomaviruses diversity of human papillomaviruses in skin lesions multiple oral carcinomas associated with a novel papillomavirus in a dog beta and gamma human papillomaviruses in anal and genital sites among men: prevalence and determinants prevalence and risk factors for anal human papillomavirus infection in human immunodeficiency virus (hiv)-positive and high-risk hiv-negative women relationship between prevalent oral and cervical human papillomavirus infections in human immunodeficiency virus-positive and -negative women high diversity of alpha, beta and gamma human papillomaviruses in genital samples from hiv-negative and hiv-positive heterosexual south african men an anal cancer screening program for msm in italy: prevalence of multiple hpv types and vaccine-targeted infections frequency and multiplicity of human papillomavirus infection in hiv- positive women in italy oral human papillomavirus dna detection in hiv-positive men: prevalence, predictors, and co-occurrence at anal site enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia mafft: a novel method for rapid multiple sequence alignment based on fast fourier transform seaview and phylo_win: two graphic tools for sequence alignment and molecular phylogeny selection of conserved blocks from multiple alignments for their use in phylogenetic analysis raxml-vi-hpc: maximum likelihood-based phylogenetic analyses with thousands of taxa and mixed models beta-hpv types in patients with head and neck pathology and in healthy subjects association between β-genus human papillomavirus and cutaneous squamous cell carcinoma in immunocompetent individuals-a metaanalysis the biology of beta human papillomaviruses the nasal mucosa contains a large spectrum of human papillomavirus types from the betapapillomavirus and gammapapillomavirus genera prevalence and concordance of cutaneous beta human papillomavirus infection at mucosal and cutaneous sites analysis of the effect of dna purificationon detection of human papillomavirus in oral rinse samples by pcr prevalence and transmission of beta and gamma human papillomavirus in heterosexual couples prevalence and multiplicity of cutaneous beta papilloma viruses in plucked hairs depend on cellular dna input crossreactivity profiles of hybrid capture ii, cobas, and aptima human papillomavirus assays: split-sample study beta-papillomavirus dna loads in hair follicles of immunocompetent people and organ transplant recipients concordance of beta-papillomavirus across anogenital and oral anatomic sites of men: the him study concordant testing results between various human papillomavirus assays in primary cervical cancer screening: systematic review continuing global improvement in human papillomavirus dna genotyping services: the and hpv labnet international proficiency studies detection of oral hpv infection -comparison of two different specimen collection methods and two hpv detection methods oral human papillomavirus infection incidence and clearance: a systematic review of the literature international standardization and classification of human papillomavirus types towards quality and order in human papillomavirus research hpviewer: sensitive and specific genotyping of human papillomavirus in metagenomic dna we are grateful to all member of virology laboratory inmi l. spallanzani for sample collection. we thank dr. mariana badescu for english editing. the authors declare that they have no competing interests. key: cord- -m va er authors: raaben, matthijs; groot koerkamp, marian ja; rottier, peter jm; de haan, cornelis am title: type i interferon receptor-independent and -dependent host transcriptional responses to mouse hepatitis coronavirus infection in vivo date: - - journal: bmc genomics doi: . / - - - sha: doc_id: cord_uid: m va er background: the role of type i ifns in protecting against coronavirus (cov) infections is not fully understood. while covs are poor inducers of type i ifns in tissue culture, several studies have demonstrated the importance of the type i ifn response in controlling mhv infection in animals. the protective effectors against mhv infection are, however, still unknown. results: in order to get more insight into the antiviral gene expression induced in the brains of mhv-infected mice, we performed whole-genome expression profiling. three different mouse strains, differing in their susceptibility to infection with mhv, were used. in balb/c mice, which display high viral loads but are able to control the infection, and genes were significantly differentially expressed (≥ . fold change) upon infection at and days post infection, respectively. functional association network analyses demonstrated a strong type i ifn response, with irf and irf as the central players. at days post infection, a type ii ifn response also becomes apparent. both the type i and ii ifn response, which were more pronounced in mice with a higher viral load, were not observed in svev mice, which are much less susceptible to infection with mhv. svev mice lacking the type i interferon receptor (ifnar-/-), however, were not able to control the infection. gene expression profiling of these mice identified type i ifn-independent responses to infection, with ifn-γ as the central player. as the balb/c and the ifnar-/- svev mice demonstrated very similar viral loads in their brains, we also compared their gene expression profiles upon infection with mhv in order to identify type i ifn-dependent transcriptional responses. many known ifn-inducible genes were detected, several of which have previously been shown to play an important protective role against virus infections. we speculate that the additional type i ifn-dependent genes that we discovered may also be important for protection against mhv infection. conclusion: transcriptional profiling of mice infected with mhv demonstrated the induction of a robust ifn response, which correlated with the viral load. profiling of ifnar-/- mice allowed us to identify type i ifn-independent and -dependent responses. overall, this study broadens our present knowledge of the type i and ii ifn-mediated effector responses during cov infection in vivo. cytokines are key regulators that dictate many aspects of innate and adaptive immunity. induction of type i interferons (ifns), a well-known subset of cytokines with antiviral activity, is triggered by a selection of cellular pattern recognition receptors, including tlrs (toll-like receptors), rig-i (retinoic acid-inducible gene i), and mda (melanoma differentiation-associated protein ). these receptors are activated in response to a range of pathogenspecific factors, which includes double-stranded rna produced during virus infection [ , ] . secreted type i ifns (i.e. ifn-α and ifn-β), subsequently induce an antiviral transcription program in the infected cell as well as in adjacent cells, thereby magnifying the "danger" signal and protecting against the infection. the role of type i ifns in controlling coronavirus (cov) infections is not well understood. a number of studies has shown that covs, like the mouse hepatitis virus (mhv) and the severe acute respiratory syndrome (sars)-cov, are poor inducers of type i ifns in cell culture, and even escape from detection by cytoplasmic pattern recognition receptors [ ] [ ] [ ] [ ] [ ] [ ] . consistently, virus-encoded ifn antagonistic functions have been described for both mhv and sars-cov [ , ] . in vivo, however, mhv infection appeared to induce the production of ifn-α in plasmacytoid dendritic cells (pdcs) by a tlr -dependent mechanism [ ] . moreover, mhv infections of primary neuronal cultures and of the central nervous system (cns) induced ifn-β gene expression, indicating that the production of type i ifns in vivo is not limited to pdcs [ , ] . furthermore, neuronal cultures infected with mhv exhibited increased expression of several type i ifninduced transcription factors [ ] . more recently, roth-cross and co-workers reported that macrophages and macrophage-like microglia cells produce ifn-β in the cns of mhv-infected mice in a mda -dependent manner [ ] . several studies have demonstrated the importance of the type i ifn response in controlling mhv infection in vivo. the exogenous delivery of type i ifns was shown to inhibit mhv infection of and spread to the mouse brain [ , ] . consistently, infection of mice lacking the functional type i ifn receptor (ifnar-/-) with mhv resulted in increased viral replication and extended tissue tropism [ , , ] . although many type i ifn-responsive genes have been identified [ ] , the protective effectors against mhv infection are yet unknown [ ] . in order to get more insight into the antiviral gene expression induced in the brains of mhv-infected mice, we performed whole-genome expression profiling. three different mouse strains (balb/c, svev and ifnar-/- svev mice), differing in their susceptibility to infec-tion with mhv, were used. previously, we have observed that svev mice are significantly more resistant to infection via the intranasal route than balb/c mice [ ] . the reason for the significant difference in susceptibility is not known, but may be related to different antiviral immune responses in these two mouse strains. furthermore, gene expression profiling of svev mice lacking the type i ifn receptor, which are not able to control the mhv infection [ ] , allowed us to identify type i ifn-independent transcriptional responses. we started by comparing the whole-genome expression profiles in the brains of the balb/c and the svev mice upon infection with mhv. to this end, mice were inoculated intranasally with tcid of mhv strain a or with pbs (control). groups of mice (n = ) were sacrificed at and days post inoculation after which the brains were harvested and total rna was isolated. the extent of virus replication was determined by quantitative reverse transcriptase (rt)-pcr targeting mhv-specific rna sequences as described earlier [ ] . previously, we demonstrated that the viral rna load correlates well with viral infectivity in tissue homogenates [ ] . while no viral rna could be detected yet at days post inoculation (data not shown), viral rna was observed in the brain of both mouse strains at day ( figure a ). as expected, the balb/ c mice displayed a much higher viral rna load than the svev mice. next, the rna extracts were processed for microarray analysis using the pbs-inoculated groups as the reference. in total, and genes were significantly differentially expressed (≥ . fold change) in balb/c mice at and days post infection, respectively. in contrast, in the svev mice, no significant induction of gene expression was observed. the results are depicted in figure b as a gene tree that was built based on the genes with a significantly altered expression level in balb/c mice at days post infection (i.e. expression-based cluster analysis). from these data we were able to identify host genes, the increased expression (≥ . fold) of which could already be detected at day (i.e. early genes; figure c ) or only at day (i.e. late genes; figure d ). the group of earlyinduced transcripts contained many ifn-inducible genes, including the well-known interferon regulatory factor (irf ), signal transducer and activator of transcription (stat ), and '- ' oligoadenylate synthetase (oas) genes (additional file a). within the cluster of "late" genes (additional file b) several chemokines (i.e. ccl , ccl , ccl , cxcl , and cxcl ) could be identified. next, in order to construct a functional association network, we applied the string . software [ ] to the list of proteins encoded by the "early" and "late" genes. we also included known interactors of our hits in this analysis, while proteins that did not demonstrate any known interactions were excluded for clarity. the results are shown in figure a and b. functional association network analysis of the proteins encoded by the "early" genes revealed two main modules. one module contained several proteins involved in antigen presentation, while the other module contained numerous proteins involved in the type i ifn response. the key player in this latter module appeared to be irf , which is the master regulator of type i ifn-dependent responses [ ] . functional association network analysis of the proteins encoded by the "late" genes revealed a large network of proteins involved in host-pathogen interactions. although the microarray analyses did not reveal the induction of ifn-γ gene expression itself, ifn-γ appeared at a central position in the network. in addition, the induction of a type i ifn response was also evident from this network as demonstrated by the presence of the transcription factors irf and irf , both of which demonstrated elevated mrna levels upon mhv infection. in conclusion, these results demonstrate that mhv infection induces a robust ifn response both at and days post infection, in which the transcription factors irf , irf , and irf appear to be the key players. at days post infection, a type ii ifn response also becomes apparent. to confirm and extend these observations, we next analyzed the induction of type i and ii ifn gene expression (i.e. ifn-α and ifn-β , and ifn-γ, respectively) by using quantitative rt-pcr. in agreement with the microarray expression profiles, significant induction of these type i and ii ifns could only be detected in the mhv-infected balb/c animals ( figure e ). the observation that the balb/c mice, unlike the svev mice, exhibited abundant expression of ifn-responsive genes upon mhv infection appears counter intuitive as the svev mice are much more resistant to the infection than the balb/c mice. apparently, the resistance of svev mice to mhv infection is not controlled by a more robust ifn response. the reason for the observed difference in susceptibility between the different mouse strains after intranasal inoculation is not known. mhv-a was recently shown to replicate efficiently in the liver of svev mice after intraperitoneal inoculation [ ] . interestingly, the resistance of svev mice after intranasal inoculation is not restricted to infection with mhv, as it was also observed for vesicular stomatitis virus [ ] . the microarray expression profiles described above suggested that the induction of an ifn response correlates with the viral load within the brain. to confirm this, we examined the data of the individual balb/c mice at days post infection in more detail. clearly, the animals with the highest viral loads (mouse and ; figure a ), also dis-played significantly higher levels of induction of type i and ii ifn expression ( figure b ). likewise, the amplitude of the gene expression profiles ( figure c and additional file ) of the individual mice also correlated with the viral loads in the brain. these observations are in agreement with results obtained by the profiling of sars-covinfected macaques [ ] . also in that study a positive correlation between virus load and the induction of gene expression was observed. a few genes (n = ), including isg , showed an inverse correlation with the viral load. we currently have no explanation for this observation as expression of isg is known to be induced by type i ifns [ , ] . interestingly, isg has been shown to exhibit antiviral activity against other viruses [ , ] . to study the role of type i ifn-independent and -dependent gene expression in the control of mhv infection in vivo in more detail, we next made use of the ifnar-/-mice [ ] . these mice are highly susceptible to mhv infection as compared to the parental svev mice [ , ] . indeed, when these mice were inoculated intranasally with tcid of mhv-a , viral rna levels in their brains became much higher than in animals from the parental strain at days post infection ( figure a ). interestingly, at this time point the viral rna levels in the ifnar-/-mice were comparable to those in the brains of the balb/c mice. however, efficient dissemination of the infection, resulting in high viral loads in the liver as determined by quantitative rt-pcr, was only observed in the ifnar-/-mice and not in the wild-type mice, which displayed viral rna levels just above background ( figure b ). thus, in agreement with previous studies, a type i ifn-dependent response is required to inhibit virus dissemination [ , ] . whole-genome expression profiling of brains of the ifnar-/-mice revealed the significantly induced expression of genes (≥ . fold) at days post infection. in contrast, at day , hardly any alterations in gene expression could be detected in these knock-out mice (additional file ). figure c shows an expression-based cluster analysis of these genes for the wild-type and ifnar-/mice. comparison of the complete expression profiles of these mice revealed that the transcriptional profile at day in the ifnar-/-mice has a larger similarity with the profile at day of the parental svev mice than with that of the knock-out mice at day post infection ( figure c ). this observation may suggest the presence of an early host response to infection with mhv in the parental mice, even though no significant induction (≥ . fold) of gene expression could be detected ( figure b) . such a response, may not be evident in transcriptional profiles of whole organs, but might only be apparent at the cellular level. we speculate that early decisive events are happening in initial target cell populations such as dcs and macro-early and late transcriptional responses to infection with mhv as the knock-out mice lack a functional type i ifn receptor, the upregulation of gene expression observed in these mice apparently occurs independently of type i ifn signalling. not much is known yet about type i ifn-independent responses to infection. the observation that the transcriptional upregulation of irf was independent of type i ifn signalling is consistent with the notion that ifn-γ can also induce expression of this gene [ , ]. the type i ifn receptor-independent expression profile within the brains of ifnar-/-mice after mhv infection likewise, we also observed increased transcription of ifitm and ifitm independent of type i ifn signalling, again corresponding with the literature [ , ] . interestingly, the expression of various genes encoding proteins involved in antigen presentation (i.e. h , b m, psmb , psmb , and ctss) was also increased in the absence of type i ifn signalling. psmb and psmb encode immunoproteasome subunits which facilitate antigen presentation to cd + t cells after virus infection, a process that is primarily regulated by ifn-γ [ ] . furthermore, also the expression of the major histocompatibility complex class ii (mhc ii) invariant chain, also called cd [ ], was increased upon infection of the knock-out mice. these data are in agreement with the observation that the induction of genes involved in antigen processing is independent of stat activation by . we also observed the transcriptional upregulation of the isoforms of metallothionein (mt , mt , and mt ), which encode proteins known to scavenge toxic metals [ ] . the induction of these genes, which was not apparent in either wild-type mice, could reflect an acute-phase reaction in the brain of mhv-infected ifnar-/-mice, which likely contributes to pathogenesis as has been shown for other viruses [ - ]. we constructed a functional association network by applying the string . software [ ] to the list of proteins encoded by the type i ifn-independent genes (additional file ). we also included known interactors of our hits in this analysis, while proteins that did not demonstrate any interactions were again excluded for clarity. the result is shown in figure . the analysis revealed ifn-γ as the central player in the type i ifn-independent antiviral network as this protein appeared to link a number of smaller modules. the induction of ifn-γ gene expression could be confirmed using quantitative rt-pcr (data not shown). the finding that ifn-γ-mediated transcriptional responses are not dramatically affected in the absence of type i ifn signalling is in agreement with reports referred to above and with a recent publication by ireland et al. [ ] , which shows that ifn-γ expression is significantly induced in the cns of mhv-infected ifnar-/-mice. while the production of ifn-γ by nk cells plays a major role in the protection against infection with mhv [ - ], the ifn-γ-mediated transcriptional responses that we observed were not protective against acute mhv infection in the ifnar-/-mice. several studies have shown that mhv [ , , ] as well as several other viruses [ - ] replicate to much higher levels (up to fold difference) in ifnar-/-mice than in their wild-type counterparts. in this study we show that a strong correlation exists between the amplitude of type i and ii ifn host responses with the viral load. the huge differences in virus replication between wild-type and ifnar-/-mice therefore do not permit a fair comparison between gene expression profiles of these mice, with the aim of identifying type i ifn-dependent responses. indeed, as no significant gene expression is observed in the wild-type svev mice, a comparison with the expression profile of the ifnar-/-mice only provides information about type i ifn-independent and not ifndependent responses. we now observe, in agreement with our previous study, that the brain of balb/c and ifnar-/ - svev mice contain very similar mhv loads at day and post infection [ ] . since the type i ifn-responsive pathway is very well conserved among many different species [ ], we considered it acceptable to compare the gene expression profiles of these mice with the aim of identifying type-i ifn-dependent responses, although comparing transcriptional profiles of wild-type and ifnar-/-mice from a different genetic background should obviously be done very cautiously. ideally, a comparison between wildtype balb/c and ifnar-/-balb/c mice would have been more accurate. while the induced expression of a number of genes was similar for the two mouse strains (i.e. type i ifn signalling-independent gene-expression), that of other genes was only observed in the balb/c mice (i.e. tentative type i ifn signalling-dependent gene expression). the expression of yet other genes appeared to be partially dependent of type i ifn signalling: increased expression of these genes was observed in the ifnar-/mice, but much more so in the balb/c mice. genes, the expression of which was upregulated (≥ . fold) in the balb/c mice but not significantly changed in the ifnar-/-mice upon infection with mhv, were tentatively designated as type i ifn-dependent. genes, the transcriptional upregulation of which was at least times higher in the balb/c mice than in the ifnar-/-mice, were also added to the list of tentative type i ifn-dependent genes. as expected, this set of genes (n = ) contained many known ifn-responsive genes like isg , ifit , ifit , isgf g, mx and ube l (additional file ). functional association network analyses showed irf and irf to be the key players in the network (additional file ). several of the tentative type i ifn-dependent genes (including mx and ube l) have previously been shown to play an important protective role against virus infections [ - ]. we speculate that other genes present in this list may also be important for full protection against mhv infection. transcriptional profiling of mice infected with mhv demonstrated the induction of a robust ifn response, which correlated with the viral load. profiling of ifnar-/-mice allowed us to identify type i ifn-independent anddependent responses. overall, this study broadens our present knowledge of the type i ifn-mediated effector responses during cov infection in vivo. [ ] [ ] [ ] week old balb/c were obtained from charles river laboratories, while type i ifn receptor knock-out mice (ifnar-/-) [ ] and the parental svev mice were obtained from b&k universal ltd. mice were inoculated intranasally with tcid of mhv strain a and sacrificed at the indicated time-points for organ dissection. control animals were treated with pbs. the study proto-col was approved by the animal ethics committee of the utrecht university, and all experiments were performed in accordance with accepted institutional and governmental policies. whole brains and livers were dissected from the mhvinfected and control mice. the tissues were added to lysing matrix d tubes (mp biomedical), containing ml of the type i ifn-independent gene expression network figure the type i ifn-independent gene expression network. the genes listed in additional file (n = ) were subjected to functional association network analysis by using the string . database as described in the legend of figure . the key player in the network, ifn-γ, is indicated in red. rnapro™ solution (q-biogene), and processed using a fastprep instrument (mp biomedical). the tissues were homogenized at , rpm for sec and immediately placed on ice. subsequently, the homogenates were centrifuged at , rpm for minutes at °c and supernatants were harvested and stored at - °c. total rna was isolated from the homogenates using the trizol reagent (invitrogen) according to the manufacturer's protocol. rna was further purified using the rneasy mini-kit with subsequent dnasei treatment on the column (qiagen). rna integrity was determined by spectrometry and by a microfluidics-based platform using a uv-mini device (shimadzu) and a bioanalyzer (agilent technologies), respectively. ], respectively), were measured by quantitative pcr using assay-on-demand reagents and equipment (pe applied biosystems), according to the manufacturer's instructions. the quantitative pcr reactions were performed in a total reaction volume of μl containing μl taqman ® universal pcr master mix ( ×), μl cdna, μl taqman ® gene expression assay mix ( ×), and μl water using an abi prism sequence detection system under the following conditions: °c for mins, followed by cycles of °c for secs and °c for min. for all assays, we performed "no-rt" (reaction using total rna as the substrate) and "no template" (reaction using water as the substrate) controls. in both cases, omitting cdna from the reaction resulted in a lack of pcr product generation. all assays were analyzed with abi prism software v . . f (pe applied biosystems). the comparative ctmethod was used to determine the fold change for each gene (primer efficiencies were similar for both the endogenous control primer set and genes of interest primer sets [data not shown]). note that the ct values of all samples were within the limits of the standard curves (data not shown). the housekeeping gene gapdh (nm_ . ) was used as a reference in all experiments, since expression of this gene was found constant among samples. the amounts of viral rna were determined by quantitative rt-pcr as described before [ ] . the microarray experiments were performed as described previously [ ] . briefly, mrna was amplified from μg of total rna by cdna synthesis with oligo(dt) doubleanchored primers, followed by in vitro transcription using a t rna polymerase kit (ambion). during transcription, -( -aminoallyl)-utp was incorporated into the single stranded crna. cy and cy nhs-esters (amersham biosciences) were coupled to μg crna. rna quality was monitored after each successive step using the equipment described above. corning ultragaps slides, printed with a mouse array-ready oligo set (operon; , spots), were hybridized with μg of each alternatively labeled crna target at °c for - h. two independent dyeswap hybridizations ( arrays) were performed for each experimental group. after hybridization the slides were washed extensively and scanned using the agilent g aa dna microarray scanner. after data extraction using imagene . software (biodiscovery), lowess normalization [ ] was performed on mean spot-intensities in order to correct for dye and printtip biases [ ] . the microarray data was analysed using anova (r version . . /maanova version . - ) http://www.r-project.org [ ] . briefly, in a fixed effect analysis, sample, array and dye effects were modelled. pvalues were determined by a permutation f -test, in which residuals were shuffled , times globally. genes with p < . after family wise error correction were considered significantly changed. cluster analysis (standard correlation) was performed with genespring gx . software (silicon genetics). when indicated, the confidence level was increased by applying a fold change cut-off. the resulting genelists were subjected to genespring . software for further analysis. arrayexpress accession numbers miame-compliant data in mage-ml format as well as complete descriptions of protocols have been submitted to the public microarray database arrayexpress http:// www.ebi.ac.uk/arrayexpress/ with the following accession numbers: microarray layout, p-umcu- ; gene expression data of mhv-infected mice, e-mexp- ; protocols for total rna isolation and mrna amplification, p-mexp- ; crna labeling, p-mexp- and p-mexp- ; hybridization and washing of slides, p-mexp- ; scanning of slides, p-mexp- ; data normalization, p-mexp- . innate immune recognition of viral infection type i interferons in host defense group coronaviruses prevent immediate early interferon induction by protection of viral rna from host cell recognition transcriptional profiling of acute cytopathic murine hepatitis virus infection in fibroblast-like cells mouse hepatitis coronavirus replication induces host translational shutoff and mrna decay, with concomitant formation of stress granules and processing bodies preferential infection of mature dendritic cells by mouse hepatitis virus strain jhm mouse hepatitis virus does not induce beta interferon synthesis and does not inhibit its induction by double-stranded rna inhibition of beta interferon induction by severe acute respiratory syndrome coronavirus suggests a two-step model for activation of interferon regulatory factor mouse hepatitis coronavirus a nucleocapsid protein is a type i interferon antagonist severe acute respiratory syndrome coronavirus nsp suppresses host gene expression, including that of type i interferon, in infected cells control of coronavirus infection through plasmacytoid dendritic-cell-derived type i interferon differential regulation of innate and adaptive immune responses in viral encephalitis inhibition of the alpha/beta interferon response by mouse hepatitis virus at multiple levels viral induction of central nervous system innate immune responses murine coronavirus mouse hepatitis virus is recognized by mda and induces type i interferon in brain macrophages/microglia protective effect of recombinant murine interferon beta against mouse hepatitis virus infection non-invasive imaging of mouse hepatitis coronavirus infection reveals determinants of viral replication and spread in vivo type i interferons are essential in controlling neurotropic coronavirus infection irrespective of functional cd t cells interferome: the database of interferon regulated genes interferon and cytokine responses to sarscoronavirus infection cyclooxygenase activity is important for efficient replication of mouse hepatitis virus at an early stage of infection this work was supported by grants from the m.w. beijerinck virology fund, royal netherlands academy of arts and sciences, and the netherlands organization for scientific research (nwo-vidi- . . ) to c.a.m. de haan. we thank connie bergmann for advice and monique oostra, marne hagemeijer, and mijke vogels for stimulating discussions. the authors declare that they have no competing interests. mr and mjagk conducted all the experiments. mr wrote the manuscript. pjmr and camdeh coordinated the research efforts and assisted with writing the manuscript. all authors read and approved the final manuscript. key: cord- -tbdsr iw authors: carvalho, c.l.; lopes de carvalho, i.; zé-zé, l.; núncio, m.s.; duarte, e.l. title: tularaemia: a challenging zoonosis date: - - journal: comp immunol microbiol infect dis doi: . /j.cimid. . . sha: doc_id: cord_uid: tbdsr iw in recent years, several emerging zoonotic vector-borne infections with potential impact on human health have been identified in europe, including tularaemia, caused by francisella tularensis. this remarkable pathogen, one of the most virulent microorganisms currently known, has been detected in increasingly new settings and in a wide range of wild species, including lagomorphs, rodents, carnivores, fish and invertebrate arthropods. also, a renewed concern has arisen with regard to f. tularensis: its potential use by bioterrorists. based on the information published concerning the latest outbreaks, the aim of this paper is to review the main features of the agent, its biology, immunology and epidemiology. moreover, special focus will be given to zoonotic aspects of the disease, as tularaemia outbreaks in human populations have been frequently associated with disease in animals. seventy-five per cent of emerging infectious diseases are zoonotic [ ] . some wildlife species have been recognised as being major reservoirs for infectious diseases and the proximity of wildlife habitats and the existence of arthropod vectors with a wide geographical spread have rendered epidemiological cycles more complex [ ] . tularaemia is a zoonosis caused by the francisella tularensis bacterium, which was first isolated in in tulare county, california, by george mccoy and charles chapin [ ] [ ] [ ] . initially termed bacterium tularense, it was allocated to a new genus and named f. tularensis in honour of the pioneer of research on the organism, edward francis [ , ] . arthropod-borne transmission of tularaemia was first demonstrated by francis in when he isolated the etiologic agent in a patient with "deer fly fever" [ , , ] . tularaemia was recognised as an important disease in the last century and since then there has been a growth in enthusiasm for research on this pathogen [ , ] . interest has arisen with regard to f. tularensis as it has emerged in new locations, populations and settings, and increasingly figured in scientific research gauging its potential use in bioterrorism [ , ] . the european centre for disease control and prevention (ecdc) surveillance report refers a total of confirmed cases of tularaemia in a number of european countries in , with sweden reporting the highest confirmed case rate, followed by finland and hungary [ ] . tularaemia is considered an unusual disease and the confirmed case rate in europe has remained stable from to . recent outbreaks of tularaemia have occurred in several european countries, presented in table , including the czech republic, kosovo, bulgaria, germany, sweden, finland, spain, turkey, france and norway [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . besides these outbreaks, sporadic case notifications have occurred in austria, estonia, italy, lithuania, poland, romania, slovakia and the united kingdom [ ] . although there are no reports of tularaemia for denmark during this period, a confirmed case of the disease in a human was recorded there in [ ] . in portugal, the bacterium has been detected in the blood of an asymptomatic man and in a dermacentor reticulatus tick by molecular methods [ ] . f. tularensis is one of the most virulent microorganisms currently known, while as few as ten microorganisms can cause potentially fatal disease in man and animals [ , ] . this high rate of infectivity has led the centre for disease control and prevention (cdc) to classify f. tularensis as a category a biowarfare agent [ ] . f. tularensis is a gram-negative, catalase-positive, pleomorphic and non-motile cocobacillus, characterised as a facultative intracellular pathogen that can grow within different types of cells including macrophages, hepatocytes and epithelial cells [ , , , ] . the cell wall of f. tularensis has an unusually high level of fatty acids with a unique profile for the genus, and wild strains have a lipid-rich capsule, with neither toxic nor immunogenic properties [ , , ] . capsule loss has been related to a decrease in virulence, although the viability or survival of the bacterium within neutrophils may remain unaltered. f. tularensis is a gamma ( )-proteobacteria of the francisellaceae family [ , , ] . f. tularensis is the most common and pathogenic species and is formally divided into three subspecies with different pathogenicities and geographic distributions: tularensis, holarctica and mediasiatica. the species francisella novicida is currently widely accepted as a fourth subspecies of f. tularensis [ , , [ ] [ ] [ ] [ ] [ ] [ ] , as it shares with f. tularensis an average of . % nucleotide identity over a . mbp of genome sequence [ , , , ] . however, some objections to the transfer of f. novicida to the subspecies rank of f. tularensis have been recorded, based on recent multiple genome sequencing results, which show divergent evolutions for f. tularensis and f. novicida populations. therefore, separate species may be retained [ ] . the f. tularensis subspecies tularensis, regarded as the most virulent subspecies and classified as type a, occurs predominantly in north america [ , , , , ] . two distinct genetic sub-populations have been identified, ai and aii, which have different geographic distributions, hosts and vectors [ , , , , , ] . sub-population ai has been additionally sub-divided into groups aia and aib [ , , , ] . the subspecies holarctica, related to milder forms of the disease and classified as type b, occurs throughout the northern hemisphere [ , , , ] . human infection with aib strains usually have a fulminant clinical progression and are associated with high mortality rates, in contrast with infections by aia and aii strains or type b tularaemia [ , , ] . recently, this subspecies has also been detected in tasmania, australia [ ] . subspecies mediasiatica presents a similar virulence to subspecies holarctica, but its geographic distribution is restricted so far to central asia [ , ] . f. novicida is less virulent and has been isolated in north america, australia and thailand [ , , [ ] [ ] [ ] [ ] [ ] ] . based on a high degree of similarity between s rrna gene sequences, other microorganisms have been classified as probable members of the francisellaceae family; these include the francisella-like endosymbionts or fles [ , , ] . fles belong to a distinct phylogenetic clade from f. tularensis species [ ] . the effect of fles, if any, on vector competency and in the transmission of f. tularensis by ticks is still unknown [ ] . fles have a worldwide distribution and are vertically transmitted by hard and soft ticks of the genera amblyomma, dermacentor, ixodes and ornithodoros [ ] [ ] [ ] [ ] . fles have been detected in ticks in north america [ , , ] a information unavailable. (texas, california, minnesota), canada (alberta) and european countries such as spain, portugal, hungary, serbia and bulgaria [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . their pathogenicity to humans is undetermined. they have recently been detected in freeliving small mammals in europe, suggesting the possible transmission of some fle types from ticks to small mammals, although, to date, attempts to demonstrate it have failed [ , , , , ] . the phylogeographic distribution of f. tularensis is given in fig. ; the geographic locations where fles have been detected in ticks are also indicated. in nature, f. tularensis has been detected in a high number of wild species including lagomorphs, rodents, insectivores, carnivores, ungulates, marsupials, birds, amphibians, fish, and invertebrates [ , , , , [ ] [ ] [ ] . lagomorphs and rodents are considered as the main reservoirs of f. tularensis [ , , ] . wild lagomorphs, such as the european brown hare (lepus europaeus), are thought to be suitable sentinels for f. tularensis and disease surveillance [ , ] . recently, there have been serological evidences that foxes and raccoon dogs could also act as biological indicators for tularaemia [ ] . natural infections with f. tularensis have also been documented in different arthropods, although only a subset of these have been identified as important in f. tularensis transmission to humans. still, few pathogens show the adaptability of f. tularensis to such a wide range of arthropod vectors capable of infection dissemination [ ] . arthropod found infected in nature include ticks of the genera amblyomma, dermacentor, ixodes and ornithodoros, mosquitoes of the genera aedes, culex, anopheles and ochlerotatus excrucians, and flies from the tabanidae family (tabanus spp., chrisozona spp. and chrisops spp.) [ , , , ] . nevertheless, vector competence has only been demonstrated in ticks of the genera dermacentor [ ] . tick-borne transmission of f. tularensis usually results in sporadic cases, although occasional outbreaks have also been reported [ ] . although regarded as merely mechanical vectors, mosquitoes have been associated with widespread epidemics of tularaemia and are capable of transient disease transmission [ , ] . both ticks and mosquitoes may be infected in the larval phase. transtadial transmission has been demonstrated in ticks although in mosquitoes evidences for transtadial transmission are only based in molecular methods [ , ] . although transovarial transmission of f. tularensis in ticks was reported [ , , ] , a recent study in dermacentor variabilis has proved otherwise [ ] . despite dissemination to ovaries and then to the oocytes, the pathogen was not recovered from the subsequently hatched larvae. tabanid flies are regarded as mechanical vectors for f. tularensis and the long-term survival of this bacterium does not occur in these arthropods [ ] . the epidemiologic characteristics of vector-borne tularaemia vary throughout the northern hemisphere and also within a given geographic location. this is thought to be related to the abundance of different vectors and host species. this could explain why, in the usa, sweden, finland and russia, the arthropod bite is a common mode of transmission to humans, whilst in western and central europe, contact with infected animals and the ingestion of contaminated food or water have been reported as more common transmission modes. differences in transmission patterns have also been recorded within the usa: in western states, both ticks and deer flies are considered to be important vectors of tularémia, while in the east only ticks are considered relevant. in sweden and finland, mosquitoes have been identified as the primary vectors [ ] . in portugal, the role of ticks and small mammals in the transmission of tularaemia is still the subject of research. a collection of mosquitoes belonging to the genus culex ( . %), ochlerotatus ( . %), anopheles ( . %), culiseta ( . %) and a small number of aedes aegypti females from the island of madeira ( . %) have been analysed, although all the results were found to be negative [ ] . so far, this is in accordance with previous findings regarding the epidemiology characteristics of vector-borne tularaemia, suggesting that, in portugal, mosquitoes have no role in the transmission of this disease. ticks are thought to be the most important vectors of tularaemia in the majority of countries where tularaemia is endemic [ ] . nevertheless, major on-going research on tularaemia, aiming at gauging the overall impact of the disease in portugal, is expected to throw further light on the main f. tularensis sources. in endemic areas, tularaemia is a seasonal disease, with higher incidence in late spring, summer and autumn, occurring annually over a -year period or unreported for more than a decade. often, the number of cases varies widely from year to another, which is thought to be due to temperature or precipitation variability. however, the association between climactic conditions and tularaemia outbreaks has yet to be demonstrated [ ] . f. tularensis has been found to be extremely resistant to environmental stress, surviving for weeks in soil, water and animal carcasses, at low temperatures [ ] . human tularaemia outbreaks are often preceded by animal outbreaks, particularly in wild lagomorphs and rodents. this is usually related to an increase in the numbers of these species, increasing the probability of exposure to infected animals [ , , , ] . the transmission of tularaemia to humans can occur either by direct contact with infected animals or indirectly due to arthropod vector bites, the ingestion of contaminated water, food or aerosols inhalation. aerosols can be dispersed by ventilators, farming, and the deposition of contaminated hay, either intentionally or unintentionally [ ] . domestic dogs and cats can also transmit tularaemia to humans after contact with an infected animal, environment or infected ticks [ ] [ ] [ ] . personto-person transmission has not been described so far [ , , , ] . tularaemia has been reported to occur in any age group. men tend to present a higher prevalence than women [ , ] . professions that are prone to contact with reservoirs or arthropod vectors have been associated with a higher infection risk: these include laboratory technicians, hunters, farmers, veterinary surgeons, and anyone handling the flesh of infected animals [ , ] . few pathogens show the adaptability of f. tularensis to varying vector, host and environmental conditions. variations occur in local transmission cycles in association with differing ecologies. both f. tularensis type a and type b are associated with different life cycles in which different animal hosts and arthropod vectors intervene [ ] . type a tularaemia is more commonly associated with the terrestrial cycle of the disease, with wild lagomorphs such as rabbits and hares acting as vertebrate hosts in which amplification of the agent occurs and where arthropods are disease-disseminating vectors [ , , , ] . type b tularaemia is more frequently associated with the aquatic cycle, although outbreaks of tick-borne tularaemia involving subspecies holarctica have been reported [ , , ] . in this life cycle, f. tularensis circulates in rodents such as beavers, muskrats and voles, and can be introduced in water courses from animal carcasses [ , , , ] . there is also evidence that f. tularensis can persist in water courses in association with amoebas [ , , ] . contaminated water can be the source of infection to humans, flies and mosquitoes [ ] . an unusual waterborne outbreak of human tularaemia has been described in spain associated with crayfish (procambarus clarkii) caught in a contaminated freshwater stream. the crayfish acted as mechanical vectors, through mud-or water-contaminated carapaces, although the presence of f. tularensis in crayfish stomach and hepatopancreas could indicate their eventual role as hosts [ ] . a diagrammatic representation of the terrestrial and aquatic cycles of tularaemia is shown in fig. . f. tularensis is a remarkable bacterial pathogen that can invade and multiply in a wide range of cell types [ , , , , ] . antigen-presenting cells (apc) such as macrophages or dendritic cells, appear to be the primary cell types targeted by the bacterium at the outset of infection [ ] . the virulence of the bacterium is directly related to its capacity to replicate within the cytosol of infected cells [ ] . f. tularensis clearly possesses several mechanisms by which it manipulates immunity. the bacterium evades detection at the point of entry in the host in three ways: (a) it has modified cell-surface structures that enable it to avoid interaction with host receptors that are associated with the induction of inflammation; (b) it targets cells that lack co-receptors which facilitate binding to receptors that might alert the host cell to invasion; (c) it utilises receptors that fail to initiate the production of proinflammatory cytokines [ ] . the entry of f. tularensis in macrophages occurs by means of a specific mechanism inherent to francisella spp. [ ] . the bacterium induces the macrophage to produce asymmetric spacious pseudopod loops in a "looping phagocytosis" process [ , ] . uptake of f. tularensis is markedly enhanced by serum opsonisation, which depends on serum intact complement factor c and host cell receptors (cr ), involving bacterial surface polysaccharides [ , ] . utilisation of cr (and of mannose receptors of dendritic cells (mr) under non-opsonising conditions) is considered to be a fairly innocuous route for entry of f. tularensis, since it is not associated with the induction of signalling cascades that result in pro-inflammatory cytokines production. when opsonised by serum, f. tularensis binds ic b and gains entry to host cells via the cr receptor [ ] . the lipopolysaccharide (lps) of subspecies tularensis is only moderately inflammatory and acts as an extremely weak toll-like receptor (tlr) agonist stimulating a reduced production of pro-inflammatory cytokines [ , ] . these is attributed to the presence of only four acyl groups on the lps that do not bind to the "lps-binding proteins", subverting tlr recognition [ , , ] . in addition to lps, f. tularensis possesses two other tlr agonists [ ] : tul and ftt lipoproteins. these interact with tlr and may alert the host cell for the presence of the bacterium prior to phagocytosis [ , , ] . tlr /myeloid differentiation primary response gene ( ) (myd ) signalling is essential for the production of pro-inflammatory cytokines and is critical for host defence against francisella infection [ , , , ] . f. novicida has been used as a model organism to study immunity to f. tularensis. nevertheless, f. novicida expresses a structurally distinct chemotype of lps that is more pro-inflammatory in mice than the dominant lps chemotype, and is expected to result in different inflammasome activations [ ] . f. novicida escapes the phagossome and replicate in the cell cytosol where it is recognised by the inflammasome signalling system [ , , , ] . inflammasome stimuli activate the protease cysteine aspartate-specific caspase- , promoting the release of potent pro-inflammatory cytokines responsible for cell apoptosis [ , ] . this results in f. novicida release from infected cells and enables the infection of new ones [ , ] . f. tularensis survival and replication within macrophages is enabled by a large set of virulence genes that include the "macrophage growth locus" (mgl) a and b and the "francisella pathogenicity island", fpi [ ] . fpi encodes for a putative type vi secretion system [ , ] and contains genes that have been demonstrated as essential for intra-cellular growth and virulence [ ] . less virulent f. novicida presents only one copy of fpi in contrast with f. tularensis subspecies tularensis and holarctica that present two copies [ , ] . genes within the fpi are regulated by mgla [ ] . although current knowledge of the gene's functions is far from complete, this is one of the most active areas of francisella research [ ] . following phagocytosis of opsonised f. tularensis by polymorphonuclear cells (pmn), the bacterium actively inhibits superoxide anion generation (ros) via nadph oxidase. this allows f. tularensis to evade the phagosome and persist in the cell cytosol. the contribution of polymorphonuclear cells seems to be related to the secretion of cytokines and chemokines that recruit effector cells to the infection site [ ] . however, an excessive recruitment of neutrophils, modulated by an increase in metaloprotease- from the matrix, plays an important role in modulating leucocyte recruitment and seems to be directly related to f. tularensis pathogenesis [ , ] . natural killer (nk) cells from the liver, spleen and lung also play an important role in the innate immune response, in particular by producing inf-␣ following primary infection by f. tularensis [ ] . as f. tularensis is an intracellular pathogen, cellular immune response is believed to be the main defence mechanism. memory effector t cells cd + and cd + are clearly important for the primary control of infection. these cells produce type th cytokines like inf-␥, tnf-␣ and il- that are critical for the initial response to f. tularensis infection [ ] . although the role of humoral immunity in f. tularensis infection is believed to be less important, some studies have demonstrated the enhanced recovery of infected humans that have received hyper-immune serum [ ] . also, infection-specific igm, iga and igg antibodies produced are good exposition indicators and may interfere with the ability of bacteria to infect host cells [ , , ] . the contribution of b cells in defence is thought to be dependent on strain virulence [ , ] . research on anti-francisella antibodies targets is expected to allow for the identification of new diagnostic or reactive antigens and the development of vaccines [ ] . furthermore, f. tularensis is capable of influencing multiple pathways, and continued research into the specific mechanisms by which f. tularensis evades, modulates and suppresses the host immune response will improve our understanding of tularaemia pathogenesis and the regulation of host immunity [ ] . relevant clinical disease has been reported with f. tularensis subsp. tularensis and holarctica. clinical manifestations of tularaemia depend on strain virulence, infective dose and infection route, the extent of systemic involvement and host immune status [ , , ] . the incubation period averages - days but ranges from to days. the disease has an acute onset, with the occurrence of fever ( - • c), chills, fatigue, generalised myalgia and headaches, resembling a flu-like syndrome [ , , ] . the subspecies tularensis (type a) causes severe disease, potentially fatal if untreated. the subspecies holarctica (type b) causes less severe disease and fatalities are rare [ ] . depending on the route of infection, the following forms of the disease are described: ulceroglandular, glandular, oculoglandular, oropharyngeal, pneumonic, typhoidal and septic [ , ] . ulceroglandular and glandular forms of the disease are the most common and frequently result from an arthropod bite or animal contact [ , , ] . in ulceroglandular tularémia, a soft, painless ulcer develops at the inoculation site and evolves to a scar [ , ] . this presentation is associated with fever, lymphadenopathy and, in type a tularaemia, pneumonia and pleural effusion can occur [ ] . in glandular tularaemia, the primary ulcer is unrecognisable [ , , , ] . direct contamination of the eye through contaminated fingers, splashes or aerosols, may be followed by oculoglandular tularaemia. unilateral conjunctivitis, with ulcers or papules in some patients, photophobia and epiphora are the main signs of this form of the disease [ , ] . oropharyngeal tularaemia is acquired by means of contaminated food or water intake and aerosol inhalation [ ] . it develops with ulcerative and exudative stomatitis and pharyngitis [ ] . pneumonic tularaemia occurs by means of contaminated aerosol inhalation but can also arise as a complication of any of the other disease forms by haematogenous generalisation [ , , ] . initial disease development is characterised by fever, cough, pleuritic chest pain and dyspnoea, along with other unspecific symptoms. type a tularaemia is associated with significantly severer and more fulminant forms of pneumonia [ , ] . typhoidal tularaemia refers to a systemic and febrile form of the disease in which no route of infection acquisition can be established [ , ] . septic tularaemia is a severe and often fatal form of the disease that can occur as a complication of the ulceroglandular form in type a tularaemia [ , ] . patients can present unspecific and neurologic symptoms, and septic shock, sirs (systemic inflammatory response syndrome), dic (disseminated intravascular coagulation), haemorrhages, sars (severe acute respiratory syndrome) and multiple organ failure [ , ] . in type b tularaemia, complications of meningitis and septicaemia have only occasionally been described [ ] . clinical manifestations largely depend on the susceptibility of animal species to f. tularensis [ ] . in wild animals, clinical signs of tularaemia are not well documented, and post-mortem findings are highly unspecific and include splenomegaly and punctual necrotic lesions in the liver and spleen [ , ] . in one experimental study in european brown hares (lepus europaeus), clinical signs developed -day postinoculation with a f. tularensis subspecies holarctica strain. these included fever, lethargy and anorexia. two of the five hares in the study succumbed to the infection on days and following inoculation. pathological findings included splenomegaly, diffuse spleen necrosis and focal liver necrosis with hepatocytes vacuolisation. the remaining three hares were euthanised and revealed no pathological lesions. both bacterial culture and mouse inoculation test failed to produce f. tularensis isolation [ ] . in a natural outbreak of tularaemia in brown hares in france, all eight hares involved presented splenomegaly, congestion and haemorrhagic lesions of several organs, tracheitis and bronchitis [ ] . a similar study carried out in hungary on european brown hares naturally infected with f. tularensis subspecies holarctica also showed very similar results [ ] . in another study, female new zealand white rabbits (oryctolagus cuniculus) were exposed to type a tularaemia aerosols, with three different doses. seven of them died while the others developed fever, anorexia and weight loss, with all infecting doses. haematological findings in six rabbits included lymphopenia, monocytopenia and thrombocytopenia. a bibasilar pneumonia and gastrointestinal tract gas distension were the only radiological findings. necropsy findings demonstrated hepatosplenomegaly with extensive spleen necrosis and small white nodules. some of the rabbits presented nodular lesions in the lungs while others showed haemorrhagic lesions [ ] . a situation of particular public health significance, given the risk of pet-to-human transmission, is associated with infected prairie dogs (cynomys ludovicianus) sold as pets in the usa and exported internationally [ , ] . a ban was put in place in the european union and other countries regarding the import of prairie dogs and other rodent species after the usa monkeypox outbreak in [ , ] . wild-caught prairie dogs are particularly susceptible to environmental stress, such as capture, transit and crowding, which can enhance disease manifestations. clinical signs include lethargy, dehydration and grossly enlarged cervical lymph nodes. prairie dogs can produce specific antibodies against f. tularensis and survive tularaemia infection, suggesting their potential role as f. tularensis reservoirs in nature. moreover, one study found that all seropositive animals harboured live infectious bacteria, suggesting persistent infection [ ] . tularaemia has also been described in domestic dogs and cats [ , ] , which may be infected by means of arthropod bites, direct contact with infected animals, their ingestion, or contaminated aerosols [ , ] . cats usually develop severe illness with unspecific clinical signs like fever, lethargy, prostration, vomiting and anorexia, dehydration, regional or generalised lymphadenopathy, splenomegaly, tongue and oropharyngeal ulceration and jaundice [ , , ] . pathological findings include multiple necrotic foci on the lymph nodes, spleen, liver and lungs. frequently, panleukopenia with toxic degeneration of the neutrophils and hyperbilirubinaemia with bilirubinuria are present [ ] . dogs are less susceptible and rarely manifest signs of the disease [ , ] . nevertheless, they can act as carrier hosts [ ] and transmit the bacterium by means their fur after contact with contaminated dead animals or soil [ ] . in most cases, infection is self-limiting and recovery is spontaneous. however, only few cases of natural infection in dogs have been reported [ , ] . in humans, samples should preferably be collected before the onset of antibiotherapy and depend on the clinical form of the disease. samples may include non-heparinised whole blood, serum, respiratory tract secretions and washes, swabs from visible lesions, lymph node aspirates or biopsies, urine, and autopsy materials [ ] . in animals, serum is the preferential sample for all disease forms, but plasma and dry blood on paper filters can also be used. blood samples should be collected at least days after the onset of the symptoms. lymph nodes or bone marrow aspirates, organs (lung, liver, spleen) and cerebrospinal fluid can also be used [ ] . in the context of an outbreak or epidemiologic studies, samples should include arthropod vectors as well as environmental samples like water, soil and rodent faeces [ , ] . culture is the gold standard for f. tularensis and must be carried out in biosecurity level facilities (bsl- ) [ , , ] . f. tularensis is a fastidious microorganism. optimal growth conditions occur at • c and ph . [ , ] . cysteine-enriched media, such as enriched chocolate agar (ca) or % cysteine heart agar with blood medium (chab) must be used for this purpose [ , , ] . growth in a chab medium enables the presumptive identification of f. tularensis by characteristic growth at - h of round and smooth green opalescent shiny colonies, - mm in diameter [ , , , , ] . antibiotic supplementation of chab is possible in order to optimise growth and inhibit contaminants [ , , ] . for cultures made from blood, the use of the bactec tm (bd) system or equivalent, bact/alert tm (biomérieux) is recommended [ , ] . liquid media is not suitable for f. tularensis growth, even when supplemented with cysteine [ , , ] . basic biochemical tests provide a presumptive identification of isolates and may be further complemented by immunological and molecular methods. some additional biochemical tests, such as the ability to ferment glucose or glycerol, or the presence of the citrulline ureidase pathway are useful for subtyping purposes [ ] . the commercial microlog microstation tm system (biolog inc., hayward, ca) based on the ability to ferment glucose has been successfully used for differentiating between subspecies tularensis and holarctica [ , ] . also, the commercially available microbial identification system (mis) and library generation system (lgs) (midi, inc, newark, nj) enables cell-wall fatty-acid analysis and can be used for the identification of francisella at the genus level. it has also enabled the identification of atypical f. tularensis strains lacking cysteine requirements [ , ] . immune based techniques have also been employed for identification: immunoblot analysis and immunofluorescence microscopy, either from grown cultures or clinical samples [ ] . antibodies against f. tularensis reach detectable levels - days post-infection [ ] . a fourfold increase in the titre between acute and convalescent sera or a titre of : or greater of agglutinating antibodies is considered for diagnostic purposes [ , , , ] . titres peak at a level of - and decline slowly [ ] . serologic methods include the whole-cell agglutination test (widal's reaction), the tube agglutination test, microagglutination assays, haemagglutination, elisa (enzyme-linked immunosorbent assay) and immunoblot [ , ] . elisa has repeatedly been more sensitive than agglutination assays, with the additional advantage of determining separately different antibody classes (igm, igg and iga) [ ] . a combination of a first elisa screening test complemented by an immunoblot confirmatory test, with higher specificity, is the current recommended two-step approach for the serological diagnosis of tularaemia [ ] . the same approach can be used for animals. serology has a limited use in highly susceptible species since death usually precedes the development of specific antibodies [ ] . however, in endemic areas, antibodies for f. tularensis are frequently detected in wild animals that have developed immunity, including foxes and coyotes. this seroconversion is suspected as being related to subspecies holarctica infection since infection by the subspecies tularensis is expected to be fatal [ , ] . molecular methods are valuable diagnostic tools whenever culture is either not possible or is negative [ , , ] . moreover, they reduce the high risk of laboratory-acquired infections over conventional biochemical typing [ , , ] . during recent years, polymerase chain reaction (pcr)based methods have been successfully used for the rapid identification and classification of francisella isolates, with increased sensitivity and specificity [ , ] . however, false positive results related to non-pathogenic closely related francisella subspecies, occurring naturally in the environment, may hamper species and subspecies identification [ ] . conventional pcr targets are tul and fopa genes, which encode for f. tularensis superficial membrane lipoproteins. both protocols show a good level of sensitivity and reasonable specificity in f. tularensis detection and may be used in blood, tissue or aerosol samples [ , , ] . pcr product specificity is confirmed by sequencing, reverse-line blotting (rlb) or restriction fragment-length polymorphism (rflp) [ ] . real time pcr for f. tularensis detection has been developed, in particular, taqman tm (applied biosystems) real time pcr multiple assay shows high specificity and sensitivity using four target genes: isftu , kda, tul and fopa [ , ] . real-time pcr for the differentiation between the subspecies tularensis and holarctica is also now available [ ] . further discrimination has been achieved using highresolution genotyping methods including pulse-field gel electrophoresis (pfge), amplified fragment-length polymorphism (aflp), ribotyping, s rdna gene sequencing, canonic insertion deletions and paired-end sequence mapping [ , , , ] . still, as f. tularensis exhibits highly conserved genomic sequences among strains of diverse origin, genetic polymorphisms allowing for individual strain typing have been difficult to find [ ] . as for other bacteria, more recent pcr-based techniques such as variable-number tandem repeats (vntr), multiple-locus vntr analysis (mlva) and short-tandem repeats (str) typing have been successfully used for identification at the subspecies level and for molecular epidemiology purposes [ , , ] . one of the most discriminatory methods for the molecular subtyping of f. tularensis is mlva, which consists of a series of vntr loci that are pcr amplified via flanking primer sites and examined for size variation [ ] . one mlva system designed for f. tularensis is based on polymorphisms of vntr loci, ft-m to ft-m . this mlva typing system has a greater discriminatory power when applied to a worldwide set of f. tularensis isolates and provides accurate classification at the subspecies level [ ] . this mlva system has recently been improved by redesigning the subset of the previously identified vntrs to produce a new optimised, multiplexed mlva system with a similar level of discrimination but with fewer time and cost requirements [ ] . ten of the previously described vntr loci were selected based on their discrimination ability within the subspecies: ft-m , ft-m , ft-m , ft-m , ft-m , ft-m , ft-m , ft-m , ft-m and ft-m . locus ft-m was split into two loci, ft-m a (which contains the originally described bp repeat and is polymorphic across subspecies) and ft-m b (which contains the insertion with its bp repeat and varies only among type a.ii and f. novicida isolates) [ ] . while providing discrimination among strains, vntrs are unsuited for determining deeper phylogenetic relationships due to mutational saturation. in this case, more accurate and alternative markers should be used, such as whole-genome sequence single nucleotide polymorphism (snps) [ ] . additional studies have shown a remarkable degree of discrimination of the f. tularensis phylogenetic structure, using a combined analysis with canonical whole-genome snps for major clade typing, and mlva for high-resolution typing [ , ] . in a different study, the combined analysis of insertion-deletion markers, for subspecies and major clade typing, along with mlva, was used [ ] . microarrays have also allowed for the differentiation of the four f. tularensis subspecies and have been proven useful for pathogenicity and virulence marker identification [ ] . tularaemia usually responds to antibiotic therapy. historically, aminoglycosides have been the drugs of choice for humans. although clinically effective, they are rarely used now due their ototoxicity and nephrotoxicity. nevertheless, gentamicin has been used for treatment of pneumonic tularaemia and aminoglycosides are now generally used in the most serious cases. chloramphenicol is effective but seldom the first choice due to its possible irreversible effects on haematopoiesis. tetracyclines have been associated with high relapse rates on withdrawal. fluoroquinolones, such as ciprofloxacin, have been shown to be highly effective in per os and are the best choice for uncomplicated tularaemia. also, ciprofloxacin has proved suitable and effective in the treatment of tularaemia in children and pregnant women [ , ] . in domestic animals, gentamicin, enrofloxacin, doxycycline and chloramphenicol are referred to as therapeutic options for dogs [ , ] . in cats, there are reports of the use of doxycycline or enrofloxacin and amoxicilin-clavulanic acid as being beneficial in the early stages of the disease [ ] . currently, there is no available licensed vaccine against f. tularensis although an attenuated type b strain, known as the live vaccine strain (lvs) was developed in the united states during the s and used to vaccinate military personnel and laboratory workers [ , , [ ] [ ] [ ] . lvs failed to uniformly protect against pneumonic tularaemia and when delivered in high titres caused mild tularaemia as an undesirable side-effect [ ] . one focus of current research work in the usa and in europe is to develop a vaccine for protection against f. tularensis intentional release [ ] . the restricted efficacy of the lvs has fostered extensive research with a view to providing alternative vaccine formulations, including the exploration of different live and killed attenuated strains and immunogenic components to produce subunit vaccines [ , ] . in view of its immunogenic antigens, an effort has been made to develop attenuated strains of schus , a representative strain of type a tularaemia, for vaccine production. in fact, between lvs and schus strains there are about genes that encode for different protein sequences, whose functions are not well defined, and may represent important immunogenic antigens. still, given the increased virulence of the schus strain, only a small number of bacteria should be required to generate effective protection against wild type f. tularensis [ ] . a recently published study demonstrated that inoculation with low doses of specific attenuated mutants of the f. tularensis strain schus provided protection against parenteral and intranasal challenge with a fully virulent wild type schus strain [ ] . this favours the role of t-cell memory response as a critical determinant of f. tularensis immunity, additionally to the humoral response. this feature is the basis of the challenges foreseen for vaccine development, aiming at identifying antigen determinants that elicit an effective cellular-mediated immune response [ , , , ] . cell-mediated immunity was found to persist three decades after tularaemia vaccination. a recent study sought to identify the t-cell responses present in immune individuals in order to characterise f. tularensis-specific immune response [ , ] . the findings showed that the production of inf-␥, macrophage inflammatory protein (mip)- ␤ and cd a (lysosomeassociated membrane protein or lamp- ) by peripheral blood mononuclear cells appeared to be a characteristic of protective immune responses and that a correlation exists between these parameters and immunity [ ] . several factors such as human demographics and behaviour, international travel and commerce, including the animal trade, climactic changes and microorganism adaptation, have a potential impact on disease ecology and the emergence of zoonosis. the same factors are thought to be related to the emergence of tularaemia. special concerns regarding this bacterium exist in relation to its high infectivity, and easy dispersion through aerosols and contaminated water, which make it a potential bioterrorism weapon. also, tularaemia presents a wide geographic distribution and has recently emerged in new settings, particularly in europe. in portugal, an on-going research project on tularaemia aims to increase our knowledge about the disease, particularly its impact in this country, which is still poorly understood, in view of the fact that there is little information available to risk population and health professionals, with the result that there is a possible underestimation of prevalence in man and animals. to this regard, efforts have been made by the national institute of health to increase awareness of the disease among risk populations, particularly hunters and health professionals. in accordance with the preliminary results, on-going research will further identify and characterise f. tularensis circulating strains and develop molecular and typing methods with increased sensitivity, specificity and discriminatory power. the role of autochthon wild lagomorphs in the f. tularensis life cycle, their involvement in animal-to-human transmission and their suitability as tularaemia sentinels will be accessed. moreover, considering the economic and social relevance of hunting-related activities in this country, with very few studies having acknowledged its relation to zoonotic disease transmission risks, research into infection in game species is of major importance. f. tularensis is also associated with a considerably wider range of hosts and vectors than most zoonotic pathogens, although there is little information on bacterium mechanisms for adaptation to such a wide diversity of arthropod vectors. despite our increasing knowledge of tularaemia and its etiological agent, many aspects of f. tularensis biology and epidemiology need to be further examined, particularly its pathogenicity and virulence, vaccine development, and the specific mechanisms by which f. tularensis evades, modulates and suppresses the host immune response. as with any zoonotic emergent disease, the role of wild and domestic animals in f. tularensis epidemiology needs to be further evaluated, in particular, those which may act as reservoirs. other epidemiologic data such as the population dynamics of susceptible animals, particularly lagomorphs and rodents in europe, should be part of surveillance programmes, as they are thought to be directly associated with disease transmission patterns. from a public health perspective, disease surveillance in animals is crucial in order to prevent and monitor human outbreaks, particularly in endemic areas, where contact between humans and wildlife reservoirs or vectors is likely. although tularaemia is not regarded as a common disease, and there is little awareness of the disease among health authorities and practitioners, its eventual future impact as an emergent zoonosis should not be neglected. the authors declare that they have no conflict of interest. emerging zoonosis and vector-borne infections affecting humans in europe douglas, and bennet's principles and practice of infectious diseases francisella tularensis: unraveling the secrets of an intracellular pathogen topley and wilson's microbiology and microbial infections francisella tularensis: an arthropod-borne pathogen tularemia: emergence/re-emergence special topic on francisella tularensis and tularemia francisella tularensis reporting on surveillance data and epidemic intelligence data. stockholm: ecdc a water-borne epidemic of tularemia in chlumcany surveillance of tularemia in kosovo investigating an airborne tularemia outbreak large tularaemia outbreak in värmland, central sweden an outbreak of holarticatype tularemia in pediatric patients tularemia outbreak in castilla y león, spain, : an update tularemia in konya region outbreak of tularemia in brown hares (lepus europaeus) in france outbreak of tularemia in central norway tularemia in denmark: identification of a francisella tularensis subsp., holarctica strain by real time pcr and high-resolution typing by multiple-locus variable number tandem repeat analysis phylogeography of francisella tularensis: global expansion of a highly fit clone description of francisella hispaniensis sp.nov., isolated from human blood, reclassification of francisella novicida francisella novicida bacteremia after a neardrowning accident francisella tularensis subsp. novicida isolated 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hyalomma spp and rhipicephalus spp. (acari: ixodidae) from bulgaria association of different genetic types of francisella-like organisms with the rocky mountain wood tick (dermacentor andersoni) and the american dog tick (dermacentor variabilis) in localities near their northern distributional limits phylogenetic analysis of the francisella-like endosymbionts of dermacentor ticks detection of a novel francisella in dermacentor reticulatus: a need for careful evaluation of pcr-based identification of francisella tularensis in eurasian ticks transovarial transmission of francisellalike endosymbionts and anaplasma phagocytophilum variants in dermacentor albipictus (acari: ixodidae) tularemia of brown european brown hare (lepus europaeus): a pathological, histopatological and immunohistochemical study biochemical responses and oxidative stress in francisella tularensis infection: a european brown hare model tularaemia seroprevalence of captured and wild animals in germany: the fox (vulpes vulpes) as a biological indicator world health organization working group on tularaemia. who guidelines on tularaemia transstadial transmission of francisella tularensis holarctica in mosquitos waterborne outbreak of tularemia associated with crayfish fishing biology of francieslla tularensis subspecies holarctica live vaccine strain in the tick vector dermacentor variabilis screening of mosquitoes as vectors of f. tularensis in portugal diagnostic procedures in tularemia with special focus on molecular and immunological techniques naturally occurring tularemia in a dog tularemia in a dog host-adaptation of francisella tularensis alters the bacteruim' surface-carbohydrates to hinder effectors of innate and adaptive immunity survival and growth of francisella tularensis in acanthamoeba castellanii the subversion of the immune system by francisella tularensis type i interferon signaling is required for activation of the inflammasome during francisella infection francisella subverts innate immune signaling: focus on pi k/akt the francisella intracellular life cycle: toward molecular mechanisms of intracellular survival and proliferation innate immune recognition of francisella tularensis: activation of type-i interferons and the inflammasome tlr signaling contributes to rapid inflammasome activation during f. novicida infection outbreak of tularemia in brown hares (lepus europaeus) in france pneumonic tularemia in rabbits resembles the human disease as illustrated by radiographic and hematological changes after infection laboratory analysis of tularemia in wild-trapped, commercially traded prairie dogs first reported prairie dog-to-human transmission european commission bans import of prairie dogs from the us and rodents and squirrels from sub-saharan africa the compendium on continuing education for the practicing veterinarian tularemia in two cats acute tularemia in three domestic cats an outbreak of airborne tularemia in france early recognition of atypical francisella tularensis strains lacking a cysteine requirement worldwide genetic relationships among francisella tularensis isolates determined by multiple-locus variable-number tandem repeat analysis increased knowledge of francisella genus diversity highlights the benefits of optimized dna-based assays an optimized, multiplexed multi-locus variable-number tandem repeat analysis system for genotyping francisella tularensis canonical insertion-deletion markers for rapid dna typing of francisella tularensis localized cutaneous infection with francisella tularensis resembling ulceroglandular tularemia in a cat clusters versus affinitybased approaches in f. tularensis whole genome search of ctl epitopes the francisella tularensis proteome and its recognition by antibodies signatures of t cells as correlates of immunity to francisella tularensis low dose vaccination with attenuated francisella tularensis strain schus mutants protects against tularemia independent of the route of vaccination persistence of cell mediated immunity three decades after vaccination with the live vaccine strain of francisella tularensis tularemia in czechoslovakia and austria during and ecological conditions of natural foci of tularemia in the czech republic changes of the epidemiology and the clinical picture of tularemia in southern moravia (the czech republic) during the period - tularemia -history, epidemiology, clinical aspects, diagnosis and therapy analysis of the incidence of tularemia in the breclav district - re-emergence of tularemia in germany: presence of francisella tularensis in different rodent species in endemic areas detection of francisella tularensis subsp landscape epidemiology of tularemia outbreaks in sweden endemic tularemia the tularemia outbreak: a case-control study of risk factors in disease-endemic and emergent areas insect-borne diseases and insect bites in finland outbreaks of tularemia in a boreal forest region depends on mosquito prevalence outbreaks of tularemia in castilla tularemia epidemic in northwestern spain: clinical description and therapeutic response characteristics of the turkish isolates of francisella tularensis two cases of glandular tularemia from turkey tularemia: are hunters really a risk group tularemia a re-emerging disease in northwest turkey: epidemiological investigation and evaluation of treatment responses comparison of the and outbreaks of tularemia in the duzce region of turkey outbreaks of tularemia in turkey human tularemia in france isolation of francisella tularensis by centrifugation of shell vial cell culture from an inoculation eschar evidence of circulation of an epidemic strain of francisella tularensis in france by multispacer typing tularaemia outbreak in northern norway five case histories of tularaemia infection in oppland and hedmark counties francisella-arthropod vector interaction and its role in patho-adaptation to infect mammals we acknowledge the work carried out by ana sofia santos, fátima amaro, hugo osório, maria joão alves and rita de sousa from the doctor ricardo jorge national health institute, i.p., centre for vectors and infectious diseases research key: cord- -rbn foj authors: hohdatsu, t.; sasamoto, t.; okada, s.; koyama, h. title: antigenic analysis of feline coronaviruses with monoclonal antibodies (mabs): preparation of mabs which discriminate between fipv strain - and fecv strain - date: - - journal: veterinary microbiology doi: . / - ( ) -x sha: doc_id: cord_uid: rbn foj abstract we prepared monoclonal antibodies (mabs) against either fipv strain - or fecv strain - , and tested them for reactivity with various coronaviruses by indirect flourescent antibody assay (ifa). sixteen mabs which reacted with all of the strains of feline coronaviruses, also reacted with canine coronavirus (ccv) and transmissible gastroenteritis virus (tgev). in many of them, the polypeptide specifity was the recognition of transmembrane (e ) protein of the virus. we succeeded in obtaining mabs which did not react with eight strains fipv type i viruses (showing cell-associated growth) but reacted with fipv type ii ( - , ku- ) and/or fecv type ii ( - ) viruses (showing non-cell associated growth). these mabs also reacted with ccv or tgev. these mabs recognized peplomer (e ) glycoprotein, and many antigenic differences were found in this e protein. these results suggest that fipv type ii and fecv type ii viruses are antigenically closer to tgev or ccv than to fipv type i viruses. furthermore, the mab prepared in this study has enabled discrimination between fipv strain - and fecv strain - , which was thought to be impossible by the previous serological method. for serological diagnosis of feline infectious peritonitis virus (fipv) infection, detection of antibody by indirect fluorescent antibody assay (ifa) is popular (pedersen, b; horzinek and osterhaus, ; scott, ) . on the other hand, feline enteric coronavirus (fecv) which antigenically crossreacts with fipv, and causes only mild enteritis without inducing fip, may be present (mckeirnan et al., ; pedersen et al., a; pedersen et al., b; pedersen et al., ) . thus, the serological diagnosis of fip and the mechanisms of its onset become more complex. pedersen et al. ( a) classified the feline coronaviruses in terms of the disease types. they divided fipv into types i and ii according to the presence or absence of the induction of fip, ability of the viruses to proliferate in cell cultures, and the antigenic relationship with porcine and canine coronaviruses. fecv has been divided into types i and ii in the same way. types i and ii of fipv in this classification can be serologically discriminated by the neutralization test. fipv type i and fecv type ii can also be distinguished by the neutralization test. cultivation of fecv type i in cells is not possible at present, and its serological position remains unclear. on the other hand, even the neutralization test cannot discriminate between fipv type ii and fecv type ii. it goes without saying that, since all feline coronaviruses show cross-reaction in ifa, it is impossible to discern the types of virus infection. there are many healthy but fipv antibody-positive cats living outdoors. as long as fipv type ii and fecv type ii cannot be distinguished serologically, the clinical diagnostic significance of antibody detection in such cats is low. in this study, we attempted to distinguish between the - strain classified as fipv type ii and the - strain classified as fecv type ii, by means of monoclonal antibodies (mabs). at the same time, we examined feline coronaviruses for antigenic differences by using the mabs. we also investigated the antigenic relationship between feline coronaviruses and canine and porcine coronaviruses. cell cultures. feline whole fetus cells (fcwf- ), crandell feline kidney cells (crfk) and swine kidney cells (cpk) were grown in eagle's minimum essential medium (mem) containing % leibovitz l- medium (l- ) % fetal calf serum, units ml-~ penicillin and #g ml-~ streptomycin. the maintenance medium was mem containing % l- and antibiotics as above. the cells were maintained in a humidified % co incubator at ° c. viruses. the coronavirus isolates used in this study and their sources are shown in table . among these virus strains, the authors isolated the strain ku- of fipv from the liver cells of a kitten with the effusive form of fip, and the strain ku- of fipv from the peritoneal cells of an adult cat, also with effusive fip. among the fipv strains used in the study, strains ucd- , nw- , ucd- , ucd- , ucd- , black, yayoi and ku- show cell-associated growth, and are therefore regarded as type i virus strains in the classification of pedersen et al. ( a) . moreover, since strain ku- , like strain - , pedersen et al., b mckeirnan et al., pedersen et al., a pedersen, a pedersen et al., a pedersen and black, pedersen and floyd, pedersen and floyd, pedersen and floyd, black, pedersen and black, hayashi et al., mckeirnan et al., pedersen et al., b furuuchi et al., harada el al., binn et al., grows well even in crfk cells in a non-cell associated manner, it is considered to be a type ii virus strain. fipv and fecv, tgev, and ccv were passaged two or three times in fcwf- cells, cpk cells, and crfk cells, respectively, and were used for the study. preparation of virus antigen. the antigen was prepared with the fipv - strain or fecv - strain grown in fcwf- cell cultures. infectious culture fluid concentrated about tenfold by ammonium sulfate precipitation was layered onto a discontinuous sucrose density gradient ( and %) in an rps rotor (hitachi koki co., ltd., japan) and centrifuged at r.p.m, for h. the virus bands formed were collected, diluted in nte buffer, ( . m nac , . m tris-hc , ph . , . m edta) and centrifuged at g for h. the virus-containing pellet was suspended in a / volume of nte buffer. production of antibody-secreting hybrydomas. balb/c mice, about weeks of age, were inoculated intraperitoneally with a mixture of /~g of the viral antigen prepared as above and cells of pertussis adjuvant. four or six weeks later the mice received an intravenous booster dose of /~g of viral antigen, and spleen cells were obtained for fusing d later. the fusion was carried out by essentially the same method described by k/shler and milstein ( ) . polyethyleneglycol- (merck, germany) was used as a fusing agent and the ratio of mouse spleen cells and mouse myeloma cells (p- /x- -ag o , , ) was : . the selective medium contained hypoxanthine ( - m), aminopterin ( × l - m) and thymidine ( . × - m). the fused cells at a concentration of . × spleen cells per ml was dispensed in /a volumes into wells of -well, flat-bottomed microplates (corning glass works, corning, ny ) and incubated at ° c in a humid atmosphere containing % co . after incubation for weeks, the wells were examined and those which contained hybridoma cultures were tested for feline coronavirus specific antibody by an indirect immunofluorescence test (see below). the colonies in antibody positive wells were passaged in -well multiplates (coming glass works, corning, ny) and incubated in medium containing hypoxanthine ( - m) and thymidine ( . × - m). the cells were then cloned by the soft agar method. body-secreting hybridoma cultures were concentrated tenfold by % saturation with ammonium sulfate and used for determination of antibody class and subclass by double diffusion in % agar gel containing . % nan . rabbit antisera against mouse immunoglobulins, igg , igg a, igg b, igg , igm and iga, and x and chains (miles laboratories, u.s.a. ) were placed in center wells and test samples were added to adjacent wells. the plates were incubated overnight at room temperature in a humidified chamber. indirect fluorescent antibody assay. hybridoma culture supernatant fluid was added to acetone-fixed infected monolayers, incubated for min at °c, washed times with phosphate buffered saline solution (pbs) and then stained with rabbit anti-mouse-igg,a,m serum conjugated with fluorescein isothiocyanate (fitc) (miles lab., u.s.a. ). after a further min incubation at °c, slides were washed in pbs. stained monolayers were mounted in buffered glycerol and examined using a fluorescence microscope. neutralization (nt) test. serial twofold dilution of the mabs were mixed with an equal volume of a virus suspension diluted so as to contain approximately tcidso/ . ml. the mixtures were incubated at °c for min. each mixture was then inoculated into cell cultures in flat-bottomed microplates, and incubated in an atmosphere of % co in air at °c for d. two wells were employed for each antibody dilution. the antibody titer was expressed as the reciprocal of the highest dilution of mab that completely inhibited cytopathic effect in the test. western immunoblotting. viral antigen separated in polyacrylamide gel by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page) were transferred to nitrocellulose sheets of . /zm pore size. the transfer was carried out electrophoretically by the method adapted from towbin et al. ( ) in a transfer-blot cell apparatus at ma and v for h using transfer buffer consisting of g -~ tris (ph . ), % methanol and . g - glycine. the nitrocellulose sheets were then cut into strips and incubated at °c for h in pbs containing % fetal-calf serum. the supernatant fluid of antibody-secreting hybridoma cultures was added in ml volumes to individual strips and incubated at °c for h. the strips were then washed times with pbs containing . % tween- , and incubated at °c for h with horseradish peroxidase-conjugated rabbit antibody against mouse igg,a,m (miles lab., u.s.a. ) diluted : with pbs containing % fetal calf serum. the strips were then washed and treated with substrate solution containing . g diaminobenzidine, # of % h in ml of . m tris-hc , ph . . when distinct bands appeared about min later, the reaction was stopped by pouring offthe substrate solution and rinsing with distilled water. determination of polypeptide specificity by enzyme-linked immunosorbent assay (elisa). the polypeptide specificity of the mabs which could not be determined by western immunoblotting was determined according to the method of . briefly, the virus antigen described above was first disrupted with % nonidet p- (np- ). this material was placed on a - % linear sucrose density gradient containing . % np- , and centrifuged at g for h. after fractionation, each fraction was diluted with nte buffer, and allowed to be absorbed by -well, flat-bottomed microelisa plates. the mabs against n, e and e proteins with polypeptide specificity clarified by western immunoblotting were delivered into the wells of each fraction, and subjected to elisa. elisa was performed according to the method of hohdatsu et al. ( ) . among the mabs which recognize each protein, fractions which reacted strongly with a single type of mab were collected. elisa was performed with these fractions used as antigens, and the polypeptide specificity of the mabs was determined. - was used as the immunogen, mabs (f - , f - , f - , f - , f - , f - , f - , f - , f - , f - , f - , f - , f - , f - , f - , f - , f - , f - , f - , f - , f - , f - , f - , f - , f - ) were obtained. in addition, with fecv strain - as the immunogen, mabs (el - , e - , e - , e - , e - , e- - ) were obtained. the polypeptide specificity, ig isotypes, and nt activity to fipv strain - of these mabs are shown in table . for most mabs, western immunoblotting could deter- mine their polypeptide specificity. however, this method failed to determine the specificity of mabs, f - , f - , f - and f - . elisa using np- disrupted, sucrose gradient-purified viral polypeptide revealed that these mabs recognise e protein. moreover, western immunoblotting and elisa using viral polypeptide yielded the same results with respect to the other mabs. figure shows examples of the western immunoblotting reaction of mabs which recognize n, e and e proteins. as shown in table , two (f - , el - ) of the mabs had nt activity to fipv strain - . we examined the mabs for reactivity with feline coronaviruses by ifa. as shown in table by means of indirect if test, with fitc-conjugated rabbit anti-mouse immunoglobulin, on each virus-infected cvfk or cpk cell culture grown on coverslips. the monoclonal antibodies were used undiluted cell culture fluids. the minus sign indicates negative reactivity. the polypeptide specificity of each of the mabs was determined by its reactivity to each of the three major structural components of the fipv virion either by immunoblotting of sds-page or by elisa. did react with the only strain of fecv. all mabs in group iv, v and vi were found to recognize e protein. we examined the mabs for reactivity with porcine and canine coronaviruses by ifa. table shows the results. all mabs in group i, which reacted with all feline coronaviruses, also reacted with ccv and tgev. mabs in groups ii and iii, which did not react with ucd- , ucd- , ucd- and black strains, did not react with ccv and tgev either. however, out of four mabs in group iv, which reacted only with the - and ku- strains of fipv and strain - of fecv, three reacted with either ccv or tgev. moreover, among the mabs in group v, which reacted only with the - and ku- strains of fipv, two reacted with the sh strain of tgev, and type reacted with the - strain of ccv. furthermore, mabs in group vi, which reacted with fecv alone, reacted with the - strain of ccv. thirty-one mabs were prepared by using strain - , classified as fipv type ii, and strain - , classified as fecv type ii, as immunogens. table shows their reactivity with strains of feline coronavirus. all mabs in group i reacted with feline coronaviruses. besides feline coronaviruses, these mabs reacted with the - strain of ccv, and the sh and to- strains of tgev. these results confirm the previous reports (pedersen et al., ; horzinek et al., ; pedersen et al., a) that these virus strains are antigenically close to each other. concerning polypeptide specificity, many mabs in this group i recognize e protein. among these viruses, many common epitopes seem to exist, particularly in the e protein. among eight virus strains with the characteristics of fipv type i, reactivities of e - and e - in group ii, and f - in group iii with the mabs were different, and all of these mabs recognized n protein. four mabs in group iv (f - , f - , f - and f - ) reacted with the - and ku- strains of fipv type ii and strain - strain of fecv type ii, but not with fipv type i viruses. of these mabs, however, f - reacted with ccv, and f - and f - reacted with ccv and tgev. similarly, among the mabs in group v, which react with fipv type ii viruses alone, f - reacted with ccv, and f - and f - reacted with the sh strain of tgev. furthermore, all mabs in group vi, which react with the - strain of fecv alone reacted with ccv. as pedersen et al. ( a) have reported from their study with polyclonal antibody, these results suggest that fipv type ii viruses and fecv type ii viruses are antigenically closer to tgev or ccv than to fipv type i viruses. at present, the authors are preparing mabs with neutralizing activity using fipv strain - as immunogen, and are determining the serological re-lationships among these viruses by the presence or absence of the neutralization epitope. moreover, since all mabs in groups iv, v and vi recognize e protein, it was assumed that there are many antigenic differences in e protein among these viruses. by using mabs, also found antigenic differences among feline coronaviruses, especially in the e protein. conventional serological methods have failed to discriminate between strain - of fipv type ii and strain - of fecv type ii. however, mabs in group v reacted with fipv type ii viruses alone, while mabs in group vi reacted with the - strain of fecv type ii alone. these mabs have enabled the discrimination of these viruses, by clearly indicating antigenic differences among them. the proportions of types i and ii of fipv and types i and ii of fecv actually present in the natural environment are not clear. as stated in the introduction, the neutralizing test can appraise infection with type i and ii of fipv at the serological level. however, in the case of infection with viruses other than fipv type i, distinction of infection particularly by fipv type ii an fecv type ii at the serological level is difficult. in the future, it will be of use to be able to distinguish infection of these viruses by competitive enzyme immunoassay (fiscus et al., ; using type-specific mabs. the mabs in groups v and vi which the authors have prepared in this study are expected to be useful as such type-specific mabs. recovery and characterization of a coronavirus from military dogs with diarrhoea recovery and in-vitro cultivation ofa coronavirus from laboratory-induced cases of feline infectious peritonitis (fi p ) antigenic comparison feline coronavirus isolates: evidence for markedly different peplomer glycoproteins competitive enzyme immunoassays for the rapid detection of antibodies to feline infectious peritonitis virus polypeptides epitope-specific antibody responses to virulent and avirulent feline infectious peritonitis virus isolates comparison between virulent and attenuated strains of transmissible gastroenteritis virus studies on transmissible gastroenteritis in pigs. iii. isolation of cytopathogenic virus and its use for serological investigation serodiagnosis for feline infectious peritonitis by immunofluorescence using infected suckling mouse brain sections antigenic variation of porcine transmissible gastroenteritis virus detected by monoclonal antibodies feline infectious peritonitis: a worldwide serosurvey antigenic relationships among homologous structural polypeptides of porcine, feline and canine coronaviruses continuous cultures of fused cells secreting antibody of predefined specificity isolation of feline coronaviruses from two cats with diverse disease manifestations morphologic and physical characteristics of feline infectious peritonitis virus and its growth in autochthonous peritoneal cell cultures serologic studies of naturally occurring feline infectious peritonitis attempted immunization of cats against feline infectious peritonitis, using avirulent live virus or sublethal amounts of virulent virus experimental studies with three new strains of feline infectious peritonitis virus: fipv-ucd , fipv-ucd and fipv-ucd antigenic relationship of feline infectious peritonitis virus to coronaviruses of other species infection studies in kittens utilizing feline infectious peritonitis virus propagated in cell culture lb. an enteric coronavirus infection of cats and its relationship to feline infectious peritonitis pathogenic differences between various feline coronavirus isolates pathogenicity studies of feline coronavirus isolates - and - fip antibody test-interpretation and recommendations electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications this work has been funded by the kitasato research foundation under grant no. . key: cord- -gs c fy authors: schreiber, gideon title: the role of type i interferons in the pathogenesis and treatment of covid- date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: gs c fy type i interferons (ifn-i) were first discovered over years ago in a classical experiment by isaacs and lindenman, who showed that ifn-is possess antiviral activity. later, it became one of the first approved protein drugs using heterologous protein expression systems, which allowed its large-scale production. it has been approved, and widely used in a pleiotropy of diseases, including multiple-sclerosis, hepatitis b and c, and some forms of cancer. preliminary clinical data has supported its effectiveness against potential pandemic pathogens such as ebola and sars. still, more efficient and specific drugs have taken its place in treating such diseases. the covid- global pandemic has again lifted the status of ifn-is to become one of the more promising drug candidates, with initial clinical trials showing promising results in reducing the severity and duration of the disease. although sars-cov- inhibits the production of ifnβ and thus obstructs the innate immune response to this virus, it is sensitive to the antiviral activity of externally administrated ifn-is. in this review i discuss the diverse modes of biological actions of ifn-is and how these are related to biophysical parameters of ifn-i–receptor interaction and cell-type specificity in light of the large variety of binding affinities of the different ifn-i subtypes towards the common interferon receptor. furthermore, i discuss how these may guide the optimized use ifn-is in combatting covid- . type i interferons (ifn-i) are a family of cytokines that bind the type i interferon receptor, constituted of two transmembrane subunits, ifnar and ifnar ( figure ). the two receptors are constituted of an extracellular domain, which binds ifn-i, a transmembrane helix and an unstructured intracellular domain (icd) that binds jaks and stats ( , ) . jak is associated with ifnar and tyk with ifnar . stat and stat (and maybe also other stats) were found to be constitutively bound to the icd of ifnar ( ) ( ) ( ) . binding results in close proximity of the intracellularly associated jaks, jak and tyk , resulting in their activation through cross phosphorylation ( figure ) ( , ) . this also results in receptor phosphorylation, which role is still under debate ( , ( ) ( ) ( ) . the phosphorylated stats dissociate from the receptor and form homo and hetero dimers, which are transported to the nucleus, where they serve as transcription factors for a large number of genes. the most prominent effects are associated with stat /stat heterodimerization, which together with irf form the interferon-stimulated gene factor (isgf ), which bind a distinct group of target genes harboring the interferonstimulated response elements (isre). in addition to this, ifn-i drives stat /stat and stat /stat homodimerization, the formation of a stat /irf binary complex and more ( , ( ) ( ) ( ) (figure ). this leads to the transcription activation or suppression of over , genes, which drive a wide range of innate and adaptive immune functions. these, in turn respond against various pathogens, act as important regulators in tumor immunity and have a role in pathophysiology and autoimmune diseases ( , ( ) ( ) ( ) ( ) ( ) ( ) . stat knockout cells still activate a stat /stat response mediated by irf , while stat knockout cells activate a stat /irf -induced response ( ) . surprisingly, no change in the gene induction relative to wildtype cells was observed in stat knockout hela cells, despite the strong ifn-i-induced phosphorylation of stat . however, as ifn-i responses are cell-type specific, a stat /stat induced response may still be found in other cells than hela. due to this wide range of physiological responses, ifn-i has provided therapeutic benefits for multiple diseases, including multiple sclerosis, some cancers and viral diseases (hepatitis b and c) ( ) ( ) ( ) . due to the efficient activation of antiviral activities by ifn-is, most viruses have contemplated mechanisms to avoid its actions ( ) ( ) ( ) . for example, the ebola virus, which outbreak in central africa killed tens of thousands of people ( , ) , avoids ifn-i activity by producing the vp protein that binds the karyopherin alpha nuclear transporter. thereby, it inhibits the nuclear transport of phosphorylated stat , rendering cells refractory to ifn-is. another example of viral mechanisms that evolved to eliminate ifn-i functions in inducing innate immunity is given by the sars corona virus, where both the production of ifnb and the ifn-i induced signaling are attenuated. recently, a more infective version of sars has emerged, sars-cov- (which causes the covid- disease). covid- cases have been first reported by the end of in china, and rapidly became a world-wide epidemic with unprecedented consequences ( , ) . sars-cov- seems to have originated from horseshoe bats. similar virus strains that circulate in bats in hubei province in china may in the future cause further new zoonotic outbreaks ( ) . sars-cov- has % homology to the sars-cov virus that also spread from china in ( ) . sars-cov- proved to be much more infectious compared to the original sars virus, resulting in a global epidemic. as ifn-i drives strong antiviral activities, the mechanisms sars-cov and sars-cov- combat ifn-i activities has been a matter of intense research, with at least proteins being identified to counteract ifn-i functions in the sars-cov virus ( ) . in addition, ifn-is were implicated in contributing to the severity of the cytokine storm, which is a major complication of sars-cov and sars-cov- and can lead to respiratory distress syndrome (ards) and death ( , ) . in this review i will describe our current knowledge on the involvement of ifn-is in the development of the covid- disease, and how this relates to the different activities associated with type i interferons. type i interferon receptors are found on all cell types, and are a major component of the innate immune system. human type i interferons include similar ifnas with % homology between them and single ifnw, k, ϵ and b, with lower homology ( - %). all of them bind the receptor complex, composed of ifnar and ifnar at the same proximal location ( , , ) . despite structural similarities among the ternary ifn-i-ifnar -ifnar complexes, ifn-is drive a range of different activities, dependent on the cell type and the interferon subtype ( ) . this apparent paradox has major implications for understanding the role of ifn-i in health and disease and its varied applications as a drug against a pleiotropy of diseases. ifn-i signaling is initiated by binding of ifn-i to its receptor. it has been suggested that cytokine receptors are pre-associated, with ligand binding activating signaling through the induction of conformational changes ( ) . however, more recent singlemolecule receptor tracking on life cells has clearly shown that for many of the cytokines, its role is to bring the receptors into close proximity, which drives signaling ( ) . this seems to be the case also for ifn-i induction, as shown both using single receptor tracking and mutational analysis ( figure ) ( , ) . while structurally, the ternary ligand-receptor complex seems to be the same for all ifn-is, the binding affinity differs by many orders of magnitude. the tightest binding ifn-i is ifnb, which binds ifnar with nm affinity and ifnar with subnanomolar affinity. the different ifna subtypes bind ifnar with . to µm affinity and ifnar with to nm affinity, with ifna being the weakest binding ifna ( , ) . even weaker binding was measured for ifnϵ, with~ -fold reduced affinity relative to ifna proteins ( ) . interestingly, ifnϵ is constitutively expressed by the reproductive tract epithelium and is regulated by hormones during the estrus cycle, reproduction, menopause and by exogenous hormones. thus, its mode of action is different from other ifn-is ( ) . these large differences in binding affinity between ifn-i subtypes were suggested to result in major differences in biological activity. to obtain a better insight into the molecular mechanisms of their actions, ifna was engineered to cover the whole range of binding affinities of natural ifn-is to both the high affinity (ifnar ) and low affinity (ifnar ) receptor chains ( ) . these studies have shown that indeed, the binding affinity to both receptors is a major determinant of ifn-i activity ( ) . using both natural and engineered ifn-is has shown that even weak binding ifn-is activate the cellular antiviral program at very low (pm) concentrations ( ) . moreover, the antiviral program was activated in all cell-lines tested. despite the -fold higher affinity of ifnb over ifna towards binding ifnar receptors, its potency to elicit an antiviral response is similar. for example, in wish cells (originally thought to be of amniotic origin, but later found to be a hela (cervix cancer) contaminant) the ec for antiviral activity of ifna is . pm, while the ec for ifnb is . pm ( ) . wish cells have been extensively used to characterize ifn-i activity, including for definition of ifn-i unit activity. an upper limit for antiviral potency was further verified by engineering an ifna variant, yns-a -tail, with fold tighter binding to ifnar and -fold tighter binding to ifnar in comparison to ifna (thereby surpassing the receptor binding affinity of natural ifnb). still, the ec for antiviral activity is only -fold lower in comparison to ifna ( , ) . conversely to antiviral activity, ifnb is much more potent in activating the antiproliferative program relative to ifna , a result that was also verified using the ifna variant, yns-a tail ( ) . the ec for antiproliferative activity on wish cells is nm for ifna , pm for ifnb and pm for yns-a -tail. a similar increase in antiproliferative potency was observed also for ovcar and hela cells. interestingly, while antiviral activity was observed in all cell lines tested, some cell lines were not susceptible to ifn-i induced antiproliferative activity (for example t d and k ), independent on the concentration and subtype of ifn-i ( ) . to better understand the molecular basis for this finding, ifn-i induced gene expression was monitored using various ifn-i subtypes or engineered mutants on the background of different cell-lines. these experiments showed that low concentrations of weaker binding interferons activate the expression of mostly antiviral genes. higher concentrations of interferons activate also other genes, many of them related to immune-modulation ( ) . examples for such genes are chemokines such as cxcl and , which are involved in chemotaxis of t cells and natural killer cells, induction of apoptosis, regulation of cell growth and more. we gave the term of "robust" for the common ifn-i induced program (including its antiviral activity) and "tunable" for the other programs induced by ifn-is, which include between others antiproliferative and immunomodulatory activities ( ) . further investigations into these two programs has shown that cells with low receptor numbers activate only the robust program, and that not all cell types execute the tunable program, conversely to the robust program that is common to all cells ( ) . tighter binding ifn-is at higher concentrations are essential for the activation of the tunable program. genes upregulated by the robust program are mostly classical antiviral genes, such as mx and mx , oas and , pkr, ifit , and , isg , and many more. figure a according to string and go analysis, the commonly upregulated genes have a strong antiviral signature. the top go terms (fdr < − ) are response to type i interferon, innate immune response, response to virus, defense response and immune system process. it is interesting to note that antiviral genes constitute most of the upregulated genes common to all cell lines. antiviral genes are also the majority of upregulated genes in k and t d cells. conversely, ovcar and hela cells have many unique upregulated genes, many of them related to immunomodulatory functions, cell cycle, apoptosys and more. ifn-i. the diagram shows that genes are commonly upregulated by all cell-lines. figure b shows string protein interaction analysis of these common genes. clearly, these form a tightly interacting mesh of gene products. gene ontology analysis shows these genes to have an extremely high signature for antiviral activity and ifn-i activation. promoter analysis of common isgs has shown them to be driven by the classical isre promoter sequence ( ) . conversely, for tunable genes no clear promoter sequence was identified. the exact mechanism of how tunable genes are upregulated by ifn-i is thus not yet fully understood. from an immunological point of view, ifn-is have three major functions: . to activate an antiviral state in infected and neighboring cells that limits spread of infection. . modulate innate immune responses, including antigen presentation and natural killer cell functions while restraining pro-inflammatory pathways. . activating the adaptive immune system for the development of high-affinity antigen-specific t and b cell responses ( ) . as ifn-is are highly active molecules, their expression and signaling potency is highly regulated. opposing augmenting and suppressive signals are induced by host factors. suppressive pathways include ifn-i activation of usp , an isg that suppresses signal transduction by reducing the ability of ifn-is to form an active receptor complex ( , ) . a second inhibitory mechanism is the induction of socs and socs , which kir domain block the substrate binding groove on jak, thereby inhibiting stat phosphorylation ( ) . a third mechanism is by rapid endocytosis and subsequent lysosomal degradation of activated ifnar complexes ( , ) resulting in reduced receptor numbers ( figure ). it has been demonstrated that a mutant in ifnar (s a and s a in human and mouse respectively), which fails in ifnar endocytosis through blocking its ubiquitination result in high incidence of inflammation ( , ) . at the transcriptional level, ifn-i response can also be regulated by mir- , which is highly induced by pattern recognition receptors and inflammatory signaling, and suppresses the expression of over genes. between them genes related to the interferon pathway. it was shown that mir- -deficient cd (+) t cells had enhanced type i interferon signaling and were more susceptible to interferon's antiproliferative effect ( ) . high basal ifn-i levels are implicated in various immunological diseases, such as systemic lupus erythematosus and more ( , , ) . however, ifn-i has also anti-inflammatory effects, as best demonstrated by their ability to suppress multiple-sclerosis ( ) . it is important to note that beneficial results in treating multiplesclerosis were observed only for ifnb but not for ifna treatment ( ) . to see whether this relates to the higher receptor binding affinity of ifnb, we established a transgenic mouse harboring the human interferon-receptors extracellular domains fussed to the mouse intracellular domains and compared the severity of eae in a mice model upon treatment with ifna , ifnb and the high-affinity engineered ifn-yns-a -tail. we found that the ifn-yns-a -tail had the strongest suppressive effect on the development of eae ( ) . the effect was further enhanced by pasylation of ifn-yns-a -tail, which extends it plasma half-life by -fold. interestingly, we found a tight relation between the increased levels of expression of pd-l in mice and the severity of the disease. these data show that tight binding ifn-is induce preferential anti-inflammatory responses, at least in this ms mouse model. another example for the immunosuppressive activity of ifn-i was shown for lcmv infection, which induces consistent ifn-i production including the immunosuppressive factors il- and pd-l ( ) . in addition to the above, interferons contribute to inflammasome activation through several different mechanisms, including caspase- expression and the ifn-i inducible gbp protein expression, which was reported to have an important role in caspase- activation and pyroptotic cell death ( ) . ifn-is have important roles in protecting the lung from spread of respiratory viruses. in addition to their direct role, ifn-is have also been found to be critical in initiating lung inflammatory responses, by inducing recruitment and activation of immune responses, which have to be kept under control. ifn-is have been shown to result in the production of chemokines such as ccl and cxcl , which play important roles in the recruitment of monocytes/macrophages, t cells, nk cells, and dcs, therefore directly influencing inflammation in the lung ( ) . this varied effect of type i ifns on t cells is partly dependent on the different stats induced by type i ifns. in the absence of ifn-is, the detection of accumulating viral rna and downstream processing of the signal is compromised, leading to viral spread and also to reduced inflammation in the lung. interestingly, there is an age-related reduction of ifn-i production and isg induction after viral infection, which may be related to the higher susceptibility of elderly population to lung infections ( ). viruses have developed many strategies to interfere with the synthesis of ifn-is or the ifn-i induced responses. one of them, is the stimulation of turnover of the interferon receptors. among other viruses implicated in accelerating the turnover of ifnar are ebv, herpes simplex virus, hepatitis c and b viruses, vesicular stomatitis virus and the sars coronavirus ( , ) . sars-cov has been shown to suppress ifn-i responses in the host through multiple mechanisms. a subdued ifn-i response diminishes antigen presentation and reduces the antiviral adaptive th- immune response. ifn-is communicate between cells against pathogens and have a critical role in the immune system, such as activating natural killer (nk) cells and macrophages. in addition, ifn-is cause flu-like symptoms, which are observed in various diseases. these symptoms may have a role in alerting a person of his/her sickness, in order to limit disease-spread to other individuals. in sars-cov and mers-cov, the induction of ifnb is suppressed altogether. this dampening approach is highly associated with the disease severity and increased mortality ( ) . in the lethal cases of sars-cov or mers-cov infections, the increased influx of inflammatory cells is always observed. in a mouse model of sars-cov infection, imbalance in ifn-i and inflammatory cells were shown as the main cause of fatal pneumonia ( ) . in addition to these, sars-cov implements strategies to evade the immune response by antagonizing ifn-i induced signaling pathways. the orf protein blocks the expression of stat activated genes ( ) . sars-cov and mers-cov encode papainlike protease (plp) that is able to impede the immune response function ( ) . in addition, sars-cov interacts with isg and antagonizes the ifn-i-mediated antiviral response ( ) . the mers-cov orf b antagonizes the antiviral ifnb production by inhibiting irf and irf ( ) . also sars-cov inhibits activation of irf / , slowing ifnb production upon infection ( ) . while irf is expressed in many different cell types, plasmacytoid dendritic cells are the only cells constitutively expressing irf ( ) . ifn-i treatment has been studied against mers-cov and sars-cov in numerous experiments, both in vitro and in vivo, and in combination or not with lopinavir/ritonavir, ribavirin, remdesivir, corticosteroids, or ifng. while ifna and b were efficient in vitro and in certain animal models, their success in humans was less convincing [for review see, ( , ) ]. it should be noted that reduction in ards mortality (not related to sars) was also found to be at best marginal upon treatment with ifn-i ( ) . still, one has to consider that mice studies have shown the timing of ifn-i administration to be critical, with positive effects being observed if ifn-i was administered shortly after infection. conversely, ifn-i failed to inhibit viral replication and resulted in unwanted side-effects when administered later in the disease circle ( , ) . these include elevated lung cytokine/chemokine levels, vascular leakage, and impaired virus-specific t cell responses. it is interesting to note that a knockout of the ifn-i receptor in mice resulted in its protection from lethal sars-cov infection. these findings have major implications on how to treat humans against sars and mers, and could have affected the outcome of the clinical studies. the covid- pandemic started in december in wuhan, china. by the summer of , thirty million cases were reported worldwide, with over , fatalities. as covid- is closely related to the sars-cov virus, the interest in the effect of interferons on its disease progression, and its potential as a drug was immediate. disease progression of covid- goes through a number of stages. the initial stage, which last from to days (usually - days) from infection is asymptomatic. a certain proportion of patients never produce any symptoms (the percentage of those is under debate, but a range of - % is most likely). of those who develop symptoms, they are mostly mild ( % of those who develop symptoms). from the remaining %, about half will develop severe symptoms, which require hospitalization in intensive care units. the mortality rate, from those developing symptoms is % to %. the numbers given above are average, and change dramatically with age. at young age most of the infected people will be asymptomatic, while over the age of about % will have symptoms. moreover, as the age progresses, symptom severity increases ( ) . the major complication of severe infection is pneumonia, which can develop into acute respiratory distress syndrome (ards). in addition, covid- has been linked to cardiovascular sequelae, such as myocardial injury, arrhythmias, cardiomyopathy and heart failure, acute kidney injury, neurological complications, and acute ischemic stroke ( ) . developing severe symptoms and death is strongly related to background conditions. the strongest relation is to age, with the risk to people under being very small, while the risk peaks for people over the age of . in addition, chronic kidney disease, chronic obstructive pulmonary disease, immunocompromised state, obesity, heart conditions and type diabetes are linked to higher incidents of sever disease ( ) . cov- is presumed to infect people mostly though inhalation of viral particles, which can be airborne, in droplets or otherwise through infection through touching infected surfaces. the spike protein on the cov- surface binds to the human ace protein, which serves as its receptor ( figure ) . the homotrimeric spike glycoprotein is made from s and s subunits. its binding and subsequent cleavage by the host protease tmprss results in the fusion between cell and viral membranes and cell entry ( ) . blocking the ace receptors by specific antibodies voids viral entry ( ) ( ) ( ) . interestingly, cov- receptor-binding domain (rbd) exhibited significantly higher binding affinity to ace than the sars-cov rbd, which was speculated to relate to the higher infectivity of covid- in relation to sars. after membrane fusion, the virus enters through the endosomal pathway and the viral rna is released into the host cell. the viral rna is then translated into viral polyproteins, which are cleaved into small products by viral proteases (papain-like protease [plpro] and the main protease [mpro]). viral proteins and genome rna are subsequently assembled into virions in the er and golgi and then transported and released out of the cell. the exact mechanism of viral self-assembly is still under intense investigation ( , ) . investigating ace and the viral entry-associated protease tmprss expression levels in lung tissue and trachea has shown that tmprss is expressed in both tissues, while ace is predominantly expressed in a transient secretory cell type ( ) . in addition, ace and tmprss co-expressing cells were found within lung type ii alveolar cells (which also release pulmonary surfactant), enterocytes, and nasal goblet secretory cells ( ) . using single-cell rna-sequencing, ace and tmprss were found to be highly expressed also in the nasal goblet and ciliated cells ( ) . the inhaled virus likely binds to epithelial cells in the nasal cavity and starts replicating. the virus propagates and migrates down the respiratory tract along the conducting airways, and a more robust innate immune response is triggered. for about % of the infected patients, the disease will be mild and mostly restricted to the upper and conducting airways. unfortunately, about % of the infected patients will progress to more severe disease and will develop pulmonary infiltrates and some of them will develop ards ( ). like many other viruses, also sars-cov and sars-cov- have evolved mechanisms to reduce their exposure to ifn-i. in both viruses, mechanisms to block the production of ifnb were identified. while the antiviral potency of ifn-is on sars-cov is moderate, sars-cov- seems to be highly sensitive to ifn-i. this is evident by the significant reduction in viral replication observed following ifn-i treatment at both and h postinfection ( ) . in sars-cov- -infected cells, ifn-i results in elevated stat levels and isg production (in contrast to sars-cov infected cells). this raises the question of why the innate immune system fails to combat sars-cov- ? the apparent answer to this is in the inhibition of ifnb production by proteins of the sars-cov- virus. within cells, rna viruses are sensed by the innate immune system through three major classes of pattern recognition receptors (prrs): toll-like receptors (i.e. tlr- , - , - ), rig-i-like receptors (rlrs), and nod-like receptors (nlrs) ( ) . to identify the molecular mechanisms that block ifnb production through activation of irf / , several research groups transfected cells individually with all the cov- viral genes and with either rig i, mda , or mavs ( , ) . among the cov- proteins transfected to cells, they identified nsp and orf as competent suppressors of ifnb. yuen et al. also identified nsp and , while lei et al. identified nsp , nsp and the m protein as potent inhibitors of the mavs pathway, leading to inhibition of ifnb production ( figure ) . orf was between the strongest suppressors of ifnb production in both studies. orf was also the only sars-cov- gene suppressing the activity of an interferon-stimulated response element (isre) promoter in both studies. lei et al. also identified nsp and nsp as potent inhibitors of the induction of an isre promotor. in another study, li et al. showed that the viral orf , orf , and nucleocapsid proteins were strong inhibitors of ifnb production, and through this of the ifn-i innate immune response ( ) . in this study, orf and orf also inhibited induction of transcription an isre promotor driving a luciferase as reporter, following ifnb treatment. in addition to the above-mentioned sars-cov- genes, orf b was implicated by konno et al. as being a potent antagonist towards ifn-i production ( ) . an interesting civet in this study is the finding that a natural variant, with a longer orf b reading frame increased disease severity in two patients. in light of the much higher than expected coding capacity of the sars-cov- genome, where many more proteins than genes were identified ( ), we may find even more proteins and peptides being involved in eliminating the innate immune response, including through inhibition of ifn-i activities. another mechanism by which sars-cov- inhibit antiviral functions of the cell is thought the activity of the papain-like protease (plpro), which is essential for viral polyprotein processing. this gene was found to preferentially cleave the ubiquitin-like modifier interferon-stimulated gene (isg ), figure | sars-cov- has multiple effects on the immune system, including inhibition of ifnb production, which results in isgs not to be produced, cd + and cd + exhaustion and increased levels of pro-inflammatory proteins (tnfa, il , nf-kb). currently, the most promising drugs against covid- include ifn-is, antiinflammatory and antiviral drugs, protease inhibitors, antibodies, sars-cov -ace (receptor) binding inhibitors and more. which is an ifn-i induced gene with strong antiviral activity ( ) . this represents another layer of attenuation of ifn-i responses by sars-cov- and is similar to the mechanism previously identified for sars-cov ( ) . inhibition of ifnb production by cov- got further confirmation from measuring the levels of different cytokines in sars-cov- -infected patients. an integrated immune analysis, including immune cell analysis, whole-blood transcriptomics and cytokine quantification on covid- patients at to days after disease onset has shown an impaired ifn-i response that is a result of low ifn-i levels ( ) . this, in turn results in the low production of interferon-stimulated genes. conversely, high levels of il and tnfa were measured ( figure ) ( , ) . this is in contrast to what is seen in patients infected with highly pathogenic influenza viruses. the high production of pro-inflammatory cytokines and low production of ifn-is during sars-cov- infection suggests effective activation of nf-kb but not irf and irf ( ) . impaired ifn-i production during severe covid- may also lead to an imbalance in the pro-inflammatory versus pro-repair functions of airway macrophages. this was indeed seen in severely ill patients with covid- . other innate immune cells such as natural killer (nk) cells are also regulated by ifn-is during coronavirus infection. severe covid- is associated with exhaustion of cd + and cd + t cells ( ) , which may be a result of deficient ifn-i production, as ifn-is promote survival of t cells. an important issue to consider is that early production of ifn-is promote efficient t cell responses, while a delayed response may inhibit t cell proliferation or their exit from lymphoid organs and thus cause their functional exhaustion. indeed, t reg cell counts in covid- patients inversely correlate with disease severity ( , ) . interestingly, transcriptomic analysis of blood, lung, and airways of cov- -infected patients showed that while ifnb was indeed not highly expressed in either, a number of ifnas were highly upregulated in the lung and airways but not in blood ( ) . moreover, a clear ifn-i-induced gene expression profile was also detected for lung and airways, but not for blood (pbmcs). a similar finding of elevated ifna but not ifnb, during covid- infection was also found by wei et al. ( ) . in this study, the elevated ifn-i response was restricted to the stage in the disease were patients were in intensive care. in another study of patients, of whom did not produce ifn-i, those patients had higher viral load, required more aggressive medical intervention and their time of stay in the intensive care unit was longer that ifn-i producing patients ( ) . pdcs are the most rapid and abundant ifn-i producers. pdcs express tlr and tlr which are important in sensing viruses. the response of pdcs to viruses, particularly ifn-i production, is significantly impaired with ageing while secretion of all other pro-inflammatory cytokines was comparable to that of younger individuals ( ) . this may relate to the master regulator for ifn-i production, irf , which expression, phosphorylation and nuclear translocation decreases with age. in addition, local neutrophil-mediated inflammation is increased with age, while cytotoxicity of nk cells induced by type i ifn-is decreases in aged mice ( ) . in addition to age, other factors were also associated with reduced interferon responses. one of them is obesity, which is related to impaired ifna and ifnb responses, which may relate to inadequate response of obese people against viral infections ( ) . clinical trials of using ifn-i for treating corona viruses has a long history. already in , intranasal human ifna was given both before and after corona virus challenge, a strain that is causing common cold. the incidence of colds, the severity of symptoms and signs, and virus replication were all reduced in subjects receiving interferon as compared with those given placebo ( ) . for sars-cov, no randomized placebo-controlled trials have been performed to test the efficacy of ifn-is, however, comparing the clinical outcome of patients treated with ifn-a (infacon- ) with patients at different locations (not a control group) that were not treated, has suggested clinical benefits ( ) . these studies have raised the hope that ifn-i may be a potent drug also against covid- . this hope was further exuberated by the observation that externally administrated ifn-i induced a strong antiviral response, much more than that observed for sars-cov ( ) . while some of the sars-cov- proteins may affect isg production (most notably, orf and , see above), the main defense of sars-cov- against ifn-i innate immunity seems to be the prevention of ifnb production, which can be substituted by external administration. a major problem in assessing the efficiency of ifn-i against covid- is the lack of a good small animal model. while such models are now under development, they are still not perfect. in a recent study, mice were infected with a replication-deficient adenovirus containing human ace , and then infected with sars-cov- . these mice developed pneumonia, severe pulmonary pathology, and high-titer virus replication in lungs. to test the role of ifn-i in disease development, ifnar ko mice were infected with sars-cov- , showing higher viral titer over time. next, the mice were treated prior to infection with poly i:c, a strong inducer of ifn-i. this resulted in significantly diminished clinical disease and induced more rapid virus clearance ( ) . these results suggest that at least in a mice model, ifn-i may benefit disease recovery. due to the lack of a good animal model, and the availability of clinically approved ifn-i therapies, multiple clinical studies have been conducted administrating different subtypes of ifn-is using different routes of administration (for summary see table ). in a preventive study, nasal drops of ifna were given to , healthy medical staff in shiyan city hospital, hubei province for days to prevent sars-cov- infections. none of them developed serious side effects or was infected with cov- . while the study lacked a control group from the same city, overall in hubei province , medical staff were diagnosed with covid- ( ) . the study thus gives an indication that ifn-i may help in preventing infection for high risk medical personal. to test the benefit of subcutaneous injection of ifnb on early stage patients, an open clinical trial was conducted with patients, were assigned to the combination of lopinavir, ritonavir, ribavirin, and three doses of million international units of ifnb, while the control group of patients were given all the above except ifnb. the median number of days from symptom onset to start of study treatment was days. patients given also ifnb had a significantly shorter median time from the start of treatment to negative nasopharyngeal swab ( - days) in comparison to the control group ( - days) . moreover, ifnb reduced viral load and number of significantly ill patients relative to the control group, this without significant side-effects ( ) . in a medical study on the effects of treatment with ifna b in a cohort of confirmed covid- patients, some of the participants were given nebulized ifna b with or without arbidol while others were given only arbidol. treatment with ifna b with or without arbidol reduced the duration of detectable virus in the upper respiratory tract and reduced duration of elevated blood levels of il and c-reactive protein, which are inflammatory markers ( ) . while the study did not include a standard care group, and all patients recovered, it still provides an indication of ifn-i efficiency. the efficiency of ifnb a subcutaneously injected three times weekly for weeks for treatment of severe covid- was tested in a randomized clinical trial. all the patients (including the control group) received standard of care, including a range of other medicines (hydroxychloroquine, antibiotics, antiviral medicine and more). while the clinical response was not significantly different between the ifnb and the control groups, the -day overall mortality was significantly lower ( % vs. %) in the ifnb treated group ( ) . in a retrospective study of patients receiving ifna through inhalation, alone or in combination with other drugs at a relative early versus late stage of the infection, it was found that those receiving ifna at an early stage had a significantly lower rate of mortality. in contrast, late interferon therapy increased mortality and delayed recovery ( ) . the study suggests a relation between the time of ifn-i treatment and its efficiency. synairgen, a uk-based company, performed a controlled clinical trial of inhaled ifnb on patients and reported that compared with placebo the odds of developing severe disease during the treatment period decreased by % for hospitalized patients receiving sng , and that patients who received sng were more than twice as likely to recover from the virus during the treatment period versus those randomized to placebo. these are between the best results achieved so far in curing covid- . more clinical trials are now under way to evaluate ifn-i efficiency, but clearly the initial trials have been encouraging. moreover, due to the many years of experience in treating patients with ifn-is, the availability of the drug and its relatively modest cost make it an excellent candidate for mass treatment, once approved. however, critical questions remain concerning the use of ifn-is for covid- and other diseases ( figure ) . these questions relate to the optimal ifn-i subtype, drug-concentration, duration of treatment, mode of treatment and at which frequency should it be given. ample experience exists with subcutaneously administration, which is almost the only route ifn-is were used in the clinic. here, non-modified ifn-is are usually administrated two to three times weekly, while pegylated ifn-is are administrated once per week or less. injection of ifn-is will result in a systemic response, where ifn-is were shown to have antiviral functions as well as pro and anti-inflammatory functions. contrary, if given by inhalation, it will directly target the epithelial, and thus replace the ifnb, which production is inhibited by the virus. administration as nasal drops of ifna may be an excellent prophylactic method for people at high risk. ideally, these questions could be answered using animal models. the problem is that the disease in those is not equivalent to that observed in humans. due to the severity of the disease and the high proven safety of ifn-is, more clinical trials on humans, testing the many open questions related to its best mode of administration may be the fastest way forwards. the subtype to use is another important question. for multiple-sclerosis, ifnb has been used for many years ( ) , as it seems to provide a better anti-inflammatory response than ifnas. this may relate to its higher binding affinity to the interferon receptors, as has been demonstrated using a tight binding ifna mutant (yns-a tail), which binding affinity even surpasses that of ifnb [see above ( ) ]. for combating viral disease, most notable hepatitis c, ifna has been most commonly used ( ) , which was later replaced by pegylated (long plasma half-life) ifna ( ) . also, for cancers ifnas were mostly used ( ) . a good clinical explanation of why specific ifn-i subtypes were used is often missing, and decisions of which interferon to use may often relate to availability rather than to efficacy. moreover, due to the specie specificity of ifn-is, one cannot deduce from mouse experiments, which ifn-i to use in humans, as the data are not transferable ( , ) . the main difference between ifnas and ifnb is that the later has a stronger potency to induce antiproliferative and immunomodulatory responses (tunable), while ifna will provide a cleaner antiviral response (robust) without the additional responses associated with ifnb. the open question is which is desired for covid- treatment, where complications arise from the exuberated immune response. another, important parameter is the time of intervention by ifn-i, in early or late-stage covid- disease. in a recent study in mice it has been shown that prolonged 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recovery from viral infection the author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © schreiber. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -wwjeu ie authors: walz, lucas; cohen, avi j.; rebaza, andre p.; vanchieri, james; slade, martin d.; dela cruz, charles s.; sharma, lokesh title: janus kinase-inhibitor and type i interferon ability to produce favorable clinical outcomes in covid- patients: a systematic review and meta-analysis date: - - journal: medrxiv doi: . / . . . sha: doc_id: cord_uid: wwjeu ie background: novel coronavirus (sars-cov- ) has infected over million. novel therapies are urgently needed. janus-kinase (jak) inhibitors and type i interferons have emerged as potential antiviral candidates for covid- patients for their proven efficacy against diseases with excessive cytokine release and by their ability to promote viral clearance in past coronaviruses, respectively. we conducted a systemic review and meta-analysis to evaluate role of these therapies in covid- patients. methods: medline and medrxiv were searched until july (th), , including studies that compared treatment outcomes of humans treated with jak-inhibitor or type i interferon against controls. inclusion necessitated data with clear risk estimates or those that permitted back-calculation. results: we searched studies, ultimately including four randomized and eleven non-randomized clinical trials. jak-inhibitor recipients had significantly reduced odds of mortality (or, . ; %ci, . – . , p= . ) and icu admission (or, . ; %ci, . – . , p= . ), and had significantly increased odds of hospital discharge (or, . ; %ci, . – . , p< . ), when compared to standard treatment group. type i interferon recipients had significantly reduced odds of mortality (or, . ; %ci, . – . , p= . ), and increased odds of discharge bordering significance (or, . ; %ci, . – . , p= . ). conclusions: jak-inhibitor treatment is significantly associated with positive clinical outcomes regarding mortality, icu admission, and discharge. type i interferon treatment is associated with positive clinical outcomes regarding mortality and discharge. while these data show promise, additional randomized clinical trials are needed to further elucidate the efficacy of jak-inhibitors and type i interferons and clinical outcomes in covid- . the spread of a highly pathogenic, novel coronavirus (sars-cov- ) has emerged as the deadliest pandemic since influenza in and has proved to be the ultimate challenge for public health organizations, health care providers, and governments at all levels. [ ] severe disease caused by sars-cov- (covid- ) has strained intensive care unit (icu) and personal protective equipment (ppe) resources around the world, [ ] leading to icu mortality rates as high as % in some population subsets. [ ] as of july th , sars-cov- has infected over million worldwide and led to the death of over , . [ ] currently, only few medications have been suggested to improve the disease outcome . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted august , . . https://doi.org/ . / . . . doi: medrxiv preprint and limit the lethal disease in susceptible populations. a small number of large-scale randomized clinical trials have been conducted so far, having demonstrated modest effectiveness for agents such remdesivir or dexamethasone. [ , ] additional therapeutics against covid- are being explored, but there remains a lack of large scale rcts of many potentially useful therapies, possibly missing some important therapeutics that can alter outcomes in covid- patients. janus-kinases (jaks) are transmembrane proteins that serve to mediate and amplify extracellular signals from growth factors and cytokines. their inhibitors have been found to be effective in treating patients with inflammatory diseases. [ ] these inhibiting drugs function by targeting specific janus kinases. both baricitinib and ruxolitinib predominantly inhibit jak and jak . [ ] jak-inhibitors may be used to control high levels of cytokines and inflammation, [ ] similar to secondary hemophagocytic lymphohistiocytosis (shlh) caused by cytokine storm, seen in patients with severe sars-cov- infection. [ ] these inhibitors have proved helpful in "off-label" indications, where excessive cytokine release plays a central role in the disease progression. [ ] while the hypothesis of jak-inhibitors successfully combating high levels of cytokine expression in sars-cov- infection has been shown in some small studies, [ ] their effect on a larger population has not been investigated. type i interferons-α/β are proteins secreted by infected cells meant to induce antiviral states in neighboring cells and stimulate cytokine production. [ ] type i and type iii interferons have potent antiviral effects. these interferons work through activation of jak/stat pathway to activate a multitude of genes that are collectively known as interferon stimulated genes (isgs). these isgs act together to block the viral life cycle at different levels. given the widespread expression of type i interferon receptors, they function as broad spectrum antivirals that can directly and indirectly inhibit the replication of rna viruses at various moments in a viral life cycle through several mechanisms. [ ] these interferons have been found to have positive therapeutic effects in the treatment of viral hepatitis, [ ] and even past coronaviruses, such as the previous sars and mers outbreaks. [ , ] additionally, a recent investigation revealed that several severe cases of covid- presented with a rare, x-chromosome loss-. cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted august , . . https://doi.org/ . / . . . doi: medrxiv preprint of-function mutation that impaired type i interferon response, [ ] while another demonstrated an association between covid- severity and type i interferon deficiency. [ ] various studies have found reasons to support the use of type i interferons in combination with other antivirals to promote positive outcomes among patients with covid- , but many are restricted by the number of patients they treated with interferon. [ ] interestingly, these interferons perform their functions by activating jak pathway. uncertainty and a lack of clinically proven prophylactic and therapeutic options have precipitated the periodic update of treatment guidelines for patients infected with covid- . as such, systematic reviews evaluating effects in larger patient populations are necessary to ascertain drug-related covid- outcomes. in this meta-analysis, we evaluate janus kinase-inhibitors and type i interferons for their efficacy and ability to produce positive outcomes in patients infected with sars-cov- . reviews and meta-analyses (prisma). [ ] search strategy and study quality assessment medline (via pubmed) and medrxiv were searched since inception throughout july th , by three investigators (lw, ac, jv). the following terms were searched in free-text fields for jak-inhibitors. for medline: "covid- " and "jak inhibitor" or "ruxolitinib" or "tofacitinib" or "fedratinib" or "baricitinib". for medrxiv: "covid- jak inhibitor" or "covid- ruxolitinib" or "covid- tofacitinib" or "covid- fedratinib" or "covid- baricitinib". the following terms were searched in free-text fields for type i interferons. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted august , . . https://doi.org/ . / . . . doi: medrxiv preprint three investigators (lw, ac, jv) independently screened titles and abstracts generated by the search. after selection, full electronic articles were then carefully evaluated for data extraction. randomized studies included in the final analyses were scored by one investigator (lw) to formally assess for risk of bias utilizing the risk of bias (rob) tool (supplementary table ). [ ] non-randomized studies included in the final analyses were scored by one investigator (lw), utilizing the newcastle-ottawa scale (nos) according to the following study characteristics: ( ) representativeness of exposed cohort, ( ) selection of nonexposed cohort, ( ) exposure assessment, ( ) outcome of interest not present at the start of the study, ( ) comparability of cohorts, ( ) outcome assessment, ( ) adequacy of length of time before followup, and ( ) adequacy of follow-up of cohorts (supplementary table we included clinical trials that utilized combination or sole jak-inhibitor or type i interferon (ifn-α, ifn-β) for the treatment of confirmed covid- infection. for inclusion, possible studies must have compared treatment outcomes of those treated with a jak-inhibitor or type i interferon against a defined control group that did not receive this treatment. selection required data with clearly indicated risk ratios or odds ratios (or), or those that permitted their back-calculation. inclusion necessitated that the trial be a human study accessible in english, and could include pediatric or adult studies, observational studies, retrospective cohorts, randomized clinical trials, and case reports. studies that utilized in vivo or animal studies, as well as those examining histological, pathological, and cellular mechanisms were excluded. duplicate studies, review articles, commentaries, and proposed protocol were also excluded. trials were excluded if they primarily examined other therapies where outcomes were unclear as to which participants received jak-inhibitors or type i interferons. finally, studies were not included if they presented outcomes considered heterogenous across the review that made statistical synthesis impossible (e.g. mean vs median). . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted august , . . https://doi.org/ . / . . . doi: medrxiv preprint each full article that met inclusion criteria was carefully reviewed with the following baseline information extracted: first author, publication year, country, study type, type of jak-inhibitor or interferon used, number of total participants, number of participants receiving jak-inhibitor or interferon, and outcome measurements ( table ). the outcome measurements consolidated included mortality, disease severity (mild/moderate vs severe/critical), mechanical ventilation, intensive care unit (icu) admission, discharge, and acute respiratory distress syndrome (supplementary table reported. heterogeneity was assessed using tau-squared and chi-squared tests for random effects and fixed effect models, respectively, as well as the i statistic. for i > %, the random effects model was used. otherwise, the fixed effects model was utilized. an alpha of . was adopted to determine significance. the meta-analysis results are presented on forest plots, with a study's calculated or plotted as a black square whose size is proportional to the weight afforded to the study. bidirectional bars stemming from these black squares correspond to the risk estimate's % ci. diamonds were used to represent the summary or; its center aligns with the or and its width represents the summary % ci. publication bias was assessed using funnel plots (supplementary figure ; supplementary figure ). the initial database search returned articles. two additional articles were added by manually searching retrieved reviews. after removing two duplicates, articles were excluded following title and abstract screening by three investigators. after comprehensive evaluation of full text articles, only . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted august , . . https://doi.org/ . / . . . doi: medrxiv preprint studies complied with the inclusion criteria. the majority of the studied excluded in the final step were excluded on the basis of not presenting outcome data in terms of those who did and did not receive jakinhibitor or interferon treatment. the remainder of excluded studied were due to a focus on jak inhibition or interferon therapy as prophylaxis or heterogeneity in reporting of time among outcomes, precluding calculating pooled measures. of the included studies, six were pre-prints. overall, the studies were comprised of four observational studies, six retrospective cohorts, four rcts, and one prospective cohort. figure presents the meta-analysis flow chart and table presents the designs and characteristics of included studies. while some studies did not report which drugs were given to which patients as standard of care, many others reported treating patients with glucocorticoids, hydroxychloroquine, chloroquine, arbidol, and lopinavir/ritonavir. all studies were conducted within a hospital setting. a total of five studies investigated the effect of jak inhibition in a controlled setting (table ) figure . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted august , . . https://doi.org/ . / . . . doi: medrxiv preprint b). finally, analysis of studies of patients, of which were treated with a jak-inhibitor, revealed that those treated with jak-inhibitor had significantly higher odds than those treated with standard care to be discharged at weeks (or, . ; % ci, . - . ; p< . ; figure e ). the analysis examining the relationship between treatment with jak-inhibition and requiring mechanical ventilation, developing ards, icu admittance, and hospital discharge demonstrated very little heterogeneity (i = ). meta-analysis of sets of studies with , , and patients receiving type i interferon therapy revealed that there were no significant associations between receiving type i interferon therapy, compared to standard of care, and icu admittance, requiring mechanical ventilation, or developing a severe or critical case of covid- , respectively (p> . ; figure b ; figure c ; figure d ).[ - ] the analyses included , , and control patients, respectively. the data exhibited very high heterogeneity in cases of icu admittance and disease severity (both i > %), but relatively low in the case of mechanical ventilation (i = %). in the analyses of the and type i interferon receiving patients, intervention therapy was respectively associated with higher odds of being discharged (or, . ; % ci, . - . ; p= . ; n= ; figure e ), and significantly lower odds of mortality (or, . ; % ci, . - . ); p= . , n= ; figure a) , when compared to standard of care. the studies included in these analyses enlisted and control patients, respectively. discharge data exhibited very low heterogeneity (i = %), while mortality data demonstrated very high heterogeneity (i = %). as sars-cov- continues to infect millions and kill thousands daily, there is an urgent need to find novel therapies that can effectively limit covid- severity. type i interferon therapy as well jak inhibitors represent paradoxical approaches to treat covid- . while type i interferon therapy aims to limit the viral replication at the early time points to limit the subsequent disease, jak-inhibitors aim to limit the overt inflammation that may be detrimental to the host and cause systemic inflammatory response. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted august , . . https://doi.org/ . / . . . doi: medrxiv preprint however, no major randomized clinical trials have been performed to determine their efficacy in limiting the disease severity in covid- . to our knowledge, this is the first systematic review and meta-analysis to investigate the role of janus kinase-inhibitor or type i interferon on clinical outcomes in patients with covid- . the results suggest a robust association between jak-inhibitor and significantly decreased odds of mortality and icu admission, as well as significantly increased odds for -day patient discharge. furthermore, a significant association between type i interferon and reduced mortality was also found, in addition to an association with hospital discharge bordering significance. these results suggest the potential benefit of these therapeutic options for covid- . furthermore, as recent findings have shown that persistent viral presence contributes to disease severity, [ ] the timing of the administration of both interventions may be of utmost importance. as jak inhibitors attenuate jak signaling and subsequent cytokine release, their administration may best be suited for patients with progressing covid- who have not yet experienced a cytokine storm. [ ] by . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted august , . . https://doi.org/ . / . . . doi: medrxiv preprint contrast, as type i interferons induce cellular antiviral states via the jak/stat pathway, its administration may be most efficacious early on in disease progression where the virus is still replicating. while the literature surrounding this is sparse, one study included in this meta-analysis concluded that early administration of interferon-alpha- b could induce positive outcomes in covid- patients compared to standard treatment, while its late administration was associated with slower recovery. [ ] it is important to highlight that this meta-analysis attempted to overcome the challenges posed by studies with insufficient power to detect an effect between jak-inhibitor or type i interferon treatment and clinical outcomes, as half of the included studies in this analysis utilized sample sizes less than .[ , - , , ] nevertheless, despite the broad range of sample sizes and populations, the screening step of our analysis predominantly resulted in low effect size heterogeneity as evidenced by the i statistics displayed in figure and figure . this study contained no restrictions regarding study type in the exclusion criteria and, as such, many of the studies included are of retrospective design. accordingly, baseline characteristics of patients cannot be ignored, especially as factors such as age, gender, and pre-existing comorbidities have been found in meta-analyses to be linked to negative clinical outcomes, including mortality, among covid- patients. [ ] one study in particular contained a large disparity in the distribution of chronic conditions across those who received type i interferon therapy and controls. [ ] in addition, these non-randomized studies are inherently limited in their ability to deduce the causality of association between the treatments of interest and clinical outcomes; they should be interpreted with caution. another limitation of this study is the aspect of drug combination. included studies varied in the drugs administered in the control arm and in addition to jak-inhibitor or type i interferon in the treatment group. types and doses of jakinhibitors and type i interferons also differed across studies. moreover, publication bias may have been present in some of the analyses conducted (supplemental figure ; supplemental figure ). the low number of studies make it difficult to assess asymmetry in funnel plot analyses. however, we attempted to mitigate this bias with the inclusion of six unpublished articles. a further limitation of this study is the . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted august , . . https://doi.org/ . / . . . doi: medrxiv preprint exclusion of a large number of studies that presented heterogenous data that precluded pooled analyses. lastly, this meta-analysis included two studies consisting of similar study teams that examined the same association, [ , ] enhancing the likelihood of bias in the same direction in analyses where both of these studies were included. in conclusion, this meta-analysis supports the value of jak-inhibitor and type i interferon therapy as antivirals in combating sars-cov- infection. this study consolidates existing data and reaffirms the conclusion that, within covid- patients, jak-inhibitor treatment is significantly associated with positive clinical outcomes in terms of mortality, icu admission, and discharge, as well as type i interferon treatment's association with positive clinical outcomes in regard to mortality and discharge. although these findings should assist physicians deciding which antivirals to administer to sars-cov- infected patients, they also point to a clear need of additional well-designed rcts examining the relationship of jak-inhibitor and type i interferon and clinical outcomes of covid- patients. lucas walz (lw); conception of investigation, planning of investigation, data retrieval, article screening, data analysis, written reporting, data interpretation. avi j. cohen (ac); planning of investigation, data retrieval, article screening, data analysis, written reporting, data interpretation. andre p. rebaza, md (ar); written reporting, data interpretation. james vanchieri (jv): data retrieval, article screening. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted august , . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted august , . . https://doi.org/ . / . . . doi: medrxiv preprint . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted august , . . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted august , . . https://doi.org/ . / . . . doi: medrxiv preprint jak-inhibitor treatment group saw significantly reduced odds of mortality and icu admission, as well as significantly higher odds of discharge, when compared to standard treatment. there was no significant difference between groups in regards to requiring mechanical ventilation, or the development of ards. type i interferon group saw significantly reduced odds of mortality, as well as increased odds of discharge that bordered significance, when compared to standard treatment. there was no significant . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted august , . . https://doi.org/ . / . . . doi: medrxiv preprint responding to covid- -a once-in-a-century pandemic? fair allocation of scarce medical resources in the time of covid- clinical characteristics of covid- in covid- dashboard by the remdesivir for the treatment of covid- -preliminary report dexamethasone in hospitalized patients with covid- -preliminary report therapeutic targeting of jaks: from hematology to rheumatology and from the first to the second generation of jak inhibitors side effects of ruxolitinib in patients with sars-cov- infection: two case reports combined il- and jak-stat inhibition therapy in covid- related shlh, potential game changer baricitinib, a drug with potential effect to prevent sars-cov- from entering target cells and control cytokine storm induced by covid- global virus outbreaks: interferons as st responders hepatitis c: therapeutic perspectives interferon alfacon- plus corticosteroids in severe acute respiratory syndromea preliminary study epub / / . eng. . van der made ci, simons a, schuurs-hoeijmakers j, et al. presence of genetic variants among young men with severe impaired type i interferon activity and inflammatory responses in severe covid- patients subcutaneous administration of interferon beta- a for covid- : a non-controlled prospective trial the prisma statement for reporting systematic reviews and meta-analyses of studies that evaluate healthcare interventions: explanation and elaboration rob : a revised tool for assessing risk of bias in randomised trials therapeutic effectiveness of interferon-alpha b against covid- : the cuban experience triple combination of interferon beta- b, lopinavirritonavir, and ribavirin in the treatment of patients admitted to hospital with covid- : an open-label, randomised, phase trial. the lancet prolonged sars-cov- viral shedding in patients with covid- was associated with delayed initiation of arbidol treatment: a retrospective cohort study epidemiological and clinical features of cases with coronavirus disease in areas adjacent to hubei, china: a double-center observational study medical treatment of patients with covid- from seven cities in northeast china who fully recovered: a single-center, retrospective, observational study retrospective multicenter cohort study shows early interferon therapy is associated with favorable clinical responses in covid- patients persistent viral presence determines the clinical course of the disease in covid- . the journal of allergy and clinical immunology in practice cytokine storm and leukocyte changes in mild versus severe sars-cov- infection: review of covid- patients in china and emerging pathogenesis and therapy concepts a comparison of mortality-related risk factors of covid- a systematic review and meta-analysis. the journal of infection interpretation; supervision.lokesh sharma, phd (ls): conception of investigation, planning of investigation, written reporting, data interpretation; supervision.. cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted august , . . https://doi.org/ . / . . . doi: medrxiv preprint pmid: pmc . eng.. cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review)the copyright holder for this preprint this version posted august , . pneumonia: a retrospective study. medrxiv. : . . . . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted august , . . https://doi.org/ . / . . . doi: medrxiv preprint difference between groups in regards to requiring icu admission, mechanical ventilation, or the development of severe or critical disease. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted august , . . https://doi.org/ . / . . . doi: medrxiv preprint . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted august , . . https://doi.org/ . / . . . doi: medrxiv preprint . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted august , . . https://doi.org/ . / . . . doi: medrxiv preprint . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review)the copyright holder for this preprint this version posted august , . . https://doi.org/ . / . . . doi: medrxiv preprint key: cord- -pzixgqcy authors: benetka, viviane; kübber-heiss, anna; kolodziejek, jolanta; nowotny, norbert; hofmann-parisot, margarete; möstl, karin title: prevalence of feline coronavirus types i and ii in cats with histopathologically verified feline infectious peritonitis date: - - journal: vet microbiol doi: . /j.vetmic. . . sha: doc_id: cord_uid: pzixgqcy feline coronaviruses (fcov) vary widely in virulence causing a spectrum of clinical manifestations reaching from subclinical course to fatal feline infectious peritonitis (fip). independent of virulence variations they are separated into two different types, type i, the original fcov, and type ii, which is closely related to canine coronavirus (ccv). the prevalence of fcov types in austrian cat populations without fip has been surveyed recently indicating that type i infections predominate. the distribution of fcov types in cats, which had succumbed to fip, however, was fairly unknown. pcr assays have been developed amplifying parts of the spike protein gene. type-specific primer pairs were designed, generating pcr products of different sizes. a total of organ pools of cats with histopathologically verified fip was tested. a clear differentiation was achieved in cats, % of them were type i positive, % type ii positive, and % were positive for both types. these findings demonstrate that in fip cases fcov type i predominates, too, nonetheless, in % of the cases fcov type ii was detected, suggesting its causative involvement in cases of fip. feline infectious peritonitis (fip) is a fatal, immune-mediated disease of domestic and wild fe-et al., ; wege et al., ) . barlough et al. ( ) showed that an infection with ccv caused seroconversion but no clinical signs in the cats examined, neither was the course of the subsequently experimentally induced fip disease more severe. in contrast to these findings, mcardle et al. ( ) demonstrated that after infection with ccv the course of fip disease was more severe, and that ccv induced in some cats similar symptoms as in the dog. furthermore, one ccv strain caused in a cat clinical symptoms which were not discernible from fip. after all, the importance of ccv for the cat remains uncertain (sparkes et al., ) . two biological types of fcovs are known, feline infectious peritonitis virus (fipv) and feline enteric coronavirus (fecv) (pedersen, b (pedersen, , (pedersen, , pedersen et al., ) . the genome of some fecv strains proved to be . kb shorter, suggesting a deletion of bp at the -end (vennema et al., ) . molecular studies showed that fipv arises by mutation from fecv (pedersen et al., ; evermann et al., ; hök, ; poland et al., ; herrewegh et al., ; vennema et al., vennema et al., , . both fipv and fecv may, depending on their virulence, cause viremia (herrewegh et al., ; fehr et al., ; gunn-moore et al., ; horzinek, ) . fcovs are separated into two different types based upon their growth ability in vitro, their antigenic relationship to ccv, their neutralisation reactivity with sprotein-specific mabs (fiscus and teramoto, a,b; hohdatsu et al., hohdatsu et al., , and upon sequence analysis of the s-protein gene (motokawa et al., ) . while type i shows no or little replication in cell culture (fipv ucd , ucd , ucd , ucd , tn- , nw , yayoi, ku- , dahlberg, fecv ucd), type ii induces a lytic cytopathic effect (fipv - , nor (df ) , cornell- , fecv - ) . the ability of an fcov strain to propagate in cell culture does not correlate with its virulence in vivo (mochizuki et al., ) . among the fcov types i and ii, both fipv and fecv strains are represented. the s-protein gene of type ii is closely related to those of tgev and ccv, showing a similarity of the nucleotide sequence of and %, respectively, but of only % with the s-protein gene of type i (motokawa et al., ) . herrewegh et al. ( ) demonstrated that fcov type ii resulted from recombination of fcov type i with ccv. recent studies indicate that type ii uses the feline aminopeptidase n (fapn), a cell-surface metalloprotease on the intestinal, lung and kidney epithelial cells, as receptor, and that fapn may also bind ccv, tgev and hcv. it is not clear whether or not this receptor specificity of type ii plays a role in the pathogenesis or pathological alterations of fip (williams et al., ; de groot and horzinek, ; hohdatsu et al., ; tresnan and holmes, ) . the prevalence of types i and ii has been surveyed in two studies from austria and japan, respectively, both suggesting that the majority of fcov infections is due to type i (hohdatsu et al., ; posch et al., posch et al., , . fcovs are ubiquitous in the cat population, highly infectious by the oronasal route and therefore endemic in multi-cat households, catteries and shelters. investigations showed that a high percentage of cats without fip symptoms from exposed environments were positive for fcov infection: - % were seropositive, - % viremic and - % excreted virus in their faeces (addie and jarrett, b; sparkes et al., ; herrewegh et al., ; foley et al., a,b; gunn-moore et al., ) . posch et al. ( posch et al. ( , found % seropositive cats in austrian cat populations without signs of fip, % of these cats tested positive for fcov nucleic acid in blood. there is strong evidence for the existence of persisting and chronic infections, with virus persisting in the intestine and other organs of healthy cats. asymptomatic carriers may excrete virus over a period of months or even years (foley et al., a; herrewegh et al., herrewegh et al., , . asymptomatic carriers and shedders represent coronavirus reservoirs and therefore the main problem in the prevention of fip in multi-cat environments (addie and jarrett, a,b; addie et al., addie et al., , foley et al., a,b; herrewegh et al., ) . approximately - % of seropositive cats develop fip, with the highest incidence in cats between months and years of age, and the majority of cases occurring in cats ≤ year of age (scott, ; addie and jarrett, a) . the higher incidence of fip among purebred cats (scott, ) , cheetahs (evermann et al., ) and cats from fip-susceptible bloodlines may be an indication for a genetic predisposition. in addition, the sex of the host may influence the outbreak of the disease. while pedersen ( a) found no generic disposition, potkay et al. ( ) and binder and hartmann ( ) observed a higher incidence of fip among males than among females. although serological testing by immunofluorescence assay (ifa) (moestl, ) or elisa (mochizuki and furukawa, ) is a helpful tool for fip diagnosis, results can only be interpreted in correlation with clinical symptoms (sparkes et al., ) . at present, the only conclusive fip diagnosis can be established by histopathological examination of a biopsy or post mortem material. the recently developed reverse transcriptase polymerase chain reaction (rt-pcr) assays, using primers targeted to highly conserved regions of the viral genome ( -utr (untranslated region) (herrewegh et al., ; fehr et al., ) , or s-protein gene (li and scott, ; gamble et al., ) ), which are common to all fcov strains, became a valuable tool for the detection of fcov nucleic acid in blood, body cavity effusions, faeces and tissue samples of infected cats. in particular the n-terminal domain of the s-protein gene allows a differentiation between the two types i and ii. posch et al. ( posch et al. ( , developed an rt-pcr using primers targeted to the s-protein gene to study the prevalence of the two fcov types in cats without fip symptoms, and showed that % of the pcr-positive cats proved positive for type i, % for type ii and % for both types. with the retrospective study presented here we investigated the prevalence of the two types of fcovs in cats with histopathologically verified fip using nested and seminested rt-pcr assays, with primers targeted as well to the s-protein gene. the aim of this study was to investigate the distribution of the two fcov types in fip diseased cats. furthermore-since fcov types i and ii may use different receptors-we wanted to investigate whether the two types are associated with differences in the clinical course of the disease and/or distinct histopathological changes. finally we intended to get more information about the importance of ccv for the cat. ccv itself may infect the cat, or it may be involved indirectly, regarding the possibility that recombinations between fcov type i and ccv may happen in the field at any time (horzinek, ) . between and a total of cats were examined at the institute of pathology and forensic veterinary medicine of the university of veterinary medicine, vienna, and of these cats were diagnosed with fip. the analysis of breed, gender and age of the cats with fip compared to the cats without fip symptoms is shown in table . the statistical evaluation of the parameters breed, gender and age in the two groups "cats with fip" and "cats without fip" was carried out by χ -test using the program spss for ms windows, version . . from of the cats with histopathologically confirmed fip organ samples (lung, liver, spleen, kidney, gut) were available, either formalin-fixed paraffinembedded tissues (pet) (n = , - ) or fresh organ samples (n = , - ) . pet samples had been fixed in buffered formaldehyde for h and were then embedded in paraffin. fresh organ samples were taken during section and either processed immediately or stored at − • c until used. the preparation of pet samples was carried out essentially as described by sorg and metzler ( ): four to six m thick sections of paraffin-embedded organs from each cat were pooled and deparaffinised by incubating for min in xylene and washing twice for min in ethanol at room temperature. after centrifugation and air drying for min at • c, - l proteinase k and - l (depending on the sample size) buffer atl (qiagen, valencia, ca, usa) were added and the samples were then incubated at • c for days. when necessary, another equivalent of proteinase k and buffer atl was added at the second or third day of incubation. after inactivation of proteinase k at • c for min and centrifugation, rna was extracted from the upper aqueous phase using a commercially available kit (qiaamp viral rna mini kit, qiagen, valencia, ca, usa). the extracts were then stored at − • c. one to three grams of each organ sample were pooled and homogenised with sterile sand, and resuspended in - ml diethyl pyrocarbonate (depc)treated water. after centrifugation the rna was the general screening for fcov was carried out by rt-and nested (n) pcr as described by herrewegh et al. ( ) using the primers p and p for rt-pcr and p and p for npcr, respectively. samples positive in these assays were submitted to further analysis employing oligonucleotide primers, which had been designed in regions of the s-protein gene allowing a differentiation between fcov types i and ii. to improve the sensitivity of the pcr assays, a second round of amplifications (npcr with primers b, seminested with primers a) was carried out following rt-pcr. the primers were selected with the help of the primer designer program (scientific and educational software, version . ) and are shown in table . rt-pcr was carried out as a single-tube assay with a reaction volume of l ( . l pcr mixture and . l template) using a commercially available kit (access rt-pcr system, promega, madison, wi, usa). the mgso concentration was optimised at and mm using the primers fecv and fecv , respectively. negative samples were re-tested by employing the one step rt-pcr kit from qiagen (valencia, ca, usa). cycler schemes were carried out following the instructions of the manufacturers. an amount of . l of the rt-pcr product was added to . l of the master mix for npcr and seminested pcr, respectively, containing mm tris-hcl (ph at • c), mm kcl, . % triton x- , m each -deoxynucleoside -triphosphate, . mm mgcl , . u taq polymerase and pm of each primer. forty-five cycles of denaturation at • c, primer annealing at • c and primer extension at • c, s each, were employed. as the possibility of false positive results due to carryover of amplification products in particular during npcr cannot be ruled out, a number of precautions were taken to minimise the risk of contamination. these included the physical separation of all pcr procedures, the use of at least four negative controls of rnase free water for each assay and of three or more primer pairs for each sample. the rt-pcr amplification product was added to the master mix for the npcr in a laboratory specifically installed for this purpose. finally sequence analysis of amplification products served as an additional control. twenty microlitres of each pcr product were analysed by electrophoresis in a % agarose gel for h min at v, and visualised by ethidium bromide staining. the bp ladder (amersham pharmacia biotech inc., piscataway, nj, usa) served as molecular weight marker. bands were visualised with uv illumination and photographed using the eagle eye tm ii uv gel imaging system (stratagene, la jolla, ca, usa). sequence analysis was performed after gel extraction of the amplified product (qia quick gel extraction kit, qiagen, valencia, ca, usa) and sequencing pcr (abi prism big dye tm terminator cycle sequencing ready reaction kit, perkin elmer, alameda, ca, usa) using the sequence analyser abi prism genetic analyser (pe applied biosystems). partial nucleotide sequences (a stretch of bp within the s-protein gene region) of selected type i and type ii positive samples, as well as one of the five samples which had tested positive for both types, were determined; their alignment is shown in fig. . extracts of cell culture supernatants from five different fcov-strains (type i: fipv ku , fipv nw ; type ii: fipv - , fecv - , fipv df ) were submitted to rt-pcr, nested and seminested pcr employing the primers fecv a, fecv b, fecv a and fecv b. gel extracts of the strains fipv ku and fipv - , containing . and . pmol/l dna, respectively, obtained after rt-pcr with the primers fecv b and fecv b, were diluted in rnase free water with a concentration of % trna, and served as template for npcr. as far as antibody titres had been recorded in the case histories, they were compared to the pcr results. the results of the pathological examination were analysed according to the following criteria: during section the presence and amount of fip typical effusion (low, medium, high amount) and of fip suspicious granulomas and pyogranulomas (granulomas yes, no and localisation) were recorded. the subsequent histopathological examination confirmed the diagnosis fip only in the presence of the typical vasculitis with central necrosis and perivascular infiltration with plasma cells, macrophages, lymphocytes and neutrophils. general data of fip-diseased cats were analysed and compared to those of cats without fip symptoms examined during the same period of time ( ) ( ) ( ) ( ) at the institute of pathology and forensic veterinary medicine of the university of veterinary medicine in vienna (table ). the statistical examination showed that the incidence of fip was significantly higher among males versus females (p = . ), among purebred versus domestic short hair cats (p = . ) and among young animals up to year (p = . ). a significantly greater number of males among fip-diseased cats was found in the age category - year (p = . ), but in cats older than year this trend could not be observed (p > . ). while of the pet samples tested negative, nucleic acid could be detected in all fresh organ samples with the primers described by herrewegh et al. ( ) . the differentiation of the two types was accomplished in of the pet samples which tested positive for fcov (the pcr result of one additional sample was questionable) and in out of fresh organ samples (one additional questionable result). in total, a differentiation was possible in samples. among these, ( %) tested positive for type i, ( %) for type ii and ( %) for both types i and ii (table ) . of the samples positive for type i, tested positive with the fecv b primers, with both the fecv a and fecv b primer pairs, and with the fecv a primers only. of the samples with a questionable pcr result, tested questionably positive with the fecv a primer pair and negative with the fecv b primers, the second one vice versa. of the samples positive for type ii, tested positive employing the fecv a primers and using the fecv b primers. of these results ( %) were already achieved after rt-pcr. extracts of cell culture supernatants of five different fcov strains, the type i strains fipv ku and fipv nw , and the type ii strains fipv - , fecv - and fipv df , were subjected to rt-pcr and nested or seminested pcr with the primer pairs fecv a, fecv b, fecv a and fecv b. employing the primers fecv a and fecv b, the type i strains ku and nw showed amplification products of the estimated size, whereas the type ii strains - , - and df tested negative (fig. , primers fecv b ). with the primers fecv a and fecv b the strains - , - and df tested positive whereas the type i strains remained negative (fig. , primers fecv b). gel extracts of the strains fipv ku and fipv - , obtained after rt-pcr with the primer pairs fecv b and fecv b, tested positive in the nested pcr assays up to a dilution of − and − , respectively. antibody titres to fcov were known from the case history for cats, of them had titres of ≥ : , of : , of : and was indicated as serologically negative. in the group of cats with fcov antibody titres of ≥ : twelve tested positive for fcov nucleic acid of type i and were negative for type ii, and one was positive for type ii but negative for type i; for seven cats a differentiation between the two types could not be achieved. all three cats with titres of : tested positive for type i and negative for type ii. the single cat with a titre of : and the sero-negative cat tested both negative for fcov nucleic acid. partial nucleotide sequences of the s-protein gene of type i and type ii positive samples as well as of one sample positive for both types were deter-mined. the sequences were compiled (resulting in a readable stretch of bp) and aligned using the sequence of the type i strain fipv ku as a reference. in the alignment, also the corresponding sequences of the fcov type ii reference strain - and of the ccv reference strain insavc- were included. the analysis of the samples revealed a nucleotide identity of - % for the type i specimens, and of - % for the type ii samples, respectively, in reference to the type i strain fipv ku (fig. ) and of and % for the type ii specimens in reference to the ccv strain insavc- . histopathologic examination exhibited no differences related to the type of fcov detected. both types were found in effusive and non-effusive fip as well as in cases with signs of both forms; a statistical evaluation was not possible due to the small number of type ii positive samples. the comparison of the two groups, cats with fip (n = ) and cats without fip symptoms (n = ) examined in the years - at the institute of pathology and forensic veterinary medicine of the veterinary university in vienna showed significant differences. the statistical evaluation using the χ -test of the parameters breed, gender and age in the two groups showed that the incidence of fip was significantly higher among purebred cats, males, and among cats year of age or younger. the percentage of purebred cats in fip-diseased cats was more than twice as high as in the comparative group ( . % versus . %, p ≤ . ). these findings are sustained by earlier studies (pedersen, ) . foley and pedersen ( ) observed a higher susceptibility for fip in purebred cats when a first degree relative succumbed to fip. due to inbreeding a genetic predisposition may have evolved in certain breeds of cats, which may allow fcov to propagate more efficiently in these cats than in cats with a wider genetic history. in the group of the cats with fip, the majority was male ( . %), only . % were female. although in the comparative group the percentage of males was slightly higher as well ( . % males versus . % females), the difference between the groups was significant (p ≤ . ). these findings are in contrast to pedersen ( a) , who did not find a sexual predisposition, but in accordance with potkay et al. ( ) and binder and hartmann ( ) , who also reported a higher incidence of fip among males. the majority of the cats with fip was -year-old or younger ( . %), in the comparative group only . % were in this age class. addie and jarrett ( a) found as well as scott ( ) a higher incidence of fip in cats of up to year. when comparing the incidence of males and females in the age groups - year and older than year between the cats with and without fip, we found that among younger cats the incidence of males was significantly higher in the cats with fip than in the comparative group. the role of sex-specific differences in the immune system, in particular the cell mediated immunity and the importance of these factors in neutered animals (hormonal influence) are still not clear. in a total of . % of the samples, fcov nucleic acid could be detected. only six pet samples tested negative, whereas all fresh organ samples tested positive. in respect to the expected lower rna concentration in the pets due to the formalin fixation procedure on one hand and due to the long storage time ( - years) on the other hand, we chose primers which amplified, compared to those employed by posch et al. ( posch et al. ( , , a smaller segment of the viral genome. with these primers we achieved a differentiation of the two types in of pet specimens and in of fresh organ samples, in addition two samples exhibited a questionable pcr result. as expected, the percentage of positive pcr results was lower in the pet samples than in the fresh organ samples. specificity was tested on five different fcov strains with four different primer pairs (figs. and , primers fecvb). we found no false positive results and all amplification products showed bands of the expected size. despite the use of different primer pairs, in some samples the pcrs remained negative, probably due to the variability in the s-protein gene of fcov. the oligonucleotide primers employed in the pcr assays exhibited high sensitivity, the fecv b primers proved to amplify specific nucleic acid up to a dilution of − , and the fecv b primers showed amplification even up to a dilution of − . since the original rna concentration was similar in both samples, these findings indicate a higher sensitivity for the detection of type ii viruses. thus, in those samples, in which the differentiating pcr was unsuccessful, fcov type i may predominate as well. on the other hand, due to the lower sensitivity of the type i pcr, we cannot exclude a causative involvement of type i in the type ii positive cats. of the samples in which a differentiation was achieved, ( %) tested positive for type i, ( %) for type ii and ( %) for both types i and ii, thus identifying type i as the causative agent in the majority of the fip cases we examined. whereas posch et al. ( posch et al. ( , identified type ii in % (including those samples with both types) of fcov-positive cats without fip symptoms, we found type ii only in % of cats with fip involved, among them % showing a double infection with both types i and ii. these findings are in contrast to the results of hohdatsu et al. ( ) in japanese cats, in which none of the healthy cats tested positive for type ii, whereas among the chronically diseased cats without fip symptoms over % and among the fip-diseased cats even more than % were infected with fcov type ii. due to their close antigenic relationship in the s-protein gene and the need to choose primers from this region for a possible differentiation between the two fcov types, an infection with ccv would also have been detected (data not shown). therefore, a causative involvement of ccv in the type ii positive fip cases cannot be ruled out in this study. the importance of ccv for the cat still remains unclear (barlough et al., ; mcardle et al., ) , but the possible recombination between fcov type i and ccv horzinek, ) requires further investigation, in particular the role of ccv in double infections with both fcov types observed especially in multi-cat households. the temperature-sensitive fipv strain used as fip vaccine is a type ii strain (fipv df ). regarding the fact that the majority of the fip cases we examined was due to type i, the question arises whether this fact contributes to some of the observed vaccine failures, and whether the inclusion of also a type i strain in the vaccine should be considered. the sequencing results show even in the very short region, which had been sequenced, a clear discrimination between fcov type i, fcov type ii and ccv strains (fig. ) . the partial sequences of the austrian fcov type i samples show significant differences, compared to the reference strain ku , which resulted in identity rates of (only) - % to the reference strain; also within the austrian fcov type i samples several nucleotide changes can be noticed, indicative of a quite high mutation rate. the fcov type ii samples form an own group with an identity to the fcov type i reference strain of only - %, respectively. ccv exhibits an identity to fcov- ku- of %; its much closer relationship to fcov type ii than to fcov type i can nicely be observed by the similarity of several nucleotide changes of fcov type ii and ccv. these findings support the observation that fcov type ii may arise from recombinations with ccv . nonetheless, ccv also exhibits several unique nucleotide changes. the sequencing data of one of the five samples showing double infections (fig. ) demonstrate clearly the plausability of such co-infections. among the cats with known antibody titres, all cats positive for fcov nucleic acid showed antibody titres of : or higher. neither during section nor in the histopathologic examination any differences related to the fcov type detected could be identified. in the group of the type i positive cats % showed signs of both forms (effusive and non-effusive) of fip, followed by % with non-effusive and % with effusive fip. in the type ii positive samples all forms of fip were represented as well. these findings do not point towards a pathogenetic importance of the receptor-specificity of type ii (hohdatsu et al., ) , but emphasises the role of the immune response and the genetic predisposition of the individual in the outbreak of the disease. our findings suggest an involvement of each of both fcov types in fip which is in accordance with earlier reports that both types of fcov are able to cause fip; they also correlate with the results obtained in healthy fcov-infected cats, supporting the predominance of fcov type i infections in both fcov-infected healthy and fip-diseased cats. fcov type ii, the probable recombination between type i and canine coronavirus, was involved in % of the fip cases investigated. it has to be assumed that these recombinations occur in the field and therefore contact with dogs excreting canine coronavirus may play a role in the emergence of new type ii fcov. however, the samples positive for fcov type ii need further investigations with respect to their relationship and even differentiation to ccv. special interest should also be paid to cats with double 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pathogenesis of coronaviruses receptor for mhv is a member of carcinoembryotic antigen family of glycoproteins we cordially thank helga lussy, claudia pallan and dr. barbara bauder for their excellent technical assistance. key: cord- -n tvglif authors: kostoff, ronald n. title: literature-related discovery: potential treatments and preventatives for sars() date: - - journal: technol forecast soc change doi: . /j.techfore. . . sha: doc_id: cord_uid: n tvglif literature-related discovery (lrd) is the linking of two or more previously disjoint concepts in order to produce novel, interesting, plausible, and intelligible connections (i.e., potential discovery). lrd has been used to identify potential treatments or preventative actions for challenging medical problems, among myriad other applications. severe acute respiratory syndrome (sars) was the first pandemic of the st century. sars was eventually controlled through increased hygienic measures (e.g., face mask protection, frequent hand washing, living quarter disinfection), travel restrictions, and quarantine. according to recent reviews of sars, none of the drugs that were used during the pandemic worked. for the present paper, sars was selected as the first application of lrd to an infectious disease. the main goal of this research was to identify non-drug non-surgical treatments that would ) prevent the occurrence, or ) reduce the progression rate, or ) stop/reverse the progression of sars. the mesh taxonomy of medline was used to restrict potential discoveries to selected semantic classes, and to identify potential discoveries efficiently. to enhance the volume of potential discovery, databases were used in addition to medline. these included the science citation index (sci) and, in contrast to previous work, a full text database. because of the richness of the full text, ‘surgical’ queries were developed that targeted the exact types of potential discovery of interest while eliminating clutter more efficiently. literature-related discovery (lrd) is the linking of two or more previously disjoint concepts in order to produce novel, interesting, plausible, and intelligible connections (i.e., potential discovery). the open discovery systems (ods) component of lrd starts with an unsolved problem, and generates solutions to that problem through potential discovery. ods lrd has been used to identify potential treatments or preventative actions for challenging medical problems, among myriad other applications. the closed discovery systems (cds) component of lrd starts with an unsolved problem and a potential solution, and generates potential mechanisms that link the solution to the problem. ods is the only approach that will be used for the present study. two points should be emphasized before proceeding further. first, linking of disjoint literatures is a necessary but not sufficient condition for discovery. there needs to be value-added in the novel concept(s) that results. second, while the term 'potential discovery' is used in this paper and throughout the lrd literature, 'hypothesis' is more accurate. what results from these lrd 'discovery' studies are hypotheses that have to be tested in the laboratory/field before they can be properly termed 'discoveries'. e-mail address: ronald.kostoff@pubpolicy.gatech.edu. the mitre corporation (retired). a review paper by the author showed that, while a number of lrd published papers claimed to have generated potential discovery, essentially none of these claims could be validated [ ] . the only published lrd potential discovery claims that could be validated as credible hypotheses were in a journal special issue devoted to lrd (e.g., [ , ] ). the four medical papers in this special issue describe the application of ods lrd to four chronic diseases: raynaud's phenomenon (rp), cataracts, parkinson's disease (pd), and multiple sclerosis (ms). the present paper presents a comprehensive approach to systematic acceleration of potential discovery and innovation, and demonstrates the generation of large amounts of potential discovery for prevention/treatment of an infectious disease: severe acute respiratory syndrome (sars). the general issues of potential discovery and innovation in the lrd context are discussed in the first paper of the special issue [ ] , and the general methodology for this discovery approach was shown in the second paper of the special issue [ ] . the sars biomedical background has been published in a detailed review article, and the interested reader is referred to that article [ ] . the present paper provides an overview of the etiology and challenges of sars, then presents a retrieval and analysis of the core sars literature and literatures related directly to the core sars literature (e.g., immune system component literatures). these related literatures might contain the seeds of potential discovery (treatments and preventive measures) for sars, and some examples of potential discovery are presented. in contrast to previous work, this paper includes full text analysis for the related literatures. this provided a substantial increase in the volume of potential discovery retrieved. also, examples of interesting but non-discovery (i.e., potential innovation) concepts from the core sars literature are presented, since they have practical value in their own right. the four previous medical papers in the lrd special issue also included potential discovery from indirectly-related literatures. the indirectly-related literatures for the present infectious disease proof-of-principle demonstration were not examined. the amount of potential discovery retrieved from the directly related literature alone (including the full text directly related literature) is voluminous. a major challenge is to select those combinations of potential discoveries that provide the maximum synergy. until these potential discoveries have been exploited, there is little practical need of going to indirectly related literatures to search for yet more potential discovery. in the practical situation, even the potential innovation as defined above has not been exploited, and accelerating these potential innovations should be the first order of business [ ] [ ] [ ] [ ] [ ] . sars is a contagious disease that resulted in the hospitalization of about people world-wide in - , and resulted in the deaths of about people. according to the author's interpretation of reviews of the pandemic, none of the drugs worked (e.g., "despite an extensive literature reporting on sars treatments, it was not possible to determine whether treatments benefited patients during the sars outbreak. some may have been harmful.") [ ] . those who recovered did so by natural means; their immune systems were sufficiently strong to contain the viral attack. many were aided by public health interventions (e.g., face mask protection, frequent hand washing, living quarter disinfection, travel restrictions, and quarantine) as well. the subject of sars was selected for study because of its pandemic nature, and its apparent intractability to all drug treatments. the main goal of this study was to identify non-drug non-surgical treatments that would ) prevent or delay the onset, or ) reduce the progression rate, or ) stop/reverse the progression, of sars. for much of the study, medline was used as the data source, and the mesh taxonomy of medline was used to restrict potential discoveries to selected semantic classes. the second goal was to generate large amounts of potential discovery in more than an order of magnitude less time than required for the rp study. to enhance the volume of potential discovery, a full text database was used in contrast to previous work. because of the richness of the full text, 'surgical' queries were developed that targeted the exact types of potential discovery of interest while eliminating clutter more efficiently. the 'surgical' nature of these queries compensated for the additional 'noise' characteristic of the more voluminous full text. however, since the full text database (science direct-sd) did not have an associated mesh taxonomy, the mesh taxonomy headings were essentially used as text phrases to restrict sars treatment and prevention potential discoveries to selected substances. the science citation index (sci) was also used to search for discovery, and again the mesh taxonomy headings were used as text phrases to restrict potential discoveries to selected substances. approximately four times as many records were retrieved from medline when mesh terms were included in the query compared to using only terms in the title or abstract, due to the greater choice of potential discovery substances. this means the sci or sd queries as presently constituted will retrieve only about % of the records that are possible using mesh to define the substance pool. before the specific approach and results are described, the medical issues for sars that served as targets for the discovery search query will be summarized. the first pandemic of the st century was the outbreak of sars caused by the sars coronavirus (sars-cov). as far as is known, this outbreak was not due to the deliberate release of the sars-cov, but rather was a naturally occurring event. the appearance of sars seems to have involved: ) a zoonotic origin for sars-cov (e.g., horseshoe bats and/or chiroptera as one wildlife reservoir [ ] ); ) transmission to intermediate hosts (e.g., civet cats, raccoon dogs [ ] ); ) human contact with these intermediate hosts in southern china [guangdong province, fall ] and subsequent cross-species transmission of the coronavirus to humans [ ] ; ) transmission of the virus through both non-hospital personal contact and hospital staff contact [ ] ; and, ) global transmission of the virus via travelers from affected regions in asia to other countries. sars was eventually controlled through increased hygienic measures (e.g., face mask protection, frequent hand washing, living quarter disinfection), travel restrictions, and quarantine. a number of recent reviews have focused on different components of the above sars etiology, with the central focus of ) identifying common characteristics of those who succumbed to the disease in order to ) develop treatment targets for future outbreaks. a careful reading of these reviews shows that the humoral and cellular components of the adaptive immune system of those who succumbed were deficient on presentation and deteriorated thereafter. there is controversy about whether adequate antiviral interferons were generated during the innate response, but there is common agreement that the switch from innate to adaptive immunity was defective [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . specifically, those who succumbed from sars-cov infection tended to have the following characteristics, based on diagnostic measurements made at the time of presentation with symptoms: • older age • male gender • presence of comorbidities • elevated ldh or c-reactive protein/high initial lactate dehydrogenase level • higher initial viral load of sars coronavirus • elevated neutrophil count/neutrophilia • high levels of chemokines cxcl and il- • high levels of il- , il- , and mcp- • significant increase in the th cytokines il- , il- , il- • increased levels of ip- , mig and il- • lower levels/deficient anti-sars spike antibody production • lymphopenia/low counts of cd and cd at presentation • reduced ace expression • reduced levels of il- p and tnf-alpha (relative to positive outcome) • positive rt-pcr on nasopharyngeal aspirate samples • elevated pulse rate • raised serum albumin • raised serum creatinine phosphokinase (cpk) levels • increased serum creatine kinase, and urea • deviated isg and immunoglobulin gene expression levels these characteristics served as the targets to be improved by potential discoveries. [ ] is a flow chart that outlines the steps used in the present study. these steps include: database selection; query development to identify the core sars literature; retrieval of the core sars literature; analysis of the retrieved core sars literature to identify the main generic themes (biomedical roadblocks); query development for, and retrieval of, literatures that represent the main generic themes; subtraction of the sars core literature from the literatures that represent the main generic themes; intersection of the net retrieved literatures for the main generic themes with the literature of desired solution types (e.g., non-drug substances); searches for potential discovery candidates in the net retrieved literature for the given classes of substances; and validation of potential discovery candidates as potential discovery. three databases were selected for source material. medline is very comprehensive in the coverage of medical issues. the sci overlaps medline strongly in coverage of the major medical journals and biology journals, but in addition covers technical disciplines well beyond the medical sphere. the sci also allows citing papers, references, and papers that share common references to be retrieved. for both these databases, titles, abstracts, and keywords were searched. in addition, it was desired to ascertain the increase in potential discovery possible from searching full text. the sd database was used for this purpose. the core sars literature is defined as those documents that the research/medical community would associate unambiguously with sars. an iterative relevance feedback approach was used, and produced the following query: "(severe-acute-respiratory-syndrome or sars-virus or (sars and (coronavirus or infect* or virus* or viral or epidemic* or epidemiology or antibodies or antibody or vaccine* or influenza or pandemic* or outbreak* or syndrome)) or sars-patient* or sars-transmission or sars-cov)". the query was inserted in the three databases to retrieve the core sars literature. multiple grouping approaches were used to identify the main generic themes of the core sars literature: document clustering, auto-correlation mapping of phrases, and factor matrix analysis of phrases, using the vantage point software package [ ] . these grouping techniques were applied to retrievals from the medline and sci databases, and no significant differences were observed. this step represents the main methodological advance over the author's previous lrd studies. whereas previous lrd approaches used boolean combinations of important biomedical terms (e.g., protein and aggregation) in the query for retrieving generic biomedical literatures, the present approach used functional proximity queries (e.g., inhibit within x words of viral within x words of entry). this type of query allowed 'surgical' targeting of relevant records, and for full text in particular was necessary to filter out the large numbers of irrelevant records that would occur with a boolean query. the query has two components: (t not s) and c. t represents the technical query terms of interest, and s represents the technical query terms defining the sars core literature (shown in section . ). t not s will retrieve those records of interest in the total database, excluding records found in the sars core literature. c mainly represents the substances/behaviors that are in the potential discovery pool. as an example, if non-drugs were the only types of discovery of interest, c would be the pool of all nondrug records. thus, all records in medline, for example, that addressed medicinal plants would be included in c. c also includes the journals whose articles tend to focus on such substances/behaviors, and thus would expand the substance pool for potential discovery beyond a simple listing of substances. the specific steps employed in determining t are as follows. as stated previously, multiple grouping approaches were used to identify the main generic themes of the core sars literature. the main problem was deciding which hierarchical level of grouping to use for the query. initially, groupings at the lowest level of detail (e.g., cd , cd , il , il , ifn-gamma, etc.) were examined. however, a detailed examination of the sars literature showed inconsistencies in the desired directions of some of these items, based on clinical observations and data. this led to examination of functional groupings at a higher level of aggregation (e.g., inhibit viral replication, enhance humoral immunity, improve cell-mediated immunity, increase th , etc.), which could accommodate different directions at the lowest level of detail while achieving the targets represented by the higher level of aggregation. to retrieve the directly related literature from which potential discovery would be extracted, this higher level functional query was applied to the search engines of three databases: sci, medline, and sd. the first two of these databases provide abstracts as the major text source, and sd provides full text. since the sd analysis took place a few months after the sci and medline analyses, the initial query was modified slightly to exploit the sd search engine features. appendix of [ ] contains the full sci and medline queries, and appendix of [ ] contains the full sd query. the unique features of each are explained in these appendices. there were two types of fundamental terms in the full query (type i and type ii) , and examples from the more abbreviated sd query will be presented here. type focused on inhibiting viral entry to healthy cells and/or inhibiting their replication, whereas type focused on improving immune system component performance. these two generic categories were identified from analysis of the retrieved sars core literature. factor analysis and document clustering of this core literature identified these key thrust areas, and further phrase frequency analysis of the retrieved sars core literature identified the biomedical term ('viral entry')functional term ('inhibit') combinations eventually selected for the query. in the proximity form of the query used below, 'a pre/ b' is a precedence relation, and means that term a precedes term b, with a spacing ranging from zero (adjacency) to five words. 'a w/ b' is a proximity relation, and means that term a is within words of term b. this proximity form of the query (as contrasted to the boolean form used in prior lrd studies) provided highly 'relevant' retrievals, where 'relevant' is defined as any article that contains a potential discovery or innovation candidate. ref [ ] contains a more detailed discussion of 'relevance' in the present context. type (((inhibit* pre/ "virus entry") or (inhibit* pre/ "viral entry") or (inhibit* w/ replicat* w/ virus) or (inhibit* w/ replicat* w/ viral)) and (potent pre/ "antiviral activity")) type (((enhanc* pre/ "humoral immun*") and (enhanc* pre/ "humoral response*") and ((enhanc* pre/ "antibod* response*") or (enhanc* pre/ "antibod* production") or (enhanc* pre/ "virus neutraliz*") or (enhanc* pre/ "viral neutraliz*"))) or ((enhanc* pre/ "cellular immun*") and (enhanc* pre/ "cell mediated immun*") and ((enhanc* pre/ cd ) or (enhanc* pre/ cd ) or (enhanc* pre/ "t cell response*") or (enhanc* pre/ "t cell immune response*"))) or ((enhanc* pre/ "innate immun*") and (enhanc* pre/ "innate antiviral") and ((enhanc* pre/ "antiviral activity") or (enhanc* pre/ "antiviral response*")))). when the type query shown above was applied to full text, reasonable numbers of relevant articles were retrieved. when the type query was applied to abstracts, the articles retrieved were highly relevant, but the numbers retrieved (as will be shown) were miniscule. to obtain more articles when searching the abstracts, a modified type query was generated. this modified form changed the 'and' (in the query above) to an 'or', a much less restrictive condition. this resulted in an order of magnitude more retrieved and relevant articles (when searching abstracts), although still small compared to the retrievals obtained from searching full text. type queries focused on enhancing the performance of the different components of the immune system. there were a number of variants of the type query shown above that were examined. appendix of ref. [ ] shows the form of the query used to generate the sci results. the type entry under the t component is for 'improv*', but the full query added blocks that replaced 'improv*' with enhanc* or induc* or stimulat* or increas* or activat* or regulat*. obviously, other such terms could be identified and used as well, but these terms were deemed adequate for the present proof-of-principle demonstration, which shows the feasibility of the methodology. the query examples provided above were for the sd database, which has intrinsic proximity query capabilities in addition to boolean query capabilities. the sci search engine does not have adjacency/proximity search capability presently. the only search capabilities are boolean/co-occurrence in a selected field or among all fields (e.g., a same b, or a and b). to overcome this deficiency, the author developed an algorithm that would provide such capability [ ] . in the algorithm, sci stopwords are used to set spacing between terms. thus, a query term of the form [improv*-of-humoral-immun*] will retrieve those records containing variants of improv* that precede variants of 'humoral-immun*' with one word intercalated. the intercalated word could be any word, not just the stopword used in the query. as can be seen from the terms in appendix of ref. [ ] , the actual query used did not go beyond precedence spacings of two words, but a larger production-oriented study could use greater spacings between the terms of interest. this would retrieve far more records, and lead to more candidate potential discoveries. in appendix of ref. [ ] , the s block of terms represents the core sars records, and its inclusion as a negation expression insures the records retrieved are disjoint from the core sars literature. in appendix of ref. [ ] , the blocks listed under c are the types of substances/behaviors considered for discovery. they consist of records from non-drug journals as shown, and records that contain non-drug substances. the value of including the journals is that their records could include substances/behaviors not identified in the substances/behaviors blocks (i.e., pre-specified lists of substances/behaviors). obviously, many more substances/behaviors could be added to the list in a more comprehensive production-oriented study. the records retrieved by the t not s query were intersected with the records retrieved by the c query, to yield records that identified non-drug substances/approaches that would produce immune system changes in the desired directions. these records were inspected visually, and sample results are presented in the next section. the purpose of this study is to identify potential discovery and innovation. for this purpose, 'relevant' is interpreted as any article that contains a potential discovery or innovation candidate. what is potential discovery? it is the linking of two or more literature concepts (that have not been linked previously in the literature; i.e., disjoint) in order to produce novel, interesting, plausible, and intelligible connections. a potential discovery candidate is an interesting linkage that has to be vetted against prior knowledge to validate disjointness. in practice, a major roadblock is defining 'prior knowledge', and in particular the databases that will be used to represent 'prior knowledge' for the vetting process and how these databases will be interpreted. to make the problem tractable, only the main source databases are selected for the 'prior knowledge' determination. in the present case, the same three major sources that were used in previous lrd medical studies have been selected for the 'prior knowledge' determination: sci; medline; and patent database as represented by derwent innovation index (dii). for each candidate potential discovery, a query was developed that intersected the candidate potential discovery (e.g., curcumin) with the sars core literature query (severe acute respiratory syndrome or .....), and the query was inserted into each of the three validation databases. if no prior records were retrieved, the concept was viewed as a potential discovery. if prior records were identified, the concept could still be viewed as a potential innovation if the judgment was made that the concept could be developed at a more accelerated pace. application of the full query to the sci/ssci database ( - , articles and reviews) yielded records. the records were sampled for 'relevancy' to discovery or innovation, using the definitions of relevancy as discussed previously. approximately % were judged to be 'relevant' (potential discovery candidates). in addition, the most recent papers in the sci that cited the records were retrieved. these citing papers covered approximately the seven year period - , and about % were judged to be relevant. in the previous lrd studies on medical topics, before the ability to retrieve all the papers that cite an initial retrieval became available in the sci, only spot checks could be done of papers that cited potential discovery candidates. not only are papers that cite potential discovery candidates good potential discovery candidates themselves, but the author's preliminary (unpublished) experiments which show many different types of citation linkages (e.g., papers that share references) to potential discovery candidates will identify good potential discovery candidates. application of the query to the medline database yielded records. while the version of the medline database used (through the web of knowledge search engine) goes back to , it effectively started in about , when abstracts were introduced. the records retrieved were sampled for 'relevancy' to discovery or innovation, using the definitions of relevancy as discussed previously. approximately % were judged to be 'relevant' (potential discovery or innovation candidates). there is much overlap between medline and the sci. application of the appropriate query from appendix of ref. [ ] to the science direct database ( - ) yielded different types of results, depending on which field was searched, and some of these findings are reported in table . the retrieval results among sci, medline, and science direct are not comparable due to the different journal coverage of each database. specific examples of potential discovery from each of these databases will be shown later in the present results section. in table , the science direct results are for the three components of the type query. the first row (inhibit*acetabularia) reflects the intersection of the terms for the 'inhibit' group (e.g., "(inhibit*-virus-entry or inhibit*-viral-entry or inhibit*-of-virusentry or inhibit*-of-viral-entry or inhibit*-of-of-virus-entry or inhibit*-of-of-viral-entry or (inhibit*-replication same (virus or viral)) or (inhibit*-of-replication same (virus or viral)) or (inhibit*-of-of-replication same (virus or viral)))" and the group of substances and behaviors that starts with 'acetabularia' (e.g., acetabularia or achlya or acupressure or acupuncture or algae or alkaloid* or allium or angiosperms or anthocerotophyta or anthocyanins.....) listed in appendix of ref. [ ] . the second and third rows substitute the euglendia and plankton groups for the acetabularia group. for the first row, the second column (unmod query -absrec) contains the number of records retrieved when the full text query was applied to the abstract field. the full text query was designed to provide 'surgical' targeting of key phrase relations in the full text, contains the intersections of a number of proximal relations, and is a rather restrictive condition. applied to the full text, the query is very effective, but it is far too restrictive for the abstract field, as the results show. very few records are retrieved, and they are all relevant (column ). essentially no records were retrieved in this column for any of the type queries, since the type queries are even more restrictive (more intersecting terms) than the type query. column (mod. query -absrec) contains records retrieved using the modified type query applied to the abstract field. all occurrences of 'and' were replaced with 'or'. on average, about an order of magnitude more records were retrieved with the modified query compared to the full text query, but the numbers are still small. the relevance of the retrieved records is still quite high. for the type query components (not shown), the column retrievals were about half as much for the 'enhance' group, about twice as much for the 'induce' group, about the same for the 'stimulate' group, and about the same for the 'increase' group. there were overlaps among the groups, and search engine limitations did not allow estimation of the degree of overlap. column is the relevance percentage of the column retrievals. it is highest for the 'inhibit' group, is about % for the 'enhance' group, and is about % for the other three (type ) groups. column (unmod. query -fltxrec) represents the retrievals for the full text query of appendix applied to the full text. they are almost an order of magnitude larger than those of column , and the relevance percentage (column ) is almost the same as that for the abstracts in column . the percentage is highest for the 'inhibit' group, is about % for the 'enhance' group, and is about % for the other three (type ) groups. column (ft/abs-norm) is the ratio of full text retrievals to abstract retrievals (normalized to the total numbers of full text and abstract records in the database) using the restricted form of the full text query for each, and column is the same ratio where the less restricted form of the query was used to search the abstracts. the bottom line is that many potential discovery candidates have been retrieved. many more are possible: increasing the substance base (through more non-drug items or by inclusion of drugs) would probably increase the number of candidates by an order of magnitude; increasing the number of terms in the query would enhance the retrieval; relaxing the proximity conditions would increase the retrieval; relaxing the intersection requirements would increase the retrieval; and adding further types of citation linkages would increase the retrieval. a major factor in the high relevance fractions achieved is the form of the query terms; they were not used in any previous lrd studies, but will become a fixture in future studies. this remainder of this section contains representative examples of potential discovery from literatures related directly to the core sars literature. before proceeding to analyses, a few illustrative examples from the core sars literature restricted to semantic classes will be presented. while these are not discovery, they nevertheless reflect the types of impact that the non-drug approaches could potentially have for delaying or preventing the onset of sars. in addition, as will be discussed later, some of these core concepts are prime candidates for innovation. for example, "aurintricarboxylic acid (ata) has been shown to inhibit the replication of viruses from several different families, including ….. the coronavirus causing severe acute respiratory syndrome. vaccinia virus replication is significantly abrogated upon ata treatment, which is associated with the inhibition of early viral gene transcription. this inhibitory effect may be attributed to two findings. first, ata blocks the phosphorylation of extracellular signal-regulated kinase / , an event shown to be essential for vaccinia virus replication. second, ata inhibits the phosphatase activity of the viral enzyme h l, which is required to initiate viral transcription. thus, ata inhibits vaccinia virus replication by targeting both cellular and viral factors essential for the early stage of replication" [ ] . as another example, "we identified that three widely used chinese medicinal herbs of the family polygonaceae inhibited the interaction of sars-cov s protein and ace . the ic values for radix et rhizoma rhei (the root tubers of rheum officinale baill.), radix polygoni multiflori (the root tubers of polygonum multiflorum thunb.), and caulis polygom multiflori (the vines of p. multiflorum thunb.) ranged from i to [lg/ml]. emodin, an anthraquinone compound derived from genus rheum and polygonum, significantly blocked the s protein and ace interaction in a dose-dependent manner. it also inhibited the infectivity of s protein-pseudotyped retrovirus to vero e cells. these findings suggested that emodin may be considered as a potential lead therapeutic agent in the treatment of sars" [ ] . more detailed descriptions of the following potential innovations and discoveries can be found in ref. [ ] . cimicifuga rhizoma, meliae cortex, coptidis rhizoma, phellodendron cortex and sophora subprostrata radix decreased the mhv production and the intracellular viral rna and protein expression, and could be potential candidates for new anti-coronavirus drugs [ ] ; betulinic acid and savinin were competitive inhibitors of sars-cov cl protease [ ] ; quercetin- -beta-galacto side was identified as an inhibitor of the protease [( cl(pro))] [ ] ; alpha,beta-unsaturated peptidomimetics, anilides, metalconjugated compounds, boronic acids, quinolinecarboxylate derivatives, thiophenecarboxylates, phthalhydrazide-substituted ketoglutamine analogs, isatin and natural products have been identified as potent inhibitors of the sars-cov main protease [ ] ; tannic acid and -isotheaflavin- -gallate were found to be inhibitive. these two compounds belong to a group of natural polyphenols found in tea, and only theaflavin- , ′-digallate (tf ) was found to be a clpro inhibitor [ ] . glf[fermentation product of chinese medicinal mushroom] up-regulated the cell-mediated immune response related cytokines (il- , ifn-gamma, and tnf-alpha) expression in different lymphoid tissues [ ] ; jacalin, an antigen-specific lectin from jackfruit seeds, has been shown to induce mitogenic responses and to block infection by hiv- in cd (+) t lymphocytes [ ] ; sulforaphane significantly downregulated the serum levels of proinflammatory cytokines such as il- beta, il- , tnf-alpha, and gm-csf during metastasis [ ] ; immunomodulatory activity of methanolic extract of m. koenigii leaves was evaluated on humoral and cell mediated immune response to ovalbumin, and the extract holds promise as immunomodulatory agent, which acts by stimulating humoral immunity and phagocytic function. [ ] ; the aqueous extract of t. cordifolia was found to enhance phagocytosis in vitro. the aqueous and ethanolic extracts also induced an increase in antibody production in vivo. [ ] ; oral intake of the fucoidan might take the protective effects through direct inhibition of viral replication and stimulation of both innate and adaptive immune defense functions. [ ] ; atractylodes macrocephala koidz (amk) markedly stimulated lymphocyte proliferation, antibody production, and cytokine secretion in mouse splenocytes, showing the ability to induce the preferential stimulation of th type, rather than th type t lymphocytes [ ] ; a potent anti-influenza virus activity was discovered in summer leaves of japanese wasabi [(wasabia japonica)], inhibiting influenza virus replication regardless of the hemagglutinin antigen type. [ ] ; purification of an antiviral peptide from seeds of sorghum bicolor l strongly inhibited the replication of herpes simplex virus type i (hsv- ) [ ] . combined treatment with pidotimod and red ginseng acidic polysaccharide has an immunostimulatory effect in a synergistic manner on antibody response to challenge with lipopolysaccharide and sheep red blood cells without toxic changes [ ] ; myrica rubra leaf ethanol extract showed anti-influenza virus activity irrespective of the hemagglutinin antigen type in the influenza virus type a (h n ), its subtype (h n ), and type b [ ] ; oral intake of l. paracasei ncc by aged mice enhanced the specific adaptive immune response to in vivo antigenic challenge without altering other cellular and humoral immune responses [ ] ; methanol extract of asarum sieboldii inhibited the h n influenza viruses from the infected cells [ ] ; caffeoyl glycoside from the roots of picrorhiza scrophulariiflora (kutki) stimulated cell proliferation of splenocytes and peritoneal macrophages, enhanced the cytotoxicity of natural killer (nk) cells, increased cd and cd cell populations, and has immunomodulatory activity by regulating expression of th and th related cytokines [ ] . korean mistletoe lectin c increased influenza-specific antibodies with dominant igg subclass in serum, igg in genital secretions and iga in saliva, and significantly enhanced influenza-specific lymphocyte proliferation and cytotoxic activity in spleens and in mediastinal lymph nodes. when kml-c was used as a mucosal adjuvant, mice were completely protected from mortality after the challenge with a homologous (h n ) mouse-adapted influenza virus [ ] ; seeds of cochinchina momordica numerically increased the antibody levels, suggesting potential to improve the immune responses by use as an adjuvant [ ] ; ag b of mycobacteria, which cross-reacts among mycobacteria species, elicits helper t-cell type (th ) immune responses as a novel adjuvant [ ] ; probiotic bacillus cereus var. toyoi-treated piglets showed a significantly lower frequency of cd (high)/cd + t cells and cd (low)/cd + t cells and a significant higher cd +/cd + ratio [ ] . different components of kefir have an in vivo role as oral biotherapeutic substances capable of stimulating immune cells of the innate immune system, to down-regulate the th immune phenotype or to promote cell-mediated immune responses against tumors and also against intracellular pathogenic infections [ ] ; lentinan possesses antiviral activity due to an induction of interferon-γ production; enhances host resistance against infections with bacteria as well as fungi, parasites, and viruses, including the agent of aids, and reduces the toxicity of azt [ ] ; a polyphenol rich extract (cystus ) from the mediterranean plant cistus incanus exerts a potent anti-influenza virus activity in a or mdck cell cultures infected with prototype avian and human influenza strains of different subtypes [ ] . because the purpose of the sars study was to demonstrate an approach, and not necessarily to be comprehensive, a number of shortcuts were taken. not all possible semantic categories for potential discoveries were identified, only the most obvious. relatively few terms were selected for the queries; many more were available. not all retrieved records were examined; only enough to demonstrate the quality of results. the potential expansion to indirectly related literatures using both text linking and citation linking described previously was not done. thus, the results obtained should be viewed as the tip of a very large iceberg. in previous medical applications of lrd, the discovery approach was to cluster the disease literature into groups of disease characteristics, generate combinatorials of intra-group characteristics (essentially synonyms), construct a discovery query from these combinatorials, and apply the query to non-drug substances/behaviors. in the present application, a somewhat different approach was taken. since the characteristics of those who had succumbed to sars tended to be sub-optimal performance of different immune system components, the query combined the immune system components at a higher aggregation level with functional terminology designed to improve this performance (e.g., enhance humoral immunity). such a query structure allowed a large number of potential discovery candidates to be identified. as the previous paragraph shows, much more is possible in terms of potential discovery volume. the picture from the handful of potential discoveries reported in this paper (and the hundreds of additional potential discoveries possible with a properly resourced study) is a synergy of lifestyle/dietary practices that could be interpreted as anti-sars. along with non-discovery items such as aurintricarboxylic acid (ata), emodin (an anthraquinone compound derived from genus rheum and polygonum), cimicifuga rhizoma, meliae cortex, coptidis rhizoma, phellodendron cortex, sophora subprostrata radix, betulinic acid, savinin, abietane-type diterpenoids and lignoids, quercetin- -beta-galacto side, d.alpha,beta-unsaturated peptidomimetics, anilides, metal-conjugated compounds, boronic acids, quinolinecarboxylate derivatives, thiophenecarboxylates, phthalhydrazide-substituted ketoglutamine analogs, isatin, tannic acid, and -isotheaflavin- -gallate (tf b) are potential discovery items such as ganoderma lucidum, jacalin, sulforaphane, methanolic extract of m. koenigii leaves, tinospora cordifolia, fucoidan, atractylodes macrocephala koidz, wasabia japonica, seeds of sorghum bicolor l, pidotimod and red ginseng acidic polysaccharide (rgap), myrica rubra leaf ethanol extract, l. paracasei ncc , methanol extract of asarum sieboldii, caffeoyl glycoside, and adjuvants korean mistletoe lectin c, cochinchina momordica seed, ag b, probiotic bacillus cereus var. toyoi, kefir, lentinan, polyphenol rich extract (cystus ) from the mediterranean plant cistus incanus. as stated above, more laboratory tests and field trials would have to be done on all these items to insure that they are anti-sars and safe, but these preliminary literature-based results offer some promise of what is possible. there is a major disconnect between the absence of therapies presently or potentially available on all the major medical web sites (and in sars mainstream journal review papers), and the potential therapies suggested by what has already been demonstrated in the core sars literature, much less what this study has generated from the related literatures. few medical web sites even mention any of the approaches shown in the sars core results section. the core literature and related literature potential discoveries and innovations have the potential to evolve into mainline medical treatments. literature-related discovery literature-related discovery (lrd): potential treatments for parkinson's disease literature-related discovery (lrd): potential treatments for multiple sclerosis literature-related discovery (lrd): introduction and background literature-related discovery (lrd): methodology the highly cited sars research literature evolution of genomes, host shifts and the geographic spread of sars-cov and related coronaviruses towards our understanding of sars-cov, an emerging and devastating but quickly conquered virus the outbreak pattern of sars cases in china as revealed by a mathematical model pathology and pathogenesis of severe acute respiratory syndrome interferon and cytokine responses to sars-coronavirus infection pathogenetic mechanisms of severe acute respiratory syndrome analysis of serum cytokines in patients with severe acute respiratory syndrome pathogenesis of severe acute respiratory syndrome clinical features, pathogenesis and immunobiology of severe acute respiratory syndrome sars coronavirus and innate immunity interferon-mediated immunopathological events are associated with atypical innate and adaptive immune responses in patients with severe acute respiratory syndrome human immunopathogenesis of severe acute respiratory syndrome (sars) mechanisms of severe acute respiratory syndrome pathogenesis and innate immunomodulation, microbiol characterization and inhibition of sars-coronavirus main protease t cell responses to whole sars coronavirus in humans severe acute respiratory syndrome coronavirus as an agent of emerging and reemerging infection molecular pathogenesis of severe acute respiratory syndrome, microbes infect adjacency and proximity searching in the science citation index and google aurintricarboxylic acid inhibits the early stage of vaccinia virus replication by targeting both cellular and viral factors emodin blocks the sars coronavirus spike protein and angiotensin-converting enzyme interaction in vitro inhibition of coronavirus replications by the traditionally used medicinal herbal extracts, cimicifuga rhizoma, meliae cortex, coptidis rhizoma, and phellodendron cortex specific plant terpenoids and lignoids possess potent antiviral activities against severe acute respiratory syndrome coronavirus binding interaction of quereetin- -beta-galactoside and its synthetic derivatives with sars-cov cl(pro): structure-activity relationship studies reveal salient pharmacophore features characterization and inhibition of sars-coronavirus main protease inhibition of sars-cov c-like protease activity by theaflavin- , '-digallate (tf ), evid. based complement effects of fermentation products of ganoderma lucidum on growth performance and immunocompetence in weanling pigs glycosylation-dependent interaction of jacalin with cd induces t lymphocyte activation and th /th cytokine secretion modulation of cell-mediated immune response in b f- melanoma-induced metastatic tumor-bearing c bl/ mice by sulforaphane immunomodulatory activity of methanolic extract of murraya koenigii (l) spreng. leaves enhanced phagocytosis and antibody production by tinospora cordifoliaa new dimension in immunomodulation defensive effects of a fucoidan from brown alga undaria pinnatifida against herpes simplex virus infection stimulating effects on mouse splenocytes of glycoproteins from the herbal medicine atractylodes macrocephala koidz anti-influenza virus activity of extract of japanese wasabi leaves discarded in summer antiviral activity and mode of action of a peptide isolated from sorghum bicolor synergistic immunostimulatory effect of pidotimod and red ginseng acidic polysaccharide on humoral immunity of immunosuppressed mice anti-influenza virus activity of myrica rubra leaf ethanol extract evaluated using madino-darby canine kidney (mdck) cells effect of lactobacillus paracasei ncc on antigenspecific t-cell mediated immune responses in aged mice screening of a natural feed additive having anti-viral activity against influenza a/h n immunopotentiation of caffeoyl glycoside from picrorhiza scrophulariiflora on activation and cytokines secretion of immunocyte in vitro intranasal immunization with influenza virus and korean mistletoe lectin c (kml-c) induces heterosubtypic immunity in mice improvement of the efficacy of influenza vaccination (h n ) in chicken by using extract of cochinchina momordica seed (ecms) innovation of vaccine adjuvants bacillus cereus var. toyoi enhanced systemic immune response in piglets effects of kefir fractions on innate immunity nutraceuticals: a piece of history, present status and outlook a polyphenol rich plant extract, cystus , exerts anti influenza virus activity in cell culture without toxic side effects or the tendency to induce viral resistance literature-related discovery: potential treatments and preventatives for sars, dtic technical report number ada , defense technical information center innovation forecasting: a case study of the management of engineering and technology literature evaluating innovation networks in emerging technologies mining ideas from textual information a compared r&d-based and patent-based cross impact analysis for identifying relationships between technologies quantitative mapping of scientific research-the case of electrical conducting polymer nanocomposite sars: systematic review of treatment effects he conducted research at bell labs and mitre corp, and managed programs at department of energy and office of naval research. he is presently a research affiliate with the school of public policy, ga tech, where he focuses on textual data mining. he is listed in who's who in america, who's who in science and engineering the research described in this paper was supported by mitre corporation internal research funds. key: cord- -eiobmxp authors: zhao, shan; li, wentao; schuurman, nancy; van kuppeveld, frank; bosch, berend-jan; egberink, herman title: serological screening for coronavirus infections in cats date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: eiobmxp coronaviruses (covs) are widespread among mammals and birds and known for their potential for cross-species transmission. in cats, infections with feline coronaviruses (fcovs) are common. several non-feline coronaviruses have been reported to infect feline cells as well as cats after experimental infection, supported by their ability to engage the feline receptor ortholog for cell entry. however, whether cats might become naturally infected with covs of other species is unknown. we analyzed coronavirus infections in cats by serological monitoring. in total cat serum samples and fcov type or type -specific antisera were screened for the presence of antibodies against the s receptor binding subunit of the cov spike protein, which is immunogenic and possesses low amino acid sequence identity among coronavirus species. seventy-eight sera were positive for antibodies that recognized one or more coronavirus s s whereas serum exclusively reacted with human coronavirus e (hcov- e) and two sera exclusively reacted with porcine delta coronavirus (pdcov). we observed antigenic cross-reactivity between s s of type and type fcovs, and between fcov type and porcine epidemic diarrhea virus (pedv). domain mapping of antibody epitopes indicated the presence of conserved epitope(s) particularly in the cd domains of s . the cross-reactivity of fcov type and pedv was also observed at the level of virus neutralization. to conclude, we provide the first evidence of antigenic cross-reactivity among s proteins of coronaviruses, which should be considered in the development of serological diagnoses. in addition, the potential role of cats in cross-species transmission of coronaviruses cannot be excluded. coronaviruses (covs) are enveloped viruses with a positive-stranded rna genome and classified into four genera (alpha-, beta-, gamma-and deltacoronavirus) within the subfamily orthocoronavirinae in the family coronaviridae of the order nidovirales. covs are found in a variety of mammals and birds, in which they can cause respiratory, enteric and systemic infections [ ] [ ] [ ] . additionally, covs have proven ability for cross-species transmission, exemplified by the emergence of severe acute respiratory syndrome (sars) coronavirus in / , and of the middle-east respiratory syndrome (mers) coronavirus in [ ] . both viruses belong to the betacoronavirus genus and have an animal origin. sars coronavirus crossed over from bats via intermediate hosts to humans, became human-adapted and quickly spread worldwide before its containment. mers coronavirus recurrently enters the human population via its dromedary camel reservoir host, with limited, non-sustained human-to-human transmission particularly in healthcare settings [ ] [ ] [ ] . apart from sars-and mers-cov, all four globally endemic human covs (hcov-oc , hcov-nl , hcov- e and hcov-hku ) originate viruses , , of from animals [ ] [ ] [ ] [ ] . in addition, cross-species transmission potential of covs is also illustrated by the occurrence of chimeric coronaviruses that resulted from recombination events between feline covs (fcov) and canine covs (ccov) [ , ] . in order to get insight into the frequency of interspecies transmission of coronaviruses within and between animal and human populations and the risk of subsequent development of a pandemic, it is useful to screen for coronavirus infections in animal species; especially those that are in close contact with humans. serological assays that can detect virus-specific antibody responses against infection play an important role in these epidemiological studies [ ] . cats live in close contact with humans and often roam around freely in the environment. hence cats are an interesting species to study for infections with coronaviruses. infections with feline coronaviruses (fcovs) are recognized and widespread [ , ] . fcovs are classified into two types, type and type , based on the genetic and antigenic difference of their spike (s) protein [ ] . in the field, the majority of fcov infections are caused by fcov type , while fcov type , derived from recombination events of type fcovs and ccovs obtaining the s gene and some flanking regions of ccovs, is less prevalent [ , ] . depending on the virulence of the fcov strain and the immune response of the cat, the clinical presentation can range from apparently asymptomatic, through diarrhea, to full-blown feline infectious peritonitis [ ] . fcovs are members of the genus alphacoronavirus, to which also hcov- e, porcine transmissible gastroenteritis virus (tgev), and ccov belong. the latter three viruses and fcov type have been proven to use feline aminopeptidase n (fapn) as a functional receptor in vitro [ ] . the receptor for type fcov has still not been identified [ ] . notably, previous studies have shown that hcov- e and ccov could infect cats after experimental inoculation, causing an asymptomatic infection [ , ] . thus, cats might potentially become naturally infected with covs of other species which may lead to virus-host adaptation e.g., mutation or recombination, resulting in emergence of novel coronaviruses and potentially new diseases [ , ] . the extent to which infections with covs of other species occur in the field, has not been explored in previous epidemiological studies of cov infections in cats [ , [ ] [ ] [ ] . being the main envelope protein of coronaviruses, the spike (s) protein mediates cell attachment and membrane fusion to allow viral entry. s functions as the main determinant of cell-, organ-and host-tropism. additionally, it is also the major target of neutralizing antibodies. spike comprises two functionally interdependent subunits, s and s , with s responsible for receptor binding and s for membrane fusion [ , ] . the s subunit is the least conserved and the most variable immunogenic antigen between coronavirus species [ ] . therefore, the s subunit is well suited as an antigen to screen for coronavirus type specific antibodies [ ] . in this study, covs infection in cats were detected through profiling antibody presence in serum samples from cats. recombinant cov spike s subunits of different animal and human covs were expressed in a mammalian expression system and used for screening of cat sera for the presence of antibodies against the respective proteins. positive samples were also tested by virus neutralization assays to support the specificity of the reaction [ ] [ ] [ ] . this investigation intends to extend our knowledge of cov epidemiology, potential reservoirs, and cross-species transmission. specific fcov type and fcov type sera were obtained from specific pathogen free (spf) cats previously infected with strain uu or rm and fipv- respectively [ , ] . in addition, for the serological survey, feline sera were retrieved from the serum bank in our lab. these had all been collected from cats in the netherlands. most of the samples (> %) were from a study on antibody titer testing for feline panleukopenia virus. the other samples were send to our lab for fip or felv-fiv diagnostics. sera of uninfected spf cats were included as negative controls. all samples were stored at − • c until analysis. african green monkey kidney cells (vero-ccl ), human hepatoma cells (huh ), pig kidney epithelial cells (llc-pk ), human embryonic kidney cells stably expressing the sv large t antigen (hek- t) were maintained in dulbecco modified eagle medium (dmem, lonza, basel, switzerland) supplemented with % fetal bovine serum (fbs, bodinco, alkmaar, the netherlands). virus strains used in this study have been described previously [ ] [ ] [ ] . briefly, recombinant porcine epidemic diarrhea virus (pedv) (rpedv-s dr -gfp) was propagated and titrated in vero cells, and hcov- e in huh cells. pdcov was propagated and titrated in llc-pk cells, but supplemented with µg/ml tpck-treated trypsin (sigma-aldrich, inc., st louis, mo, usa) in dmem. synthetic sequences of coronavirus spike s subunits (hcov-hku (gb: yp_ . ), mers-cov (gb:yp_ . ), sars-cov (gb: aax . ), hcov-oc (gb: aar . ), hcov- e (gb: np_ . ), hcov-nl (gb: yp_ . ), tgev (gb: abg . ), pedv (gb: aog . ), bcov (gb: p . ), pdcov (gb: aml . ), fcov type (gb: fj . ), fcov type (gb: ay . )) and different domains of pedv s subunit (s and s a-d , as identified and described in [ ] ) were cloned into pcaggs expression plasmids as described previously [ ] . similarly, the expression constructs encoding chimeric proteins in which s s were fused to the fc domain of mouse igg a. for protein production, hek- t cells were transfected with plasmid dna conjugated to polyethyleneimine (polysciences, inc., warrington, pa, usa). at - h post transfection, inoculum was removed and the transfection mixture was replaced by sfm ii expression medium (gibco®, life technologies inc., grand island, ny, usa). at - days post transfection, cell supernatants were harvested and proteins were collected by protein a sepharose beads (ge healthcare bio-sciences ab, uppsala, sweden). proteins were then eluted with . m citric acid, ph . and neutralized with m tris-hcl, ph . . concentrations of proteins were assessed by nanodrop spectrophotometry (thermofisher scientific inc., waltham, ma, usa) and confirmed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (sds-page) with bovine serum albumin (bsa, bioivt, west sussex, uk) as standard. typical yields for proteins were . - . mg/ml. for long term storage, proteins were stored at − • c upon usage. to study the potential cross-reaction between fcov type and in more detail, models of fcov type (strain: uu ; genbank accession no.: fj . ) and fcov type (strain: - ; genbank accession no.: ay . ) s proteins were generated via the automated protein structure swiss-model homology modelling server (https://swissmodel.expasy.org/) [ ] using the elucidated hcov-nl cryo-em structure (pdb code: szs) as the input model. figures were made with pymol (the pymol molecular graphics system, version . schrödinger, llc.). fcov s domains of both type and , namely s -cd , were expressed as murine fc fusion proteins in hek- t cells as described above. high binding microtiter plates (greiner bio-one bv, alphen aan den rijn, the netherlands) were coated overnight at • c with equal molar amount of protein ( . pmol per well, diluted in phosphate buffered saline (pbs, ph . )). after three washes with washing buffer (pbs containing . % tween- ), the plates were blocked for h at • c with blocking buffer (pbs containing % milk powder (protifar, nutricia, zoetermeer, the netherlands), . % tween- ). protein coating efficiency was assessed by binding of anti-mouse igg antibodies in a direct elisa, and confirmed the equimolar coatings of all proteins. to detect antigenic reaction with serum samples, sera were tested in duplicate at a : dilution in blocking buffer, and then incubated in the plates at • c for h. after washing, plates were incubated with a : diluted horseradish peroxidase (hrp)-conjugated goat anti cat igg (rockland immunochemicals, inc., pottstown, pa, usa) at • c for h. the peroxidase reaction was then visualized via adding tmb super slow one component hrp microwell substrate (biofx®, surmodics ivd, inc., eden prairie, mn, usa) for min. reaction was stopped with . % sulfuric acid and optical densities (od) were measured at nm. negative sera (from uninfected spf cats) were included to determine the elisa cut-off values; sera with od values higher than -fold the od of negative sera were considered positive. all s proteins were coated on the same elisa plates making it easy to screen and compare the od values of individual sera in one assay. hereby we excluded the sera that give high background od values against all proteins being considered false positive. neutralization assays were performed with some of the covs to support the specificity of elisa results. cat sera were serially diluted -fold in dmem and mixed : with rpedv-s dr -gfp, hcov- e or pdcov ( % tissue culture infective doses [tcid ]/ml). these mixtures were then incubated at • c for h, and µl of each mixture was used for inoculation with vero, huh and llc-pk cell monolayers in -well plates, respectively. for pdcov infection, tpck-treated trypsin (sigma-aldrich, inc., st louis, mo, usa) was supplied to llc-pk tissue culture medium at a final concentration of µg/ml. at - days post infection, cytopathic effect (cpe) could be observed via microscopy. virus neutralization titers (vnt) were expressed as the highest serum dilution resulting in % reduction of cytopathic effect (hcov- e and pdcov) or virus-induced fluorescent cells (pedv). before virus neutralization, sera were inactivated through incubation at • c for min. experiments were performed in triplicate. feline sera (n = ) were screened by indirect elisa for antibody reactivity against cov s antigens. the od values against these antigens are shown in figure . in total, of the sera ( . %) contained anti-cov antibodies, while sera showed reactivity against more than one cov s antigen. none of the samples had to be discarded because of reactivity against all of the proteins indicating a potential false positive result. the frequency of different combinations of cov-s reactive samples is summarized in table . reactivity against eight out of cov s antigens could be observed, whereas none of the sera recognized the s protein of hcov-hku , mers-cov, sars-cov and hcov-oc . different s antigens were grouped by amino acid sequence phylogeny (left panel) using mega . each column of the heat map represents an individual sample, and columns were arranged in a descending order based on elisa-reactivity against feline coronavirus (fcov) type . table . numbers of positive cat samples and different combinations of reactivity found. the number of positive sera against each individual s is shown in the bottom row. positive elisa reactions are colored in orange. cut-off value was determined as the -fold over the od of negative sera. fcov type -s fcov type -s pdcov-s tgev-s e-s bcov-s different s antigens were grouped by amino acid sequence phylogeny (left panel) using mega . each column of the heat map represents an individual sample, and columns were arranged in a descending order based on elisa-reactivity against feline coronavirus (fcov) type . table . numbers of positive cat samples and different combinations of reactivity found. the number of positive sera against each individual s is shown in the bottom row. positive elisa reactions are colored in orange. cut-off value was determined as the -fold over the od of negative sera. number of cats (total = ) fcov type -s fcov type -s pedv-s pdcov-s tgev-s e-s nl -s bcov-s as expected, many sera were positive for fcov s , with sera ( . %) positive for fcov type and sera ( . %) for fcov type s ( figure ). all of the fcov type s positive sera also tested positive for fcov type s , while of fcov type s positive sera also reacted with tgev s . the fcov type s and tgev s elisa reactivities showed a strong nonparametric spearman correlation (spearman r = . , p < . ). with respect to this, we suggest that the tgev s positivity was due to cross-reactivity of fcov type s , as fcov type shows close antigenic and genetic relationship with tgev (s shares . % amino acid sequence identity). the remaining fcov type s positive but tgev s negative sera do react with fcov type s . an explanation might be the cross-reactivity between fcov type and type . remarkably, feline sera were reactive with s proteins from human, porcine and bovine covs (table ) , including hcov- e ( / ), hcov-nl ( / ), pedv ( / ), pdcov ( / ) and bcov ( / ). od values of feline sera positive for hcov-nl s and bcov s were relatively low ( figure a ). elisa reactivity towards non-feline cov s proteins might be explained by infection with the respective or related covs or by the presence of cross-reacting antibodies, although there was low sequence identity (< . %) between s proteins of fcov type and related non-feline coronaviruses (for the complete comparison of s sequence identities, see table ). yet, all of pedv-s positive sera were also positive for fcov type s ( figure b , table ). the elisa results of fcov type s and pedv s showed a strong nonparametric spearman correlation (spearman r = . , p < . ). thus, this might indicate the occurrence of antibody cross-reactivity against fcov type and pedv s antigens. many of the hcov- e and pdcov s positive sera also reacted with fcov type s , but no strong nonparametric spearman correlation was observed (hcov- e, r = . ; pdcov, r = . ). one feline serum only reacted with hcov- e viruses , , of s , and two feline sera only recognized pdcov s . ( figure b , table ). this observation led us to hypothesize that cross-reactivity may not play a role in elisa reactivity of these three sera, but that the three cats had been infected with these viruses or related viruses. fcov type -s (fj . ) fcov type -s (ay . ) . pedv-s (aog . ) . . pdcov-s (aml . in our screening, samples were shown to be positive for two or more s proteins including fcov type . the data prompted us to test different hypotheses which may explain this phenomenon: specific reaction through natural virus infection or reaction due to cross-reactivity with fcov-s antigens. to explore this further, we employed fcov type specific sera derived from specific pathogen free (spf) cats that had been experimentally infected with fcov type i strain rm (n = ) or strain uu (n = ). these sera were tested for their elisa reactivity against seven cov s proteins (excluding tgev s ) that showed positive reactivity in the previous serological screening. as expected, all sera were positive for fcov type s in our elisa; interestingly, four samples also reacted with fcov type s , and five samples with pedv s . no positive elisa-reactivity was detected with s of hcov- e, pdcov, hcov-nl or bcov (table s ). thus, fcov type infection could lead to the generation of antibodies that cross-react in the s -elisa with fcov type and pedv s proteins. the elisa cross-reactivity of fcov type specific sera with fcov type s antigens prompted us to map the domains responsible for cross-reaction within the s subunit. hence, to identify domain borders within s , we built homology-based models of both fcov type and type spike using the related elucidated hcov-nl cryo-em structure as the template model. as shown in figure a , continuous structural domains can be identified for the s subunit of both spikes, namely s , and s a through s d . amino acid sequence identities of these domains between fcov type and type differ, ranging from . % to . % ( figure b ). several s proteins for both type and type fcov-s comprising one or two domains were expressed and purified ( figure c ). fcov type specific sera (n = for strain rm and n = for strain uu ) and type (n = , strain - ) specific sera were then tested against these proteins in elisa format. the four fcov type specific sera that cross-reacted with s of fcov type again showed binding to fcov type s . but the fcov type specific sera showed little to no reactivity against fcov type s . as shown in figure , the type specific antisera reacted with all of the homologous s domains, with s and s b of both type and type displaying the strongest reaction. interestingly, the cd domain showed the highest level of cross-reactivity between fcov type and , in agreement with its highest sequence identity among s domains (figure ) . the other three domains showed little to no cross-reactivity. antisera reacted with all of the homologous s domains, with s and s b of both type and type displaying the strongest reaction. interestingly, the cd domain showed the highest level of crossreactivity between fcov type and , in agreement with its highest sequence identity among s domains ( figure ). the other three domains showed little to no cross-reactivity. the s subunit of one protomer are colored, with s shown in cyan, s a in blue, s b in green, and the domains s cd in red. the s part of the protomer is marked in light gray. (b) schematic presentation of the fcov type (strain uu ) and type (stain - ) s protein with the signal peptide (sp), the s subunit (the domains are colored as described in the legend of figure a ) and the s subunit (the c-terminal transmembrane domain is indicated by a black box). amino acid sequence identities between fcov type and type s domains are indicated. (c) diagram of the different s subdomains sequence. all s subdomains were c-terminally tagged with the fc part of mouse igg a (not shown in the figure) and expressed as fc fusion proteins. the s subunit of one protomer are colored, with s shown in cyan, s a in blue, s b in green, and the domains s cd in red. the s part of the protomer is marked in light gray. (b) schematic presentation of the fcov type (strain uu ) and type (stain - ) s protein with the signal peptide (sp), the s subunit (the domains are colored as described in the legend of figure a ) and the s subunit (the c-terminal transmembrane domain is indicated by a black box). amino acid sequence identities between fcov type and type s domains are indicated. (c) diagram of the different s subdomains sequence. all s subdomains were c-terminally tagged with the fc part of mouse igg a (not shown in the figure) and expressed as fc fusion proteins. viruses , , x for peer review of figure . elisa-reactivity of fcov specific antisera against different s subdomains of fcov type and . equimolar amount of purified s proteins and the four s subdomains were coated onto well plates and antibody binding was determined by elisa. the fcov type and specific antisera used in the screening were derived from experimentally infected specific pathogen free (spf) cats and are indicated at the right side of each panel, absorbance values and antigens in use are shown on the y-and x-axis, respectively. graphs represent the mean values from three independently performed experiments. standard deviations are indicated as error bars. because the fcov type specific cat sera also showed elisa reactivity with pedv-s (table s ), we analyzed the reaction of the five pedv-s positive cats in more detail. samples were analyzed via elisa using antigens comprising different pedv-s domains, as described in our previous study [ ] . cat sera taken pre-and post-fcov infection were collected and tested. as indicated in figure , all five cats had developed pedv-s reactivity to different extent after fcov type inoculation. noticeably, all sera showed the highest od values with the cd domain, while the other domains, including the s b containing the presumed receptor binding domain (rbd) [ ] , were non-reactive ( figure ). on the other hand, the swine pedv positive control serum exhibits strong reactivity against all pedv-s domains. the next question we asked was whether fcov type specific sera could neutralize pedv infection in tissue culture, as they showed no reactivity with the s b of pedv spike. as shown in figure , pedv neutralizing antibodies were detected in three out of five fcov type i specific cat sera. figure . elisa-reactivity of fcov specific antisera against different s subdomains of fcov type and . equimolar amount of purified s proteins and the four s subdomains were coated onto -well plates and antibody binding was determined by elisa. the fcov type and specific antisera used in the screening were derived from experimentally infected specific pathogen free (spf) cats and are indicated at the right side of each panel, absorbance values and antigens in use are shown on the y-and x-axis, respectively. graphs represent the mean values from three independently performed experiments. standard deviations are indicated as error bars. because the fcov type specific cat sera also showed elisa reactivity with pedv-s (table s ) , we analyzed the reaction of the five pedv-s positive cats in more detail. samples were analyzed via elisa using antigens comprising different pedv-s domains, as described in our previous study [ ] . cat sera taken pre-and post-fcov infection were collected and tested. as indicated in figure , all five cats had developed pedv-s reactivity to different extent after fcov type inoculation. noticeably, all sera showed the highest od values with the cd domain, while the other domains, including the s b containing the presumed receptor binding domain (rbd) [ ] , were non-reactive (figure ). on the other hand, the swine pedv positive control serum exhibits strong reactivity against all pedv-s domains. the next question we asked was whether fcov type specific sera could neutralize pedv infection in tissue culture, as they showed no reactivity with the s b of pedv spike. as shown in figure , pedv neutralizing antibodies were detected in three out of five fcov type i specific cat sera. the experiment was carried out in duplicate and repeated three times. error bars indicate standard deviations. sera were collected from spf cats prior (cat - p) and after (cat - ) experimentally inoculated with fcov type . positive serum: pedv positive swine serum collected from the field; negative serum: serum from fcov negative spf cat. several serum samples from field cats, but not virus-specific serum samples from fcov inoculated spf cats, were found to be elisa positive for hcov- e (n = ) and pdcov s (n = ) ( figure a) . also, a few feline sera displayed unique elisa positivity for s of hcov- e (n = ) or pdcov (n = ) ( figure b, table ). this could indicate that these antibodies were induced upon infection with these specific viruses. to corroborate the possibility of a natural infection in these cats with hcov- e or hcov- e-like viruses, we tested sera neutralization antibody titers. the results showed that one of the hcov- e s reactive feline sera was able to neutralize hcov- e infection (vnt = ); no neutralization of pdcov was detected for the all pdcov-s positive sera. coronavirus infections are endemic and ubiquitous in feline populations. two viral types, type and , are distinguished and both of them could well sustain themselves in the cat reservoir [ , ] . both have been shown to have worldwide distribution, with the seropositivity rate up to % among animal shelter populations and in multi-cat households [ , ] . the majority of natural infections are caused by type fcovs, while in the field type fcovs are less common and mainly occur in asia [ , , [ ] [ ] [ ] . covs are generally considered to be host-specific; however, cross-species transmission does occur which may lead to incidental infections like the spillover of mers-cov from dromedary camel to humans, where humans function as an incidental and ultimately dead-end host [ ] . but covs might also adapt to the new host exemplified by the animal origin of all four endemic human covs (hcov-oc , hcov-nl , hcov- e and hcov-hku ) [ ] [ ] [ ] [ ] . whereas in cats infections with fcov are well recognized, studies regarding possible natural infections with other animal and human coronaviruses are lacking to the best of our knowledge. knowing the genetic variability of coronaviruses and the use of orthologous receptors by non-feline covs, studies on cross-species transmission are desirable. this may provide insight regarding whether cross-species transmission does occur. in the present study we used the highly immunogenic s antigens to screen cat sera for the presence of antibodies against feline and non-feline coronaviruses, as a first indication of possible infections with these viruses. in our study, of the cat sera were shown to be seropositive for coronaviruses. the seropositive rate ( . %) against s of fcov type is consistent with previous studies [ , ] . all of the fcov type s positive sera of naturally infected cats were also positive for fcov type s , which might be the result of cross reaction between the two proteins, despite their low amino acid identity. elisa with specific antisera from experimentally fcov type and type infected cats showed that sera of several fcov type infected cats could cross-react with fcov type s . domain mapping elisa results showed that fcov type specific sera react to different levels with the s domains of fcov type s protein, and also reacts with fcov type s cd . vice versa, fcov type specific sera also reacted with s cd of fcov type . these observations pose a potential two-way cross-reactivity between s cd domains. interestingly, in parallel with our findings on feline coronaviruses, we identified a number of samples that were seropositive against the s of pedv, a viral pathogen that mainly replicates in the porcine intestinal epithelium. to study the possibility of cross-reaction, samples derived from preand post-fcov infected cats were screened against pedv s in elisa. the reactivity found against pedv s with fcov specific sera shows that cross-reaction can occur at the level of domain s cd ; the other pedv s domains showed no reaction with the fcov positive sera. judging from these observations, it seems that s cd plays an important role in cross-reaction between fcov type and , and also fcov and pedv. as s cd is the most conserved domain among fcov and also between fcov and other alphacoronaviruses (for a systematic assessment of sequence identities, see table ), it is reasonable to hypothesize that antibodies can develop against conserved epitopes within this region and subsequently cause cross-reaction. this should be taken into account when developing and interpreting serological assays. table . identities of amino acid sequences of fcov type (strain: uu ) s and s domains compared with the amino acid sequences of other alphacoronaviruses. (identities are shown in %; na: not available) the genbank accession numbers of these viruses are as follows: fcov type (uu ), fj . ; fcov type (rm), fj . ; fcov type , ay . ; tgev, abg . ; pedv, aog . ; hcov- e, np_ . ; hcov-nl , yp_ . . amino acid % identity to fcov type (uu ) s s s a s b s cd noticeably, elisa reactivity among cat sera towards the n-terminal fcov s domains and a was less consistent and generally lower compared to whole s , which seems to correlate with the higher antigenic variation in those domains found among fcov type strains [ ] (figure ). especially the sera from fcov-rm infected cats (cat , , and ) showed lower od values against s a . this phenomenon could be explained as the samples displaying higher reactivity were from cats inoculated with fcov-uu (cat , and ), the particular strain from which the s region was used as an antigen in the elisa studies. in the meantime, the possibility of the variable elisa reactivity might be due to the difference in individual antibody levels. in principle, the distinct antigenic reactivity of s and s a between the two fcov types might facilitate the development of a specific elisa method which allows the serological discrimination of fcov type and type infections in cats. in order to provide further insight regarding cross-reactivity between fcov type and pedv, we performed virus neutralization assays. cross-neutralization of pedv infection could be observed for some of the feline fcov type post-infection sera, in contrast to the pre-infection serum counterparts. since fcov specific pedv neutralizing sera did not react with pedv s , s a or s b , it is likely that the cross-neutralizing antibodies are targeting conserved epitopes in the s cd domain or the s subunit of the pedv spike protein [ ] . given the unknown tgev infection background of the pedv positive pigs, the cross-reaction of pedv specific sera against fcov type could not be explored in our study, as tgev positive pig samples would certainly influence the outcome [ , , ] . of note, our findings cannot exclude the possibility that field cats might incidentally get naturally infected with pedv or pedv-like viruses, as there had been one report showing the detection of pedv in one stray cat via pcr assay [ ] . it would be interesting to include more sera of cats from pig farms in future studies. considering the fact that cats play an important role in human society and have constant interaction with humans, it is of interest to conduct serological surveys for possible reverse zoonosis of human pathogens. in our study s antigens of several human coronaviruses were included and this led us to identify hcov- e seropositive feline samples in our elisa survey (table ) ; one serum in particular reacted solely with hcov- e s but not with any other coronavirus. of the hcov- e s reactive feline sera one showed low neutralizing activity against hcov- e infection. this might suggest that positive cats were indeed exposed to hcov- e or related viruses. mers seropositivity is also seen in other species besides the dromedary host [ ] . rare cases of seropositivity might be considered as spill-over infections from the dromedary camel reservoir. similar (perhaps dead-end) spill-over infections of e from the human reservoir to cats might also occur. a similar principle could also apply for pdcov, a porcine pathogen that emerged rather recently. both hcov- e and pdcov use apn as their receptor and have been reported to also be able to use feline apn for cellular entry [ , ] . although reports are lacking regarding the natural infection of these two viruses in cats, hcov- e was shown to cause a priming effect of fcov antibody in experimentally fcov infected cats suggesting that infection occurred [ ] . therefore, the detection of antibodies against s of hcov- e in a portion of the cats might be specific and due to the exposure to hcov- e through daily interaction with humans. eight cats were seropositive for pdcov of which two cats were seropositive only for pdcov and not for any other covs. this could be caused by infection with pdcov or pdcov-related viruses through avian sources, considering the fact that cats are natural avian predators and the presumed avian origin of pdcov [ , ] . our findings emphasize the potential role of cats as incidental hosts for non-feline coronaviruses and the need of in-depth study of naturally infected pathogens in cats. besides serological studies, efforts should also focus on isolation and identification of these viruses in cats. in conclusion, we presented a thorough serological survey in cats using s proteins of different animal and human coronaviruses. we demonstrated, despite the low amino acid identity, cross-reactivity between s proteins of fcov type and , and between that of fcov type and pedv. this should be considered when developing fcov serological assays as well as interpreting the results. our observation that some feline sera displayed 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smit s.; ponnapalli, anoosha; seedahmed, elfateh; berrou, mohammed; bachuwa, ghassan; chandran, arul title: a -year-old male with a fatal case of covid- acute respiratory distress syndrome (cards) and ventilator-induced lung injury (vili) date: - - journal: am j case rep doi: . /ajcr. sha: doc_id: cord_uid: bnjupaik patient: male, -year-old final diagnosis: acute respiratory distress syndrome (ards) • covid- •multi organ failure/septic shock • pneumothorax symptoms: cough • dyspnea • fatigue • myalgia medication:— clinical procedure: mechanical ventilation • thoracentesis specialty: critical care medicine objective: unknown ethiology background: covid- patients that develop acute respiratory distress syndrome (ards) “cards” behave differently compared to patients with classic forms of ards. recently cards phenotypes have been described, type l and type h. most patients stabilize at the milder form, type l, while an unknown subset progress to type h, resembling full-blown ards. if uncorrected, phenotypic conversion can induce a rapid downward spiral towards progressive lung injury, vasoplegia, and pulmonary shrinkage, risking ventilator-induced lung injury (vili) known as the “vili vortex”. no cases of in-hospital phenotypic conversion have been reported, while ventilation strategies in these patients differ from the lung-protective approaches seen in classic ards. case report: a -year old male was admitted with covid- pneumonia complicated by severe ards, multi-organ failure, cytokine release syndrome, and coagulopathy during his admission. he initially resembled cards type l case, although refractory hypoxemia, fevers, and a high viral burden prompted conversion to type h within days. despite ventilation strategies, neuromuscular blockade, inhalation therapy, and vitamin c, he remained asynchronous to the ventilator with volumes and pressures beyond accepted thresholds, eventually developing a fatal tension pneumothorax. conclusions: patients that convert to type h can quickly enter a spiral of hypoxemia, shunting, and dead-space ventilation towards full-blown ards. understanding its nuances is vital to interrupting phenotypic conversion and entry into vili vortex. tension pneumothorax represents a poor outcome in patients with cards. further research into monitoring lung dynamics, modifying ventilation strategies, and understanding response to various modes of ventilation in cards are required to mitigate these adverse outcomes. the cluster of pneumonia cases associated with the novel coronavirus (covid- ) or severe acute respiratory syndrome coronavirus (sars-cov- ) that emerged from wuhan, china and spread rapidly across continents was labeled by the world health organization (who) as a global pandemic. as of may , , . million cases of covid- were present in the united states (us), with roughly deaths [ ] . covid- pneumonia seems to behave differently from other viral types of pneumonia, with large swings in respiratory functioning, inferring that not all previous practices can be adopted, and new strategies are needed to mitigate the high mortality rates ( % to %) seen in advanced cases [ , ] . supplemental oxygen use was seen in . % of infected patients, with . % requiring mechanical ventilation and less than % requiring advanced therapies such as extracorporeal membrane oxygenation (ecmo); however, these numbers may likely be underrepresented with preventative measures such as social distancing and stay-at-home executive orders leading to reluctance and delay in receiving care [ ] . the progression of covid- to ards ("cards") represents a life-threatening sequela, with its ability to lower blood oxygenation levels and induce systemic hypoxemia and multi-organ failure [ , ] . despite cards meeting the berlin diagnostic criteria, its trajectory is characterized by severe hypoxemia with near-normal respiratory compliance, unlike its classic form [ , ] . patients with cards can present within a broad spectrum from perceived normal breathing ("silent hypoxemia") to floored respiratory compromise with a wide array of overlapping features in between [ ] . recently, cards phenotypes have been parsed out, type l and type h, with each one having its distinct pathophysiological pathway. understanding these nuances are vital to providing appropriate treatment and avoiding sub-optimal outcomes [ ] . we present a case of cards with subsequent sequelae and numerous challenges in management. we aim to strengthen the existing literature, explore the cards phenotypes, and discuss therapeutic and ventilator strategies to counteract the unique lung injury seen in covid- pneumonia that progress to ards. a -year-old male with a history of asthma, previous gunshot wound, and obesity, presented to the hospital with dyspnea, cough, fatigue, and myalgias. he used tobacco products and worked at an auto-parts manufacturing unit. on arrival, he was febrile, tachycardic, and tachypneic requiring supplemental oxygen. he appeared ill with a high work of breathing and a productive cough. workup revealed lymphopenia to . k/ul, thrombocytopenia to k/ul, and an unremarkable chest radiograph ( figure ); the patient was transferred to the intensive care unit (icu) with a high suspicion for covid- . testing for sars-cov- was completed using a nasopharyngeal swab transported in an m viral tube to the state department of health and human services. samples were tested on the sars-cov- real-time polymerase chain reaction (rt-pcr) abbott id now™ point-of-care system under the food and drug administration (fda) emergency use authorization (eua). computed tomography (ct) of the chest could not be performed due to concern for virus transmission and environmental contamination due to high demands and short downtime for decontamination. by day , the patient required higher flow rates on a non-rebreather mask, and by day , persistent fevers, tachypnea, and new consolidative changes in the right middle and lower lung zones were noted. he was empirically started on broad-spectrum antibiotics with hydroxychloroquine. foregoing bi-pap or c-pap due to concerns for aerosolization, he was placed on mechanical ventilation. his chest x-ray by day revealed extensive consolidative infiltrates bilaterally and a pao /fio (pf) ratio of consistent with severe acute respiratory distress syndrome (ards) (figure ). testing for sars-cov- came back positive, confirming covid- pneumonia. over the following days, he went into septic shock requiring vasopressor support, while previously sent cytokine labs, including an interleukin- (il- ) of pg/ml, were consistent with cytokine release syndrome. he was given a dose of tocilizumab mg. on day , extensive acute deep vein thromboses (dvts) was discovered in his left upper extremity with d-dimer levels over µg/ml. his previous prophylactic dose of enoxaparin was increased to a therapeutic dose. his fevers did not abate, requiring cooling, neuromuscular blockade, and deep sedation. ards strategies, including low tidal volumes, proning, recruitment maneuvers, diuretics, nitric oxide, and vitamin c, were used despite his rising pressures. during these periods, he exhibited high plateau pressures, often over cm of h o. worsening status prompted consideration of transfer to a specialized ecmo center. however, surrounding centers had limited the inflow of patients adhering to strict infection control measures, while judicious resource allocation and logistical challenges made transportation unfeasible. on day , after a sudden episode of desaturation, a chest x-ray revealed left-sided tension pneumothorax in the left mid and lower lung fields ( figure ) with a chest tube draining ml of serosanguineous fluid mixed with blood clots intermittently blocking output with persistent air leaks. fluid characteristics were not obtained, and his overall clinical trajectory began declining. a family discussion was held to discuss his poor prognosis and address the goals of care. his code status was changed to do-not-resuscitate (dnr) with an emphasis on comfort measures. he eventually desaturated and went into asystole, passing away after spending days in the hospital. trends in oxygenation in our patient can be seen in table . ards can be mitigated by opening collapsed alveoli with higher positive end-expiratory pressures (peeps), recruiting maneuvers, or proning. in contrast, these high pressures are poorly tolerated, leading to the use of low tidal volumes to minimize ventilator-induced lung injury (vili). despite this, high rates of barotrauma were reported from the previous sars epidemic [ ] . covid- patients complicated by ards ("cards") can present despite lacking traditional risk factors such as advancing age, pre-existing co-morbidities, or an advanced lung pathology [ ] . despite the initial insult being the inoculation of sars-cov- , cards can occur from injuries from either the gas or vascular side of the alveoli [ ] . approaching cards from the gas side cards is defined by phenotypes based on its clinical trajectory. "type l" has a relatively compensated clinical state while "type h" resembles full-blown ards ( table ). our patient, initially a type l, had scattered infiltrates with rising minute ventilation over days. type l patients are perceived to be breathing normally ("silent hypoxemia") and often respond to supplemental oxygen. at the same time, deep swings in respiration can induce patient self-inflicted lung injury (p-sili), triggering an inflammation cascade and a rapid downward spiral towards progressive pulmonary injury and pulmonary shrinkage, known as the 'vili vortex' towards full-blown ards [ ] . evidence of transformation to type h by day was noted by his rising plateau and driving pressures, lower static compliance, and low pf ratios signifying a bulkier lung. refractory hypoxia, fevers, and systemic insults led to a dependency on high peeps and fio to maintain oxygen saturation above %. post-mortem studies reveal numerous thromboses in covid- patients with d-dimer serving as a surrogate marker of pulmonary endothelial damage, promoting ventilation-perfusion mismatches and subsequent hypoxemia [ , , ] . dvts were noted in over % of patients, suggesting that coagulopathy may be an independent risk factor for poor prognosis [ ] . our patient reflected coagulopathy and vasoplegia with rising d-dimer levels in the days leading up to his dvt, while the development of cytokine release syndrome and coagulopathy signaled his growing disease burden. once this occurs, vasoregulation is altered due to the failure of hypoxic vasoconstriction from the endothelial damage resulting in significant hypoxemia. if the respiratory drive is not altered by oxygen administration, the generated inspiratory increases transpulmonary pressures across vascular channels, risking vili. this can also serve as a learning point, that early intubation strategies can help minimize the powerful respiratory effort leading to vasoplegia. if uncorrected, high plateau pressures reflecting high trans alveolar pressures can increase thoracic compliance leading to high oxygen and peep requirements that can redirect blood to damaged parts with high permeability affecting hemodynamics and contributing to type h conversion. the accepted thresholds for vili protection include a plateau pressure of cmh developed spontaneous pneumothorax with a % drop in his pf ratio because of his high plateau pressures, respiratory swings, and inflammation. the incidence of pneumothorax has been reported to be roughly % in covid- patients [ ] , although these represented spontaneous cases from the community, unlike our case which was a consequence of progressive lung injury and the inability to liberate the patient from the vili vortex. the development of pneumothorax after intubation can portend a poor prognosis in patients with cards [ ] [ ] [ ] . categorizing lung injury from covid- into cards type l or type h can help unwanted practices and initiate targeted ventilator approaches to correct the underlying mismatch. the surviving sepsis campaign recommended that mechanically ventilated covid- patients be managed similarly to other patients with respiratory failure in the icu, although with the growing body of evidence, cards is displaying a distinct course [ ] . the essential contributors to cards and entry into the vili vortex are ) sars-cov- burden, ) ventilator responsiveness, and ) time of symptom onset [ ] . the paucity of reported cases brings into light the importance of isolated reports in guiding therapy in the current climate. this case represents the first reported cards patient that developed a pneumothorax as a consequence of his phenotype conversion. in previous cases of sars patients, pneumothorax was noted at - days after the initial diagnosis [ ] , suggesting that a sustained period of lung inflammation serves as a pre-requisite, a similar time course as our patient recently a scoring system was proposed to predict the risk of developing critical illness in covid- , allowing early interventions and resource allocation to mitigate the high disease burden [ ] . covid- patients that develop ards ("cards") come in phenotypes: type l and h. type l is often stable while type h presents like full-blown ards. these patients require different ventilator strategies with the goal of avoiding conversion to type h and limiting vili. in these cases, pneumothorax may represent an indicator of a poor outcome. an interactive web-based dashboard to track covid- in real time respiratory support in novel coronavirus disease (covid- ) patients, with a focus on resource-limited settings respiratory conditions in coronavirus disease (covid- ): important considerations regarding novel treatment strategies to reduce mortality covid- : timing is important management of covid- respiratory distress covid- pneumonia: different respiratory treatments for different phenotypes? critically ill patients with severe acute respiratory syndrome clinical phenotypes of sars-cov- : implications for clinicians and researchers autopsy findings and venous thromboembolism in patients with covid- driving pressure and mechanical power: new targets for vili prevention a comparison of oesophageal and central venous pressures in the measurement of transpulmonary pressure change emergency tracheal intubation in patients with covid- in wuhan, china: lessons learnt and international expert recommendations sars-cov- infection associated with spontaneous pneumothorax covid- with spontaneous pneumothorax, pneumomediastinum and subcutaneous emphysema surviving sepsis campaign: guidelines on the management of critically ill adults with coronavirus disease (covid- ) severe acute respiratory syndrome complicated by spontaneous pneumothorax development and validation of a clinical risk score to predict the occurrence of critical illness in hospitalized patients with covid- none. key: cord- -gft phd authors: lawrence, c.; seckold, r.; smart, c.; king, b. r.; howley, p.; feltrin, r.; smith, t. a.; roy, r.; lopez, p. title: increased paediatric presentations of severe diabetic ketoacidosis in an australian tertiary centre during the covid‐ pandemic date: - - journal: diabet med doi: . /dme. sha: doc_id: cord_uid: gft phd aims: to determine if the frequency of severe diabetic ketoacidosis at presentation of new‐onset type diabetes to an australian tertiary centre increased during the initial period of restrictions resulting from the covid‐ pandemic (march to may ). methods: data were collected on presentations of newly diagnosed type diabetes as well as on all paediatric presentations to the emergency department of a tertiary centre between and . data from the period of initial covid restrictions in australia (march to may ) were compared to the period march to may of the previous years (pre‐pandemic periods). results: the number of new diagnoses of type diabetes was comparable in the pandemic period and pre‐pandemic periods ( in vs range – in – ). the frequency of severe diabetic ketoacidosis was significantly higher in the pandemic period compared to the pre‐pandemic periods ( % vs %; p < . ), odds ratio . ( % ci . , . ). the overall frequency of diabetic ketoacidosis was also significantly higher during the pandemic period ( % vs %; p < . ), odds ratio . ( % ci . , . ). none of the individuals tested positive for covid‐ . presentations of people aged < years to the emergency department decreased by % in the pandemic period compared to the average of the pre‐pandemic periods ( vs ; range to ). conclusions: a significant increase in the frequency of severe diabetic ketoacidosis at presentation of type diabetes was observed during the initial period of covid‐ restrictions. we hypothesize that concern about presenting to hospital during a pandemic led to a delay in diagnosis. these data have important implications for advocacy of seeking healthcare for non‐pandemic‐related conditions during a global pandemic. march . restrictions enacted in australia included school shutdowns and the closure of non-essential businesses, while the majority of outpatient health appointments were conducted by telehealth. these restrictions successfully flattened the curve with the expected surge of covid- cases and uncontrolled community spread not seen in australia during the time period described in this study. however, concern has emerged that, with fewer presentations to general practitioners and emergency departments , other serious health concerns may have gone undiagnosed. the prevalence of diabetic ketoacidosis (dka) at diagnosis of type diabetes in the paediatric population in australia was recently reported as . % . dka is an avoidable complication if symptoms and signs of diabetes mellitus are recognized early . dka places increased burden on the healthcare system and may require intensive care support in tertiary care settings for management. additionally the presence of dka at diagnosis has been associated with poor long-term glycaemic control . during a pandemic, it is important to minimize avoidable admissions to intensive care units when resources may be limited. emerging research during the covid- pandemic has shown an increase in children with type diabetes presenting with dka in countries such as italy and the usa . a recent study of the german population found the frequency of severe dka was significantly higher during the covid- pandemic, and that children under the age of years were at the highest risk . we report one australian centre's experience of presentations of newly diagnosed type diabetes in a paediatric cohort during the covid- pandemic restrictions (march to may inclusive) compared to pre-pandemic presentations in the march to may periods of the years to . all children and adolescents aged < years with the initial diagnosis of type diabetes treated at the john hunter children's hospital between january and may were identified from the hospital paediatric endocrine database and digital medical records. age, date of diagnosis, hba c , initial venous blood gas result (ph, bicarbonate and glucose), c-peptide, type diabetes-associated antibody status (presence of glutamic acid decarboxylase, islet antigen , islet cell and insulin antibodies) and the results of covid- tests were collected. in addition, the total number of presentations of children and young people aged < years to the john hunter hospital emergency department was collected for the march to may period in to . we defined data collected from march to may as the 'pandemic period' and the corresponding march to may periods in the years prior to this as 'pre-pandemic' conditions. diabetic ketoacidosis was defined as a venous ph < . on presentation, with severe dka represented by a ph < . as per the international society for paediatric and adolescent diabetes (ispad) guidelines . a diagnosis of type diabetes was defined as the presence of dka or the presence of hyperglycaemia and autoantibodies associated with type diabetes . odds ratios, fisher's exact test and anova were used to test for differences between the pandemic and pre-pandemic groups. ethics approval was granted by the hunter new england health research ethics committee (approval number: au - ). the age of onset, gender, total presentations and classification by severity of dka are presented in table and fig. for each of the march to may periods in to . data collection was complete, with no missing data. the numbers of new presentations of type diabetes in the march to may period across to were fairly consistent; the prepandemic number ranged from to (median ) across to , compared with in . there was no significant difference in age or gender across the groups. the frequency of severe dka at the time of new type diabetes diagnosis was significantly higher during the period of pandemic restrictions compared to pre-pandemic ( % vs %; p < . ), odds ratio of . ( % ci . , . ). the frequency of dka (mild, moderate or severe dka) was also significantly higher during the period of pandemic restrictions compared to pre-pandemic conditions ( % vs %; p • diabetic ketoacidosis is an avoidable complication of type diabetes, associated with significant morbidity and, rarely, mortality. • during covid- restrictions, healthcare utilization has changed significantly. • we report a significant increase in the frequency of severe diabetic ketoacidosis at presentation of new-onset type diabetes during the covid- pandemic at our tertiary centre. this study reports a significant increase in the frequency of children and adolescents presenting with severe dka at onset of type diabetes during the covid- pandemic. the reason for this is unclear. this was not attributable to covid- infection in the individuals. however, we hypothesize that presentations with new-onset type diabetes may have been delayed as a result of concern regarding covid- and the restrictions put in place to combat the covid- pandemic. during the pandemic period, people were advised to minimize contact with others. children had less contact with people outside of their primary household, with online learning rather than face-to-face schooling, a reduction in contact with friends and family and an increase in virtual medical appointments. unemployment rates rose and many workers had to adapt to working from home. disruption to everyday routine could easily have led to the symptoms of type diabetes being missed. additionally, the media showed images of people lined up outside hospitals to be tested for covid- and overseas infection rates and deaths were widely reported. it is possible that people felt that going to their doctor or the emergency department would have placed them at risk of contracting covid- . this is supported by the fact that emergency department presentations to the john hunter children's hospital fell during this time period. the increase in presentations of children and adolescents with severe dka at diagnosis of type diabetes during the covid- pandemic is a major concern. not only is severe dka life-threatening, but it also requires the use of intensive care beds and resources during a period of potential high demand. previous research has shown that delayed recognition of type diabetes symptoms is associated with dka at disease onset . however, early diagnosis of type diabetes requires people to have contact with health professionals and present to hospitals. interventions aimed at raising community awareness of the signs and symptoms of type diabetes have been shown to reduce the frequency of dka at diagnosis , . during a pandemic where schools are in shutdown and many primary care consultations occur via telehealth, alternative ways of raising awareness need to be considered. it is vital that in the eventuality of a subsequent wave of covid- , strategies are implemented in order to avoid a similar increase in severe dka. these could include social media campaigns, traditional media advertising, mailouts or advertising in local primary health services. this observational study adds to the limited existing literature describing an increased frequency of severe dka during the covid- pandemic. although this study reports the experience of a single centre, the john hunter children's hospital diabetes centre provides care for all children with type diabetes over the large geographic region of newcastle and the greater hunter. our experience during the period of pandemic restrictions was anomalous in comparison with the preceding years at our centre. the results reported in this study are likely to be representative of other australian tertiary centres, most of which have experienced some degree of lockdown. performing similar studies in centres with differing case rates of covid- and severity of restrictions enacted would help determine if our experience is universal. limitations of this study include the small sample size, which precludes stratified analyses and increases the chance of type ii errors. this study is hypothesis-generating; additional studies that evaluate the reasons for delayed presentations of type diabetes could be used to target campaigns more effectively to reduce this delay. in summary, in the present study we report a significant increase in presentations of severe dka in a paediatric population with newly diagnosed type diabetes during the covid- pandemic when social distancing restrictions were enacted. this illustrates the need to encourage children and their families to continue to seek and receive healthcare for non-pandemic-related health concerns during a global pandemic. detai l/ - - -state ment-on-the-secon d-meeti ng-of-the-inter natio nal-healt h-regul ation s-( )-emerg ency-commi ttee-regar ding-the-outbr eak-of-novel -coron aviru s-( -ncov) australian government department of health. australian health sector emergency response plan for novel coronavirus wa confirms first novel coronavirus death royal australian college of general practitioners. racgp launches nationwide campaign to stop people neglecting their health due to covid- temporal trends in diabetic ketoacidosis at diagnosis of paediatric type diabetes between and : results from countries in three continents is diabetic ketoacidosis at disease onset a result of missed diagnosis? diabetic ketoacidosis at diagnosis of type diabetes predicts poor long-term glycemic control diabetes study group of the italian society for pediatric endocrinology and diabetes* et al. has covid- delayed the diagnosis and worsened the presentation of type diabetes in children? diabetes care unintended consequences of covid- : remember general pediatrics ketoacidosis in children and adolescents with newly diagnosed type diabetes during the covid- pandemic in germany diabetic ketoacidosis and the hyperglycemic hyperosmolar state definition, epidemiology, and classification of diabetes in children and adolescents effectiveness of a prevention program for diabetic ketoacidosis in children. an -year study in schools and private practices a diabetes awareness campaign prevents diabetic ketoacidosis in children at their initial presentation with type diabetes: population awareness prevents dka the authors would like to thank dr michael anscombe, director of paediatric emergency, and the john hunter none declared. children's hospital paediatric emergency department team for their assistance in data collection and care of acutely unwell children with type diabetes. the authors would also like to thank prof. patricia crock, dr komal vora and the john hunter children's hospital paediatric diabetes team for caring for children with type diabetes in the hunter region. the authors would particularly like to acknowledge the children and families cared for by the john hunter children's hospital. c. lawrence https://orcid.org/ - - - c. smart https://orcid.org/ - - - b. r. king https://orcid.org/ - - - p. lopez https://orcid.org/ - - - x key: cord- -ef i bo authors: sciberras, justine; camilleri, lara maria; cuschieri, sarah title: the burden of type diabetes pre-and during the covid- pandemic – a review date: - - journal: j diabetes metab disord doi: . /s - - - sha: doc_id: cord_uid: ef i bo introduction: diabetes mellitus is a chronic disease and a global epidemic. it is a known fact that co-morbidities, including diabetes mellitus, pose a higher risk of infection by covid- . additionally, the outcomes following infection are far worse than in people without such co-morbities. factors contributing to the development of type diabetes mellitus (t dm) have long been established, yet this disease still bestows a substantial global burden. the aim was to provide a comprehensive review of the burden of diabetes pre-covid- and the additional impact sustained by the diabetes population and healthcare systems during the covid- pandemic, while providing recommendations of how this burden can be subsided. methodology: literature searches were carried out on ‘google scholar’ and ‘pubmed’ to identify relevant articles for the scope of this review. information was also collected from reliable sources such as the world health organisation and the international diabetes federation. results: t dm presented with economic, social and health burdens prior to covid- with an significant ‘disability adjusted life years’ impact. whilst people with diabetes are more susceptible to covid- , enforcing lockdown regulations set by the public health department to reduce risk of infection brought about its own challenges to t dm management. through recommendations and adapting to new methods of management such as telehealth, these challenges and potential consequences of mismanagement are kept to a minimum whilst safeguarding the healthcare system. conclusion: by understanding the challenges and burdens faced by this population both evident pre-covid and during, targeted healthcare can be provided during the covid- pandemic. furthermore, implementation of targeted action plans and recommendations ensures the care provided is done in a safe and effective environment. electronic supplementary material: the online version of this article ( . /s - - - ) contains supplementary material, which is available to authorized users. diabetes mellitus is a chronic disease resulting from the reduction in the body's response to insulin production by the pancreas either due to an increase in insulin resistance or due to decreased insulin production [ ] . in the past three decades, incidence of diabetes has quadrupled worldwide [ ] . diabetes has also classified as the th leading cause of death globally in [ ] . according to the recent data published by the international diabetes federation, million adults ( - years) suffered from diabetes in . if not adequately controlled, the global diabetes prevalence is expected to increase by approximately % in [ ] . the contributing factors for the development of type diabetes (t dm) can be broadly divided into genetic and environmental factors. electronic supplementary material the online version of this article (https://doi.org/ . /s - - - ) contains supplementary material, which is available to authorized users. some specific risk factors include, obesity, smoking, leading a sedentary lifestyle, age and also the presence of metabolic syndrome [ , ] . the pathophysiology and the underlying risk factors have long been established, yet the incidence of diabetes is still on a progressive incline [ ] the coronavirus sars-cov pandemic has further increased the burden on the diabetes population, those at risk of dyglcyaemic changes as well as the healthcare services [ ] . the aim of this article was to provide a comprehensive review of the burden of diabetes pre-covid- and the additional impact sustained by the diabetes population and healthcare systems during the covid- pandemic, while providing recommendations of how this burden can be subsided. a literature search was carried out in july through 'pubmed' and 'google scholar' using the keywords 't dm', 'covid- ' and 'public health'. articles were then filtered using several inclusion criteria including; english language, human studies and literature type. the latter mainly included systematic reviews, meta-analysis and literature reviews. the authors then filtered the resulting articles by title and abstract and the remaining articles which fitted the aim of this review were thus considered. additionally, information was also collected from reliable reports such as those of the world health organization (who) and international diabetes federation (idf). having an understanding of the impact of t dm at an individual, community and population level is paramount for public health authorities and policymakers alike. the burden of a disease can be quantified in terms of the quality of life, morbidity, premature mortality, economic and healthcare impact [ ] . public health policies and plans for provision of services all depend on the general population's state of health and comorbid diseases which change over time. developed in the s, the dalys metric is used to gauge the total burden of a disease by considering the number of years lost to a disease, premature mortality, or disability. it is also used to compare health and life expectancy globally. such a calculation gives policy makers a better understanding of the overall duration of life in comparison to duration spent in poor or good health [ ] . a global observation of the incline in dalys across years comparing individuals at different age categories ( - years, - years vs + years) adapted from the global burden of disease website can be seen in figs. , and respectively. [ ] . as seen in the graphs, the higher incidence of t dm at a younger age range is contributing to the increase in dalys. this will place a strain on healthcare costs and economic healthcare services as well as [ ] decreased work productivity and increased likelihood of early retirement or mortality. this will ultimately be a burden on the country's economy. the universal rise in life expectancy has left policy makers questioning whether individuals maintain a good quality of life during these additional years, as reported by the global burden of disease (gbd) study and illustrated in figs. , and . such evaluations are extremely relevant to decisions involving extension of retirement ages and health care stipulations. namely to increase efforts for risk prevention of non-communicable diseases such as t dm from early stage of the disease. great inequalities between the burden of a disease and healthy life expectancy are present globally irrespective of a country's quintile on the socio-demographic index or between sexes [ ] . this implies that quantity is more prevalent than quality of life worldwide. the disabling outcomes of a disease such as t dm has considerable implications for the health care system plans and disbursements [ ] . economic status and healthcare t dm presents with economic, social and health burdens not only for the individual but also for families and careers as well as the healthcare system. additionally, employment is another social factor which is often impacted, leading to further strain on the country's economy [ ] . a country's ability to prevent t dm lies in the presence of an identification and targeting strategy aimed at high risk individuals. this is dependent on the infrastructure and human resources available with a consequential effect on the management plan of the diagnosed individuals [ ] . furthermore, statistical data regarding epidemiology would be essential for health care providers in the identification of the risk factors contributing to t dm at a country level. this would aid in surveilling, diagnosing, monitoring as well as treating t dm. in previous studies diabetes was reported to be more common with individuals with high socio-economic status [ ] [ ] [ ] . in contrast, a recent study reported that a higher t dm prevalence was associated with individuals having a lower socioeconomic status due to limited access to health care and [ ] . moreover, this factor was also observed in low and middle income asian countries experiencing fast economic advancement [ ] . coronaviruses are enveloped viruses known to cause respiratory infections in humans. whilst most of these viruses are harmless and cause mild symptoms, a novel virus known as sars (severe acute respiratory syndrome) -cov as well as covid- emerged in december of , which proved to be more harmful than the previously known coronaviruses [ ] . it is now a known fact that co-morbidities such as obesity, diabetes mellitus (dm), hypertension as well as advanced age all increase the chances of being infected with covid- [ ] . additionally, reports from the centres for disease control and prevention stated that patients with diabetes and metabolic syndrome might be times more likely to die due to covid- [ ] . there are several possible mechanisms which can make diabetic patients more susceptible to covid- . some of these mechanisms include; impaired macrophage activity; impaired neutrophil recruitment and cytokine storm. however, the one mechanism which seems to be considered most is the increased viral load due to the virus entering the cells efficiently. in fact, the receptor which this virus uses is the angiotensin-converting enzyme (ace ) receptor which can be found expressed by various tissues including lungs, kidneys, pancreas and the heart [ , ] . firstly, the sars cov- spike protein bind to the ace cell surface where the s protein is then primed by the cellular proteases such as tmprss and furin. priming involves cleaving the s protein at the s /s domains, allowing the virus to fuse to the cell surface [ ] . virions are then taken up into endosomes where the sars cov is cleaved and possibly activated by cathepsin l [ ] . inside the cell sars cov replicates itself whilst ace catalyzes the conversion of angiotensin i to angiotensin ii and ace converts angiotensin ii to ang - [ ] . since ace receptors are also found in the pancreas, the entry of coronavirus in the pancreatic cells may result in acute beta cell dysfunction [ ] . finally this may lead to a state of acute hyperglycaemia which if left uncontrolled predisposes the diabetic individual to a greater risk of infection and also a higher chance of mortality [ , ] . certain medications prescribed to diabetic patients such as glp- agonists, angiotensin receptor blockers (arb's) and angiotensin converting enzyme inhibitors (acei's) are thought to upregulate ace expression [ ] . acei initially inhibits the angiotensin converting enzyme (ace) leading to decreased angiotensin i levels. this possibly causes a negative feedback loop that ultimately upregulates more ace receptor which can now interact with the decreased angiotensin i substrate available [ , ] . additionally, evidence of a -fold increase in ace levels with lisinopril and a -fold increase in ace levels with losartan was also published [ , ] . therefore, due to the ace receptor being expressed in various tissues as well as due to the upregulation of ace receptor there is thus an increase in potential binding sites for sars-cov- . this mechanism takes place in patients with diabetes and/or hypertension since they usually take acei or arb's. hence, infection by covid- may be more severe in these patients [ , ] . whilst the above mentioned mechanism seems to confirm that arb's and acei's upregulate ace expression, other studies which contradict this have been published. these studies claim that the administration of these medicines is actually beneficial to patients infected with covid [ ] [ ] [ ] [ ] . lack of exercise one of the many mitigation legislations put forward by governments along with public health authorities to contain the spread of covid- was to institute social-distancing restrictions along with the closure of gyms and parks [ ] . furthermore, the population was advised to limit going out of their homes unnecessarily [ , ] . exercise has long been established to be an important requisite as part of the diabetes management and prevention plans [ ] . several studies carried out over the years found that lifestyle interventions including min/week of physical activity and diet-induced weight loss of - % reduced the risk of progression from impaired glucose tolerance (igt) to type diabetes by % [ ] [ ] [ ] . a systematic review and meta-analysis carried out on structured exercise interventions also concluded that structured exercise programs had a statistically and clinically significant beneficial effect on glycaemic control [ , ] . consequently, the world health organization (who) released a guideline called 'stay physically active during quarantine' which contains possible ways to stay active during covid- . the use of online classes and videos were encouraged as were frequent walking breaks around the house [ ] . the mandated lockdowns resulted in the limited access to fresh fruit and vegetables. individuals including those with diabetes might have resorted to the consumption of long shelf-life canned or packaged foods that are typically high in calories and/or fats, with a potential increase in the consumption of carbohydrates [ ] [ ] [ ] . such food consumption increases the risk of weight gain and impose a higher cardiovascular, thrombotic and respiratory complications [ , ] . the concurrent presence of obesity within the diabetes population poses additional detrimental effects on the functioning capabilities of the lungs lead to a decrease forced expiratory volume (fev) and forced vital capacity (fvc) [ ] . additionally, it has been hypothesized that pulmonary lipofibroblasts together with normal adipocytes play a role in the pathogenic response of covid- . this is believed to be brought about by the increased expression of the ace- receptors which turns the adipocytes into reservoirs for the virus. moreover, the adipocytes aid in the transdifferentiation of lipofibroblasts into myofibroblasts leading to pulmonary fibrosis. consequently, the presence of fibrosis leads to severe outcomes of the covid- infection among the diabetesobese population [ ] . a recurrent issue during lockdown appeared to be an increased 'mental stress' and changes in sleeping habits [ ] . anxiety mainly stemmed from contracting the virus, being restricted to the place of residence for a long period of time and also not being able to meet with loved ones [ ] . the increased levels of anxiety were reported by more than % of the participants from north india who stated that they were worried about covid- , out of which . % reported difficulties in sleeping [ ] . another study carried out in china reported that . % of participants sustained a moderate to severe impact on their mental health due to covid- pandemic [ ] . fig. in the supplement material is a guideline released by the national diabetes service scheme (australia) intended in helping with management of worries and anxiety related to covid- and diabetes [ ] . similarly, the european country of malta also released a set of recommendations to help the local diabetic population in managing their condition as well as to reduce anxiety related to covid- [ ] the national health service (nhs) also published 'guidance for: supporting people with diabetes during the covid- pandemic' which compiles informative websites that the diabetic population might need to access during these difficult times [ ] .apart from these guidelines, a number of countries including the european country of malta, set up designated helpline to provide aid to all those experiencing mental health issues including the diabetes population [ , [ ] [ ] [ ] . whilst covid- and the subsequent stress can be a source of sleep disturbance, one has to also take into account diet; lifestyle and diseases [ ] . in fact, shorter sleep duration and unstable sleeping patterns have been linked to obesity and cardiovascular problems [ , ] . an association was also found between sleep disorders and patients with t dm, where increased rates of insomnia, excessive sleeping during the day and a more frequent use of sleep medications were reported [ ] . these changes in sleeping patterns may be due to the t dm itself as well as due to complications which come with it such as polyuria and peripheral nephropathy [ ] . lockdown restrictions challenged individuals including those with diabetes with inadequate vitamin d levels due to low sunlight exposure during this pandemic [ ] . vitamin d deficiency can lead to an increased mortality and morbidity due to covid- [ ] vitamin d supplementation is not only thought to decrease the risk of infection but it is also being suggested as a cure for infection patients [ ] vitamin d has numerous mechanisms through which it decreases the risks of microbial infections and death. these mechanisms can be grouped into three main categories; physical barrier, cellular natural immunity and adaptive immunity [ ] . it was observed that infected elderly with diabetes had an elevated fasting blood glucose as opposed to their hba c which remained stable [ ] . however, during the acute phase of the covid- infection it is essential that strict glucose control is maintained to prevent the occurrence of complications [ , ] . a number of healthcare recommendations and guidelines have been issued during these unprecedent times by different stakeholders including the institute for healthcare excellence on managing the diabetic population pre-covid- [ ] . examples of these recommendations can be found as part of the supplement material (supplement tables , change in healthcare services due to covid- individuals with diabetes are not always able to self-cafe and modify drug doses, especially those in marginalised and disadvantaged populations as well as elderly deprived of social support. these populations are dependent on health professionals [ ] . in such cases, where no designated point of reference is available, managing their own condition can place further psychological stress on the patients, which might have been the case during the covid- lockdown periods. complications arising from poorly managed blood glucose such as diabetic ketoacidosis raises the risk for morbidity and mortality. this will not only put a strain on an individual and the family unit but also on the health care system [ ] . most outpatient services were temporary halted during the pandemic whilst those that continued their services were challenged due to staff reduction as these were deployment to frontline duties or illnesses [ ] . hence, ensuring that delivery of care does not cease during this pandemic was a great feat. virtual care was a tool employed by many countries in an attempt to continue provision of service whilst also preventing nosocomial exposure to covid- . telehealth was consequently beneficial for countries, such as usa,uk and india, when providing a service in distant locations with shortage of staff [ ] [ ] [ ] . using such technologies enabled imparted education to individuals with diabetes about changes in insulin dosing as well as general self-care. the ongoing communication empower individuals and allow them to independently manage their condition. studies carried out prior to the pandemic indicated that virtual communication can successfully lower hb a c [ ] . practitioners through telemedicine can further emphasize the importance of controlling glucose levels as well as relate the potentially improved outcomes of covid- if encountered [ ] . however, such a tool is not always viable due to limited accessibility, acceptance and knowledge on the use of technology. in fact, some individuals still requested to be seen in the traditional face-to-face setting [ ] . moreover, practitioners in developing countries should always consider financial implications of therapies on an individual. simple treatment regimens and low-cost therapy should ideally be prescribed especially to underprivileged populations [ ] . the guidelines observed in supplement fig. have formulated by the british national health system (nhs) to assess the risk of covid- susceptibility before setting up an outpatient assessment or follow up [ ] . healthcare professionals can potentially encounter clients who are awaiting result or have been confirmed as covid- positive. hence it is essential to encourage staff to wear ppes whilst also adhering to recommended sanitisation procedures; especially in aerosol generating practices. such procedures should also be enforced in hospital routine activities such as waste, food, utensil and laundry handling. bornstein et al., compiled a list of guidelines for healthcare workers to follow when dealing with diabetic patients in different scenarios. these guidelines can be found in fig. in the supplementary material [ ] . easy and practical recommendations that were compiled by wang et al., ( ) that can be relayed to patients are listed in fig. which can be accessed in the supplement material [ ] . the extensive impacts on health revealed by this pandemic has demonstrated the vulnerability of individuals with noncommunicable diseases (ncds) [ ] . a study carried out in italy showed that % of patients that died in hospitals had previous comorbidities, with t dm being second highest amongst hypertension, malignant tumours, cardiac and respiratory diseases [ ] . the link between ncd and covid- mortality has also been made in usa, china and spain [ , , ] . measures undertaken for ncds included quarantine and physical distancing. this could potentially result in poor management of the condition by both the patient -through behavioural risk factors -and the healthcare professional [ ] . rescheduling of routine medical tests and appointments can further hinder management as well as limited access to primary healthcare centres, pharmacies and transport. all these factors will make it tougher to ensure continuity of care. research from other pandemics indicates that exacerbation of ncds occurs without proper healthcare management [ ] . this is due to stress that is brought about by changes in routine, uncertain economic situations and new regulations which will ultimately raise rates of disability, morbidity and mortality in patients with ncds [ ] . the importance of t dm management to avoid serious repercussions on health and overall economy is not a new concept. hence it is important to equip patients with the right knowledge about the current pandemic and its possible effects on their overall health. it is crucial, now more than ever, to ensure that patients have direct contact with a healthcare practitioner to mitigate any queries or concerns that they may have. this will ultimately empower individuals to adhere to recommendations whilst also avoiding extra stressors which may exacerbate hyperglycaemic effects such as kidney failure, amputation, nerve damage and heart disease [ ] . t dm has been a global burden for decades; however, additional burden has been imposed with the onset of covid- pandemic. consequently, at a global level, healthcare systems as well as the diabetes population were impacted during this pandemic. mitigation restrictions that were aimed to curb the spread may have imposed a higher burden on the diabetes population. having an understanding of the different challenges and subsequent burden faced by this vulnerable population will enable healthcare professional, healthcare provision and policy makers to provide targeted action plans. funding no funding was received to conduct this study. 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non-communicable diseases in emergencies difficulties to treatment adherence according to the perception of people living with type diabetes publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations key: cord- -le t l g authors: nan title: pathological society of great britain and ireland. rd meeting, – july date: - - journal: j pathol doi: . /path. sha: doc_id: cord_uid: le t l g nan claire m allen, d m hansell. mary n sheppard depanmenls of diagnosbc radrology and lung palhalogy royal bromplon nahonal hean and lung hosprlal, sydney slree:. london sw np percutaneous fine needle biopsy s an established diagnostic technique for lung lesion^ afirm diagnosisofbenign versus malignant is often achieved but histological interpretation of small fragments or groups of cells is difficult manual cutting (twcut) needles provide asuperior histological specimen but are associated with a high complication rate and have been mainly used for pleural lesions this s the first prospective study to assess the feasibility of obtaining histological samplesfrom lung lesionswing apoweredcuning needle (biopty gun) we have biopsied patients using biopty gun there were no major complications histological diagnoses were obtained in patients ( malignant, benign) the malignant lesions identified included nan-small cell carcinomas. adenocarcmamas. squamous cell carcinomas, bronchiolaalveolar cell carcinomas, -cell lymphomas, small cell carcinoma. atypical camnoid and metastatic breast carcmoma. the benign es ons included sarcoidosis. clyptogenic organising pneumonia. wegener's, resolving pneumonias with chronic inflammation the rad~ologistls assessmentaftheamauntoftissueobtainedconelated withgood histology. thethreefalsenegativespecimens obtained the radtdogist noted the inadequacy of the samples ~n two cases and the third was a geographic miss percutaneous biopsy using the biopty gun is a simple and effective means of obtaining good quality histological material from lung parenchyma with a high degree of diagnostic accuracy for bath benign and malignant lesions inorganic particulate matter in "normal" lung: a study using light microscopy (lm), scanning electron microscopy (sem), and energy dispersive x-ray analysis (edxa) london, onlano. l partmeni of palhology soulh sireel london onlano n a g canada h e possible association between inhaled inorganic matter and some cases of usual inter tltial flbrosis (uipi has been of longstanding interest to pathologists and cllnoians. unfortunately. most analytical techniques are impracticable ~n the context of a pathology sewice laboratory in an anempt to find a practicable s utm to this problem and to establish a baseline for inorganic particle load in a "normal" population, a study was undertaken using techniqueswhichwauld beavailable ~nmostlargepathologylaboratories ninecaseswereselectedfram the case(centra andperipheralparliansaf lower. middleand upperlobesjandexamined with thesemand edxaand compared with the lm appearance to determine the panicle type and distrlbutm m a "normal" population. a wide range of inorganic matter was ldentlfied canespanding to siiicb. aluminium and magnesium s cate , rutile and alumina "like" particles varying from < p to p in size in addition. trace elements including zinc. cadmium and increase in number of particles wasalso noted in areas of fibrosis which were present in two cases (old mflammatory disease) preseumably related to problems in particle clearance the findings of this pilot study suggest that although the sem and edxa will likely prove useful tools in the evaluation of lung biopsy soeclmens. the findinq of lnorqanic material in cases of uip must be interpreted with caution autopsy senicb whlch had no known expcsure to lnorganlc dust sectlons were taken from the nght lung each pulmonary adenomatosis has been described as adl~tinctive pathologlcal change seen in the lungsolexperimental area of replacement of normal alveolar lining cells by a taller more glandular type of eplthelwm. usually wlthwt significant cytological atypia. we describe cases i" whlch a smtlar change was seen as an mdental fcndlng ~n resection specimensfor primary pulmonary adenocaranoma. the lesions (usually multlpleand each mm orless m diameter) were identified in lung parenchymaat a distance from the tumour and consisted of thickened alveolar walls lined by prominent, distinctly atypical cells morphologically slmllar to type i pneumacytes and cytologically different to the associated turnour reactive changes " lung involved by obstrmtive pneumonitis were not included !n thts sews all of the associated tumwra were peripheral adenocarcinamas and all showed a pattern of alveolar wall spread at the tumour periphery clinically of the patients were female and all were smokers or ex-smokers the slgnlflcance of this lesion in the histogenesis of primary pulmonary ademcarcinoma is. as yet, unclear animals often i" assoclatlon wlth exposure to inhaled camnogens morphologically the lesion is a clrcumscrlbed departmen ofli~slopafhology sf richard's hosprlal. chrcheslei wesf sussex. po se a series of consecutive personally conducted autopsies in patlenfsdying suddenly outside hospnal and where the death was reported to h m coroner presented cot deaths were excluded. in cases the death had been reported because theattendingdoctarwas unwillingtoissueadeathcertlficate. in theother cases. deathwasnot duetonaturalcausesandfallowedsuicidearan accident oftheformetgroup, therewere t unsuspected malor findings cn patients ( . %). that s to say findings which were either the cause of death or would have led to admission to hospital far assessment and possible treatment they had been discovered in life the largest group were cardiovascular ( ) but there were cases of unsuspected malignant disease there were only five malor unsuspected findings in the group of unnatural deaths years). these results highlight the loss of teaching material in the coroner's system, at a time when hospnal postmortem rates are in universal decline. this matenal would be of value to medical students and to pathologists in traming. and largely comes from cases of lmle or no medico-legal significance. %) and these were in older sublects (mean age . a total of postwnortem specimens refened to the royal victoria ho~pdal electron microscopy unit dunng the years - havebeen reviewed thkscompnsed . vo of thetotalnumber of sewre-relatedcaaesreferred to theunit.oulof ocasesreferred. wereexaminedandrecorded indetail theremainderdidnotundergoelectron microscopic examination for various reasons, such as a concius~ve diagnosis being reached by light microscopy alone. and semi-thin sections showing severe tissue mtoiysis. the most common tssues referred for examination were lung, kidney. liver, brain and head the range of e m. studies carried out included transmission electron microscopy item). scanning electron microscopy (semi and x-ray microanalysis on sem. theelectron micrographs wererevlewedwlth respect totlssuepresewatlanand th wa correlatedwlth the timeintervalbetweendeathand autopsy. electron microscopy was considered, an review. to have been diagnostically useful in % of cases an which it was deployed c s herringtan, a k graham, k cooper, j mcgee it was shown prev usiy that the discrimination of human papillamavirus (hpv) types and by nlsh in archival biopsy matenal requires dlfferent conditions from those predicted by conventional solufion kinetic analysis the parameter tm' (tissue tm) was defined in order to describe these differences in this study. these pnnciples were extended to the discrimination of hpv . and in casesol cln the results of nlsh analysis werecompared with both immunohistochemistry for viral capsid protein and pcr typing these data demonstrate that crosshybndisatm of high nsk viral types occurs ~n clinical lesions under conventional hybridisation and stnngency washmgcondit ns thiscross hybridisation isnot due to thepresenceofviralcapsidproteinand sm relikelyto be areflectionoftheendpoint usedinn sh.i e thepresenceofvisiblesignal.practcally.multiplenlshsignalsduet closelyrelatedprobesinarchival materialarenot indicativeofmultiplehpvinfectionunlesstheyarepresent either ~n morphologically discrete areas of the biopsy or their presence has been confirmed by another molecular technique. motegenerally, thepresenceof asignal in nlsh usingaparticularprobedoes not implythattheidentity of the target nucleic acid is that of the probe medical students in the autopsy room shows that % thought faces should be covered during autopsy, and % thought that genitals should be covered. about half the group were indifferent to both proposals % thought the patient's identrty should be concealed from observers three students thought there should be no conversation at all. % thought that conversation should be limned to procedures and findings and % thought it should be limited to professional maners;conversely. % thought thereshwldbenolimitson thetopicsdiscwaed nearlyso% thoughtstudents should beencouragedtoassistbutnotpressuredintadoingsa. . % thoughtthatallshould becompelledtoassist, but % thought that students should onlv obsewe. and not be allowed to ass s.t. % of students thought that they should oni!iobsene autopsies on patients they had clerked. whereas % thought they should onlfbe on autopsies on patients they had not clerked. fmally. % thought that relations should give spmfic perm on before students observed autopsies. whereas . % thought not a quantitative study of the effects of fibroblast growth factor on wound strength and cellularity fibroblast growih factor (fgf) has potent angiogenic and fibrogenlc effects and is lmpllcated in the formatlon of granulation tissueand healing few attempts have been made toquantity theseeffectsmnvo. we havestudled arat skinwound modelusing red cellghostsasan fgfvehicle tensiometv ofthewoundsshowed amaxlmaleffectof the fgf after seven days when the wound strength was % above that m controls (p < ) this effect had disappeared by fourteen days. computerized image analys~ using a joyce lo& mini magiscan measured total nuclear content of areas i" the wounds. permitting a topographic analysis of cellulanty versus distance from the wound centre cellularlty effects showed adifferent time course from wound strength. a % increase at fouldays anda % decreaseat sevendays. relative tocontrols(bothp < . ) attwelvedaysthecellularityeffectwasstill sqnlflcant at a vo decrease but by twenw-one days it had dwappeared the results suggest that fgf causes an early transient increase in cellulardy and more rapid increase in wound strength: most of these cells are macrophages and fibroblasts suggesting a connection between thesetwo obsenat ns. the adhesion molecule$ integrin (cd ). integrin (cdl ). and intercellular adhesion molecule- (icam- , cd ) are essential to the intimate co-operation of antigen presenting cells (apcs), t cells and keratinocytes cyclosponn. which is an effective treatment for psonas~s, may cause immunosuppression by altering antigen presentation we have pertormed a quantitative immunohistochemical assessment of the effect of low dose ~yclosporin on the expression of p -integnn. p -integnn and icam-f m the epidermis in chronic plaque psonas s. staininglevels werecompared withclinicalresponseasassessed bythepsoriasisareaseverihllndex(pas score) pt-lntegnnand cam expressionon keratioocyteswerenotaltered bytherapy buttherewasasignificantdecrease inthemeanlevelsof p positivelargedendriticcells(apc )within theepidermis.bz-integnn wasnot expressed by keratinacytes there was a strong correlation between p expression and pas score after three months on cyclosporln and one month off therapy these results lndlcate that p stalnlng on large dendrltlc epidermal cells previous studies enumerating silver stained n~c l~l a r organiser regions in problematic cutaneous melanocytic lesions have yielded inconsistent. but generally favourable, resulis. it seems probable that such inconsistencies. arise largely from differences ~n fixation. staining and counting strategies. our group, having devised improved methods of agnor staining and counting. is now able to re-examine the potential role agnors in borderline lesions pilot work demonstrated potentially significant differences in agnor dispersal between benign and malignant lesions and i" this study bath agnor numbers and dispersal patiems have been evaluated a range of melanocytic lesions including banal naev~. dysplastic naevi. typical melanomas, spiu naevi, atypical spih le~ions and minimal deviation melanomas were collected virtualty all of the unusual lesions had been diagnosed by specialists in dermatopathology. using a combined assessment of agnor numbers and dispersal, it proved possible to discriminate borderlip lesions from banal naevl and typical melanomas. benign borderline lesions -such as spit naevi ~ possess numerous nor$ but display tight clustenng in contrast to malignant melanomas wherenordispersa isaprominentfeature discriminatinganetypeaf borderlinelesionfromanathercauld not be achieved however, in practice this distinction is probably less important than assigning a aoirary specimen to a benign or malignant group a larger prospective trial of agnors in melanocytic lesions currently in progress with a mean total exposure of pack-years. alihough p was expressed more commonly in adenocarcinoma ( rof lo)andsquamouscarc~noma( ~~of )than~nsmallcelltumours(l %of o),thiscouldbeaccountedfor by the smoking history. since patients with non-small cell carcinoma smoked more (a mean af pack-yeas) than those with small cell lesions (mean of pack-years). there was no relationship between p expression and sunlyal. s. a. sun. j. r. ~a s n e y depaitmenl ofpathology university of liverpol, p box , l!vemooi. l bx activation of thec-myconcqene with overexpression of itsancopratein product ocurs in bronchlal malignancles ofalltypes, but has beenmost extensivelystudied insmall celicarcmoma, wherensaverexpression~n culturedllnes has beenassociatedwithdevelopmentoffast-growingvanants whlchlosemuchofthelrendocrlnephenohlpeand aner their morphology. it has been suggested that these vanant lines might be the equlvalent of the large cell bronchial endocrine carcinomas sometimes seen i " yiyo, but this is not proven. we have used th myc - e monoclonal antibody and the avidin-biotin technique to study the panem of expression of the p oncoprotein product of the c-myc gene in turnour deposits of twelve subjects coming to necropsy with disseminated small cell carcinoma in an attempt to relate l o morphological vanablity and metastatic site. anhough expression bore no relationship to morphological vanation. it ohen differed markedly horn site to slte. whereas parts of the primary t m w r strongly overexpressed the protein ~n all but one wbfect, there was considerable vanability between secondary deposits. oossiblv mdicatina arelation~hi~ between c-mycex~resslon and propensltvfor metastasls to certain iocations. tumour growth rate is a key parameter of neoplastic aggression. and is determined by the balance of cell gain and loss.apoptos~sisama]ormadeoftumourcellloss, but linleis knownof itsregulation. dieremesin turnourgrowth conferred by hpvtypes and werestudied in aratfibroblast modelsystem. immortaiisedcells weretransfected with hpv and expression vectors. either alone or with activated c-ha-rasl. monoclonal cell lines were established. and their vector dna content was confirmed by pcr tumour cell ddfered in their growth properties m wvo and m vilro. hpv containing turnours were larger and showed less apoptosis than those containing hpv , although bothshowed moreapoptasls than thenodulestormed bytheparentfibroblastsalone. in all turnours the presence of ras greatly reduced apoplosis and increased the growth rate. very similar propetlies wereobswvedin culture, and apoptoticratesshowed astranglnveneconelatlonwlthratesofnet cellgrowth. hpvs appeared iostim"latetumourcei apoptasis, butthiswas suppressed byras. melowerapaploslsassoclatedwilh hpv compared with hpv may partly explain the more aggressive phenotype of cervical c a n m containing hpv . it hasbecomeapparent thatanumberof maleculesmaybeexpressed byrestingorqu~escentcellsandarelostwith transition into the cell cycle. in addition to being of biological interest, such molecules may prove useful as operatcanal mahers of quiescent cell populations in histological material and allow the further characterisation of cellular subpopulations. one such molecule is statin. a kd protein previously reported to be expressed only by cells ~n go. we have shown bylaserconfocalfluarescence microscopythat thestatin antigen is associated with the nuclear envelope with a dstnbution m l ar to that of nuclear lamins. using a monoclonal antibody ( - ) that recognises slatin we have defined the tissue distribution of statin immunoreactivlty in a range of continuously renewing, conditionaily renewing and non-renewing tissues. the distribution of immunoreactivlty s essentially as would be expected of a maher of quiescent ceils. in contrast. in established pancreatic carcinoma and other epithelial ceil lines. we have found $latin immunoreactivity ~n cycling cells using biotinylated k slatin double labelling techniques. in conciusion. statin lmmunoreactivitv in normal tissues correlates with auiescence but this realtionship is lost, at least in vifro. loss of cell-cell and cell-substratum adhesion are imponantfactors during turnourprogression. tumour promoten are compounds which although not carcinogenic themsews increase the frequency of turnour development in animals previously exposed to carcinogens. we have used the turnour promoter tpa on cultured human renal epithelia ~ells mimic nwplastlc transformation. following tpa treatment we haveexamined thedistribution of vlnculin, b integrin and actin within thetreatedcells byfluorescencemicroscopy.treatment of therenal epithelium by tpacauses arounding up of the cells and a loss of adhesion toeither laminin or fibronecttn substrata fiuorescent microscopic examination reveals a progressive loss of reactivity for vinculin and b inlegrin within local conlacts. these changes are accompanied by a redistribution of the actin microfilaments from orientated bundles of stress fibres to a circumferential arrangement these changesofareduction in focal contact componentsand disarganised actin cytoskeietan mimic the changes we have previously described in renal carcinoma. glutathione s-transferases are a diverse group of enzymes with an important role !n the metabolism faelgn compounds including some carcinogens and cancer chemotherapeutic agents. increased expression of gst pi is seen in manyanimal and humancancersandisassociated with resistancetocalboplatinandcisplatin in humanlung cancet cell iines. gsts are involved m steroid hormone transport and metabolism and have a role in the complex metabolic relationships between settoli ceils and germ cells. we have studied gst isoenzymne expression by an immunohistochemical method in stage i teratomata. stage i tetatomata, stage i seminoma, cases of intratubulargerm cell neaplasia( tgcn) and a groupof cryptorchid and normal testes. the stage i teratomata had beentreated with cisplatin based therapyand bothprimmaryandpost-therapymetaftatictumourtissuewerestudied. gstalphaexpression correlated with morphological evidence of epithelial differentiation in teratomata.therewas no difference in gst expression between stage i and stage i pnmary testicular iumours nor between primary testicularand post-therapy metastatic tumours. gstexpression did not correlate with survwal. gst pi was strongly expressed in the neoplastic germ cells of itgcn but was weak or negative in normal germ cells. this may be significant in view the potential for later contralateral turnour development tn patients treated by cisplatin based therapy. in summary, gstexpression in testicular germ cell turnours reflectedlheirdifferentiationandappsared to be unrelated l o therapy and subsequent survival. a case illustrating the usefulness of electron-microscopic examination of fine needle aspirates s described fine needleaspiration wasperlormedon asubcutanwusnoduleinthechsstwall ofaneldedyman whowassuspmted tobesuffering from bronchogeniccaronomaanclinicaland radiologicalfindings.mesmears werenotdiagnan~c. but ~n view of the history af asbestos exposure, the needle washings were submined for eiectron-microscopic examination, which showed mesathelial differentiation and a diagnosis of metastatic malignant mesotheiioma was suggested. an autopsy perlormed eight months later confirmed the diagnosis of malignant mesotheboma. t. dorman, a ti ismail, b cunan, m. leader pathology department hoyal college of surgeons m ireland, dublln many histologi~allymalignantfeaturessuch as hypercellularltyand high mitoticcounts, but clinically followa benign course. demoid tumours and fibromafoses can be densely cellular but usually quite bland histologically and are associated wdh infiltrative marginsand repeated local recurrences. in addtion irradiated tissue often contains many cellswithcytologicalfeaturesol malignancy thisstudyexaminesthepioidyofsuch lesionsbyboth olthecunently availabletechniques using dlsaggregated formalin fixed paraffin embedded tissue sixteen cases nf, dfsts. dts. fibromatoses( nci~ding palmar, plantar. retrcpentonealand soh tissue) and miscellaneouscases including leiomyoblastoma, inflammatory pseudosarcoma and plexiform histlocytama. one malignant fibrous histimytoma and t paragangliama were included as possible poslive controls. cases containing numerous irradiation fibroblasts were alm analysed all 'pseudosarcwnas' were euploid by both image analysis and flow cytometry the malignant fibrous histiocytoma was aneuploid by image analysis and flow cytometry and the paragangliomawasaneuplaid by imageanalysis and tetraploid by flow cytometv themndwonsof thisstudv are that pseudosarcomas are euploid and aneuploidy would appear to be confined to malignant tumours of mesenchymal ongm. other sites we collected cases to study their morphology and antigenic profile after staining for the epithelial markefficam . flow molecularweight keratins)andlp (high molecular wefghtkeratins), thegeneralmelanoma and neural markers s w. neurone specific enoias (nse). protein gene product . (pgp) and the melanoma s w i f l c marker hmb , the neuroepithelia markers leu and glial fibnllary acidic protein (gfap), and the intermediate ftlaments vimentln vim) and nsurofilament (nr the following turnour types emerged: i pure spindle celltype(n = ), ,purepolyganalcelltumaun(n = )comp sedofpleomorphiccells(n = ).undwmcellswith an alveolar in = ) or sheet arrangement (n = ). and mixed pleomorphic spindle and polygonal cell tumouffi (n = ). two tumouffi proved to be lymphomas an intraepithelial melanocytic component could be established in cases within adjacent respiratory or squamous melaplastic epithelium. in cases n e w trunks a~mclated with tumour contained increased numbers of atypical schwann cells. consistently expressed were s w ( and colon ovanan tumour antigen (cota). csais a heat stablemucin associated antigen present m normal colonic epithelial cells and is expressed m greater quantitites tn colon c adenmarcinomas cota is a heat stable antigen present n colanicneoplasiaand mumowwananturnours but not ~nnormalcalonicepithelium psmionsfrom to primary adenocarcinomas ( ovanan, colo-rectal, gastric. breast. oesophageal. prostatlc. pancreattc, endometnal and t gallb adder)and two adenmarcinomas metastatic tothe liverwere incubated with anti csa and anti cota antibodies using the p.a.p technique with positive and negative controls. while anti csa positivitywasseenin d ~ loni~aden carcin ma~( wsakand lo~tr~"g).~twasalsaseen n af ovanan ( weekand strong). of gastric ( weakand strong), of breast( weak and strong). of oesophageal ( weak and strong). of prostatic (all weak). of pancreatic ( weak and strong). of endometnal (all weak) and the gallbladder ademcarcinoma. both liver metastatic adenocarcinomas were negative in additim while anti cota staining was positive m of colonic adenocarcinomas ( weak and strong) and of ovarian adenocarcinomas.~twasalsapositive~n af gastnc( weakandzstrong). al lobreast( weakand stlmg), all esophageal(allweak) of pr ~tati~(allweak), pa"creatic(low akand t strong),all endometnal(all weak) and the case of gallbladder endocarcinoma (strons) both liver metastatic adenocarcinomas were negative. m e c o~c~u s~o~ of this study s that anti csa and anti cota are not adequately specific in the identification of a ~olonlc or ovarian origin af an adenocarcinoma and cannot reliably be applied to the identification of a metastatic adenocarcinoma of unknown primary site the early intfa-epithelial changes of adenocarcinoma of the nose and paranasal sinuses were sought in the histological sections from high grade adenocarcinomas and cyclindric cell carcinomas from this region none of which had had previous radiotherapy treatment. of the cases came from wwd-workers. i r m a polishw and from a plasterer attention was directed to the non-neoplastic epithellm of the surface and of the sera-muclnous glands. in casesofadenocarcinomaand t of cylindriccell carcinoma surfacechaogeswere detected these took the form of hyperplasm of goblet cells of the rsspiratq epithelwm accompanied by dysplastic changes of other epithelial cdls: m the latter these became enlarged and irregular m shape with large hyperchromatic nude and prominent nucleoli. lnthsmajorityof iesionsthesechangeswereisolatedin theepithelium. changesof thesetypes were never seen in sefomucinous gland epithelium. in addition similar changes were not noted m the coverlng respiratoryepithelium in casesofnasal polyps evenwhen severely inflamed thesemsmchangesso described haematoporphynn derivative sensitised tumwr tissue. s underwing evaluaton " widesptead and m i s t a n t papillary tumoursand ,n casesof rvldespreadseveredysplasra/carnnoma,nufu". arewewof spallents ( , m, ages years. pre-and post therapy) has revealed no change ~n histological grade and stage ~n patients, progression to invasion in t case and a redunion to mild urothellal atypla alone in case. local tissue changes following treatment were limnedto oedema and an acute lntlammatoryreactlon. no metaplastlc. stromalfibroblast or nene changes were seen variations ~n bladdw size. both increased and dlmtnlshed, wwe encountered the histological grading of tcc following pot shows little improvement. but studies are in progress to improve lhght delivery in the bladder, and hence improve treatment outcome. a armour. a. s. jack bpartment of pathology, umverslty of i d s . leeds. lsz jt follicular lymphoma is a disease characterised by widespread lymph node involvement usually at the time of presentation proper tie^ of lymphocyte homing and circulation appear to be mediated by a variety of cell adhesion molecules. the a m of this study was to compare mrmal nodal lymphocytes with the neoplastic populatlm m folllcularlymphoma. hall casesalloftheneaplasticfolliclesexpressed lcaml but thisyanedfromonlyafewcellsto % of cells within a follicle this dosltlvlty included dendmic retic~l~rn cells. germinal centre cells and neoplastic lymphocytes. leu (lymphocyte homing receptor) showed an inverse pattern of expression in all but cases the phenotype (icaml + l a is a feature of germinal centres and scanwed paracontcal blasts in reaclive nodes anhough there is uniformity of expression of lcaml in germinal centres which is not apparent in any folllcular lyumphoma case. this study showed a loss of lcamt and increased leu expression by neoplastic lymphocytes within follicles this may relate to the propensity of this disease to spread widely throughout the lymphatic system activation leads to an alteredfunction and aconcomitant aneration in the chemistry ofthe cell surface, which ~s a site nchincalbohydrate. rat lymph nodelymphacytes.eitherunactivatedoraclivatedfot days namixedlymphocyte reanlon. were treated with biotinylated iectins from a large panel. chosen to probe surface glycans lmlns were revealed with awdln-phycoerythnn and cell populations were analysed in a fluorescence activated cell-sorter double-stalnlng with fitc-monoclonal antlbodles defined the functional lymphocyte subsets. uea- and lta boundtonoce sof anytype. unactivatedb-cells boundall theremaininglectins.savempa. toagreaterextentthan did unactlvatedt-cellsand b-cell actlvatlon produced no change i" glycan sxpresslon. unactlvated and actlvatedtcells all expressed a . -linked sialyl residues, but o- . -linked sialyl expression was hetrogeneous, did not conespond toanysubsets,and wa~unchangedonanlvatian.alargegroupof lectinsshowedlow wno binding to unactlvatedt-cells, but boundonactivatianexactly in parallel to l- receptorexpressionctacantigen) thes~ngle structure gawgalnacal, galpl, glcnac-) galigalnacat , gal~ , glcnac-) r could account for their binding the presence of neoplastic (light chain restricted) b cell follicles in low grade b c d gastrolntestlnal (gi) mmphomaof although malt s not present in normal human gastric mucosa, lymphold t,ssue ,* acqulr& ,n response lo colonisationof themucosa by helrcob~cleipyioii. we have investigated the possibility that thisacquiied lymphoid t ssue is malt type whlch may povovlde the background i" which lymphoma can arl~e w e examined gast,,c b~ p ~e fr m casesafh~l~~~bacrerassaciatedgastntis.and biopsyandresectionspecimensfram casesaf t"mours has contr'buted to a peklstence of ihe more "iew arefofl'cle (fee) gastrlc cell lymphomaof malt in cases of hebcobaciergastritis prominent lymphoid follicles were idenfilled in aftheee b cell clusters were identified within the gastric epithelium. reminiscent oltheteatures seen the dome epithelium of sm intestinal peyer'z patch. thls b cell-eplthelh assoclatlon was not assaclated with the eplthelm cell changes or the glandular destructm seen ~n lymphoeplthellal i~s i o~s malt lymphomas. in / cases of gastric maltlymphoma helicobaclercould be identified oltheremaining cases. weiegastrecfamy specimens n which specimen washing may have contributed to the negativefindings. we suggest that gastrlc malt s acqulred in response to local immunological stimulation as a result of mucosal colonisation by hel~cobaclerpylon, and that the development of malt lymphoma is a subsequent event mucosa associated hmphold tlssue (malti has been explained on the bas of speclftc colonization at reactlve b lymphomasbe included in lhecatqoryof malt lymphoma but the frequent presenceotafoll~culapanem in these foltic es by ihe neaplastlc cantrocyte-like(ccl) it has been low grade cellthyro d we have ihe and 'nvesflgated the and genotype of' of primary low gradebcelllymphomasofthethyro'd alsodemonstratedfeatures ofmalt ymphama'ncludtng cclce and l y m p h~' t h~' a l l~' o n s . m e appearances and immunoh'sto'og~ofthefo'l'c'es werethoseofto'l'cularcalon'rat'on described 'ngimaltmmphomarather thanfccfoll'cular'~mphoma thepredomlnant iwg ~ane'naffoll~cularcolon~rat~~nconformed tothatdeslgnated cclcells show'ng astnk'ngly h'gh prol'feratton late no evidence of the '( ' ' transiocat'an was found in any on dna extracted from fresh (n = ) or paraffln embedded (n = ) t sue mese ilndlngs argue against a fcc lineage lor primary thyroid lymphomas and support their ncius on " the maltcategory we have used a panel of antibode$ to demonstrate stages of granulocyte maturation by immunoh~st chemistry i" decalcified. wax-embedded bane marrow trephine biopsies antibodies reactive with muramidase. u- -ant~tryps,n. neutraphil elaslase and cd react with early granulocyte precursors. cd and calgranulin identity later granulocytes. ihdiylduai antibodies dlfier rn the populations cells ldentlfled theantlbcdtes also react with cells of mwlocyte llneageand provide information concerning theorganlsatlon afmonopolesls about whlch liffle is known in normal marrow granulopoiesis is zonal wlth maturation occurring radially around trabeculae and blood vessels this pattern is exaggerate in reactive hyperplasia and chronic granulocytic leukaemia (cgl) there is marked disruption of this zonal organisation in myeloproliferative and myelodysplastic states, with considerable overlap patterns between these conditions in chronic mvelomonacvtic leukaemia (cmml) there is comdlete absence of zonal arrangement of granulopaiesis possibly due to monocytic proliferation obscuring the underlying marrow spaces and that by analogy with cgl cmml represents an exaggeration of this normal panern granulopoletlc panern we hypotheslse that monopoles normally occurs " a randomly dispersed fashion within as previously shown by us in animals untreated with cyclosparin a. the cell birth rate fell from an initial prelmmunisation valueof cellsit cellsihour to cells celldhouran day followed by a rise cellsit cellslhour on day however, in cyclosporin a treated animals the cell birth rate ( ce s/looo cells/hour) was signiticantlydepressed belowthecontrol pre-immunisation levelandremained suppressedupfaday fallowed by an abrupt rise these results are consisten with the hypothesis that t lymphocytes or their pmducts not only drwe the morphological appearancescompnsed lymphoepithelia lesions (in onecase). numerous lymphoid follicles and a diffuse infiltrate of monocytoid cells (centrocyte like cells) there was striking plasma cell differentiation and colonisation of lymphad folllcks by monacytoid cell?. and neoplastic plasma cells. lmmunahistochemistry convincingly demonstrated heavy and light chain restriction ~n all the biopsies from both the cases the bladder s developmentally related to the hind gut and this manifests " the variety of metaplastic epithelium seen at this site circulating cells. encauntenng the endothelial surface. make contacts an environment rich n glycan. a large panel of biotinylated lectins was used to probe far variations in the glycans expressed on endathelia of artenes. veins, arterioles, venules. capillanes, high endothelia vessels and lymphatics m a range of normal and pathological human tissues formalin-fixed, paraffln embedded specimens from the files of manchester royal infirmary and manchester royal eye hospital were used lectins were revealed with an avidin-peraxidasesystem no differences werefound between arterial. arteriolar, venular, veinous or lymphatic endothelia all expressed abundant complex-type n-linked glycans. of several subtypes. capillaries were highly variable and showed heterogeneity ~n their expression of ) outer chain sequences fn n-linked glycansand ) mucin-type sequences. both between different. normal organs and within an organ. implying that the surrounding tissue probably had a regulatory effect where endothelium was reactive, additional aneratiom wete seen and actively growmg endothelium in granulation tissue expressed hqhmannose siwctwes. high endolhelial ybss~is showed a much lower density and narrower range of glycan expression thandidad~acenlnormalcapillanes,despite theirknownvery highrateofglycansynthesisand secretion the iymphocyteiln-homing receplor(s) would be a component of this restricted porlfolio of glycan monocyte margination in atherosclerosis associated with immunological injury n. j. combs, p j gallagher. p. s bass clinical and experimental evidence indicates that immunological injury is associated with accelerated atherosclerosis. allograft recipients may develop accelerated atheroscleross and anlmalsgwen serum sickness and a high fat diet develop more extensive atherosclerosis than controls fed the diet alone we have tested the hypothesis that atherosclerosis ~n these animals is associated with increased adhesion of monocytes to the aortic endothelium. chronicswum sickness was induced in genetically hyperlipidaemic rabbits with nativeanionic bovine serum albumin (nbsa) or highly cationised protein (cbsa) rabbits given nbsa showed a spectrum of glomerular endocapillary prollferativechange thasegiven cbsa developed early membranous glomerulopathy in controls the numberofmanocytesadherent to theendothelium ranged from y isq mm and m animals given senm sickness - isq mm (nbsa)or ~ isqmm(cbsa) as therewerenosignificant intergroupdifferences theseiesultsdo not support the hypothesis that immunological injury increases monocyte adhesion to the aortic endothelium departmen! oipatholcgy, souihampfon universily hospdals soufhamplon. so xy amyloidcan beidentified nupta % of elderty hearts,especiallyin themyocardium oftheatrialappendages in a small prapanionofth%lecasesamylaid~salsapresent inthecardiacvalve~ butisolatedvalvulardepos,tsarerare. a year old male presented with bilateral leg weakness and an intradural turnour. he died days after spinal surgeryanddeposltsofaglloblaslomamunlforme wereldentlfledm both thecerebrumand thecord therewasno hlstory of cardiac disease but all four valves showed translucent verrucous thrkenlngs. these had a uniform eosnophilc hi~lologicalappearance. stainedwithcongo red and had ultrastnucturalfeatures of amyloid amyloid p proteinwas identified immunohistochemically ~n thesedeposits but negative multswere obtained withantibodies t o m . aland a proteins. mwewas no evidenceof cerebrovascular ormyocardial amyloidosis the involvementof all four cardiac valves. the stnking absence of amyloid i" other organs and the a~~o~l a t i o n with a widespread glioblastoma are unusuai but unexplained features of this case the ima s used as a bypass of narrowed coronary arteries it is said to be less prone than vein gratk to develop subsequent occlusivedisease. thisstudy of pairs of imasfrom subjects of vario~s ageswas to see if the histological structure ofthevessel might explain immunityto graft disease sixteen imas were fixed in distension byformalln at cm of water pressure and secllon~ taken at the level of each nb (first to stxth). all arteries undelwent similar changes along thelr length. wlth no slgniflcant difference between left and rlght lntimal changes were minor conssting of lbro-elastic muscular thickening in all age groups and in those who had died from vascular disease theinternalelastic lamellawaswelldefinedat all levels. buttheexternallamellawasclearlydefinedonlyatthelevel ofthefmhandslxthnbs(i.e. mthedetalartwyj. medral thrckne~~decreasedalongthelengthofthearter~esandinall cases changes from an elastic to a muscular structure, generally at the level of the fourth or fiflh rib the ratio of medial thickness to numbw of lmdlae in~reases along the artery. notably at its point of change muscle fibre orientation changes from inter-mixed cir~ular and longitudinal in the elastic part to predominantly circumferential in the muscular pan. the pronounced stnctural differences of the ima compared to the similar sired epicardia coronary arteries. which are muscular. may be of relevance in explaining their markedly different incidence of atheroma. naomi carter, s variend fatty change of the heart is a pwrly defined pathological entity which in the adult hean can be caused by severe hypoxia nutritional disorders. poisoning by selected drugs and catecholaminerelease it is most commonly seen ~n association with coronary artery disease little data exists with regard to the paediatric hean in pediatric deaths coming to post mortem over a year period. there were cases of myocardial fatty change of varying seventy and distribution detected ~n red -stained sections infection and congenital disordenwereimplicated n deaths infectionand cangenitaldisordenwereimplicated n deaths. fallcasesoffarmchangeand % of all deaths seven ofthe e~ases had acombined ~nfecti~u~andcangennalaetiology othercausesofdeath included turnour, traumaandcomplicationsofbinh % of casesofsudden infant dealhsyndrome(s ds) hada fatty heart only one case of the total cases showed deflnlte hlstaloglcal evldence of ischaemic myocardial damage. insome instances, thedegreeaffattychangemayberelatedtathedurationand seventyoftheundertyng condition someofthesechildren may havean occunnutritional orenlymedisarderwhich ~sexpressedatacellular level ~n the form o! fatty change and that contibutes to their early death. we havecampared thereparting oftemporalarterybiopsiesbetween - ( cases)and ( cases). the overall incidence of positive biopsies was . % and . % in each period the number of patients with clear clinical evidence of cranial arteritis was % but in - , % and in , % of these had positive temporal artery biopsies when the histology was reviewed approximately % of biopsies in each period had been erroneou~ly reported as healed or atypical artentis. in contrast. a true histological diagnosis of artetitis was missed in~nlya~inglepatient approximately % ofall patientswithaclinicaldiagnosisof giant cellarteritisdevelopedan additional symptom or pathological change associated with steroid treatment frequent final clinical diagnoses in patients with negative temporal artery biopsies were transient ischaemic attacks, cerebrovascular accidents. unexplained headache or migraine. polyarteritis or polymyalgia hwmatica these resuns confirm that one third of patients with deflnite clinical evidence of cranial arteritis will have negative biopsies pathologists continue to misinterpret normal arterial ageing changes as evidence of healed or atypical arteritis depanmenf hslopafhology and depanmen! resp raioiy medicne. sl banholomew s hospifal london ecla /be adhesion ~fepithelium to extracellularmatrices is mediated partly byafamily of heterodimeric molecules known as megrins we haveexamined theexpressionofthealpha- toalpha- integnn subunits in~unured human branchia epithelial cells. and in bronchial biops es from normal subjects and atopic asthamtics. we have also studied the expression of intercellular adhesion molecule-t (icam- , cd , rhinovirus receptor) br~nchialepithelia cells from surgical specimens were grown as explant cultures on glass covenlips. bronchial biopsies were taken from right upper and middle lobe carinas lmmunastaining was performed on acetone fixed cells and frozen tissue sections using alkaline phosphatase and immunoperoxidase techniques all biopsies showed strong positive staining of epithelium for alphe?, alpha~ and alpha- integnns. stainingforalpha- was weak or negativeand epithelium was negative foralpha- and alpha- except in twoasthmaticswhere itwas weakhi alpha- posittve. in contrast. cultured bronchial epithelial cellswere positive for all these mtegrinsexcept alpha- . epithelium was positive for cam- in asthmatics but negative all other biopsies. cultured cells were ~trongly positive for this molecule it concluded that expression of some adhesion molecules bv bronchial emhelium may vaw ~n relation to the cellular environment and that ths may be imponant tn disease l%panmen! of pafhology univeisrty of birmingham and departmenrs o! 'immunology and his!opalho!ogy eas! blood eosinophils are in a relatively inactive state with migration into tissues eosinophils became more activated these activated cells are hypodense compared to most blood eosnoph s low affinity receptors for both ige (fcerii. cd and igg (fcgriii. cd ) have been documented on activated. hypodense eosinophils this study assessed the expression of these proteins on ttssue eosmophk derived from nasal wlyps the blood and nasal polyps of seven patients undergoing nasal polypectomy were studied nornodense and hypodense eosinophils were isolated from venous blood by centrifugation an a discontinuous percoll gradient percoll wassimilarly used to obtainaneosinophilrichpreparationfromcellsearactedoutafthenasalpolyps cytospin preparationswetem?.de of these samples frozen sections of each polyp were also prepared lmmunostaining using an alkaline phosphataseianti-alkalln phosphatase detection system demonstrated that neither blood nor nasal polyp eosinophilsexpressed detectablecd orcdt l t i~p~~~i b l e t h a t e~~i n~p h l l s o f na~alp~lyp~aresimilarto blood eosinophils and are in a relatively inactive state lavage and biopsy studies. the relationships between mucosal inflammation. bronchospasm and bronchial hyperreactivity are unclear since bronchial smwth muscle has an essential rolein the pathophydogyof asthma. we haveexamined theextentto which it ~sin~ol~ed~nallergicinflammation bi p~ie~in~l~ding~moothmu clefr m asthmatics (age range who did not use steroids and controls (age range ) were embedded i" araldite and stainedformast cells ~monocional ant bodyaat)and eosinophils(monoclona antibody eg ). mast cell numben in the lamina propria and smooth muscle were similar for both asthmatic and control sub e m (mean values. astham lamina propna mm . smooth muscle timm , controls /mm and blmm'respectively) eosinophil numbers in the lamina propria were increased -fold in the asthmatics (p = ) but there was no significant increase in the number of eosinaphils rn the bronchial smooth muscle eosinophil numbers in the asthmatics correlated positively with fev, we conclude that the role of muco~al inflammation in the pathophysiology of asthma has yet to be determined we present cases ofextra-pulmonary pneumocystosis diagnosed on routinesurgical specimens (two biopsiesof liver. and one each of gastric mucosa. small intestine and a pen-anal mass). in each case. the histological features were similar to those seen in the lung. and as in other material from cases of aids, munlple pathology was often found extra-pulmonaly pneumocystasis s now being reported from a widerangeof clinical specialties. one reason forthismcreasemaybe that impraved patient suiyiy~i with"topicay inhalatiooalpentamidinetherapyallowsvisceral foci of infection to become clinically apparent this hypothesis is supported by the finding that ofour cases were taking nebulised pentamidme inthesehypersensltivitystatestothatobselved mbronchiectasis (a chronic suppurative lung condition not thought to in~olve a hypersensitivity aetiology) and in smokers and non-smokers with no evidence of active pulmonary inflammation ourresuns havesh wnthattheb:tlymphocyteratioisnodifferent ineaaandsarcoidfromthat seen ~n bronchiectasis and in normal lungs. we believe that thns is further evidence to suggest that bal is an unrepiesentativetechnlquetorlhestudyof interstitial lung diseasesand that morecansiderationshould begivento the possibility of humoral immune components in the pathogenesis of ea determine its effects upon the number of pulmonary neuroendocrine cells and their peptides. in one experiment. the concentration of noradrenaline in the lung was estimated by chromatography, and that of the peptides bombesm. neurotensin and caknonin gene-related peptide (cgrp) by radiaimmunoassay. there was significantly ies noradrenaline and bornbesin in the lungs of test rats than in controls but the levels of neurotensin and cgrp were unchanged. in a second experiment, pulmonary neuroendocrlne cells in histological sections were labelled with antiserato bombesin,calc,t~tonin,cgrp andproteingenepmduct [pgp s ) a~d =~" " t~d . t h~, e w =~ nochange in the number of labelled neuroendocrine cells expressed per unit area of lung or per unit length of airway between test and control rats for calcitonln. cgrp or pgp . . bornbesin-containing cells could not be identified in either group. an increase ~n pulmonary neuroendocrine cells could not be identified ~n either group. an increase in pulmonary neuroendacrine cells immunoreactive for bomtesin and calcitonin occu~s in the early stages of plexogenicpulmonaryartertopathy in man. theabsence ofsucha change in monocrotallnedulmonanhydertenslon in the rat suggests that this is a poor model for the human disease. this preliminary study was carried out to assess the quantitative expression of neuroendocrine and mast cells in adun humanlungsofcasesofasbestos-relateddisease.tencaseseachofasbestasis, pleuralplaques,carcinoma and mesothelioma were studied m comparison with ten normals wnh no history exposure to asbestos the lung sections were stained lor neuroendocrine markers neurone specific enolase (nso and chromagranln. and, chloroacstate esterase and toluidine blue for mast cells. there was a notable variation in the number of neuroendocrine and mast cells between the control and asbestos-related disease group the variation was also seen between the various asbestos-related diseases. though not statistically sbgnificant. the trend of the vanatton indicated that the individual diseases follow a particular pattern a n lhe expresston of these two cell popuipioiis. we have studied fibrous turnours of the pleura using morphology. immunohistwhemislry and eleclronmaroscopy. the findings were compared and contrasted with reactive pleural fibrosis and desmoplastlc mesothelioma. the fibroustumwn hadarangeof histologicalappearancesand % weremalignant ~nnature.theimmunophemtype was uniform and consistent with positive staining for vimentin and alpha m w t h muscle actin. this was ~n sharp contrast to findings ~n reactive pleural fibrosis and desmoplastic mesothelioma. uttrastructural appearances of the fibrous tumours of the pleura were supportive of a myofibroblastic ongin. we propose that fibrous tumours of the pleura arise from the submesothelial myofibroblast. the malignant fibrous turnours have a distinct immunohistochemical profile and electron microscopic features to differentiate l from themalignant mesenchymai mesothelioma. a study was undenaken to evaluate the use of immunaperoxidase stains on paraffin embedded tissue lo define the cell type in routine lung cancer preparations. and in particular to identdy a subgroup of turnours showing neuroendocrinedifferentiation. forty lour consecutivethoracotamycaseswere selected. following apilot study of cases. to assess digestion times and potentiaily useful antibodies, the remaining cases were processed using a battery of monoclonal antibodies: cytokeratr iaelfae , e ). neuron specitic enolase (nse), chromogranin. and beta microglobulm. in addition to the carcinoid turnours and oat cell carcinoma in the study large cell carcinomas and adenmarcinomas demonstrated positive neuroendocrine markers. uiirastnrcturally, dense core granules could be demonstrated in only / of the large cell carcinomas and in / adenocarcmomas. the discrepancy between the lmmunoperoxldase staining and electron microscopic features likely reflects the heterogeneityafthese turnours. in thisstudy noneof the turnours co-expressed neuroendocrine markersand beta microglabulm. however, the staining panern was inconsistent m the remaining cases high molecular weight cytokeratin ( pe ) was stronglyposltlve m all casesot squamouscell carcinoma and negativein everyihing else. in summary. monoclonal nse and chromogranin appeared to provide sufficient information to identity neuroendocrinedifterentiatian in thecasesexamined m this study. high molecularweight cytokeratinwasfound to bea usefuldiscriminaforforsquamouscelicarcinoma. beta microglobulin was negativemall the turnoursshowing neuroendocrine differentiation. butthe inconsistency of staining m non neuroendocrinetumaurs. made it less helpful for routine laboratory use although endocrine differentiation is the essence of small cell carcinoma of the bronchus, its occurrence m other morphological ("on-small cell) types of bronchial umour (large cell, squamous and adenmarcdnoma) is welldescribed however, its prevalence m such tumours is uncertam, estimates differing from study to rudy and accordingto how it is sought. we have examined, byimmunalabellmg, expression offiveendocrine markerproteins (neuron-specific enolase (nso, protein gene product [pgp) . , the bb isoenzyme of creatine kinase [ck-bb), synaptophysinands-t protein)m bronchoscopictissue biopsiesof "on-smallcellcarcinomaandasse~ns varlabiltty within and between tumour deposits in subjects coming to necropsy with disseminated disease exactly half of the tissue biopsy specimens immunolabelled for one or more marken; one for four, four for three, twenty lor two and five for one. possibly indicating an element of endocrine dtflerentiation inapparent from their morphology. expression was even more prevalent amongst the extensively-sampled turnours at necropsy, but since theinlraductionofthenewgeneralpractnianercontractmapril ,lhere hasbeenasignlicant increasein thenurnbeisof skin biopsiesreceivedmhistopathologydepa~ments in ourdepartment there has beenalhreefold increase in numbers of general practiioner skin biopsies. the aims of this study were to crnlcally appraise these biopsiesand comparethem tosimilarlysized skin biapsiesreceivedfrom hospital in-patientsviageneraland plastic surgeons for the six months prior to and aner st april . data collected included numbers received, ranae of pathological diagnoses, quality information supplied on the request card, accuracy clinical diagnoses. adequacy of excision, age. sex and sites of lesions the resuns showed a similar range of pathological diagnoses. the quality of clinical information supplied was comparable in the two groups as was the age and sex of patients. general practitioner biopsies were less common from the face. clinical recognition of lesions was somewhat less accurate amongst general practitioners than amongst hospital surgeons inadequate excision was more common m general practnioner cases. . % of general practitioner lesions were found unexpectedly to be premalignant or malignant (eight cases) and all ofthesewere inadequatelyexclsed. important implicationsemergingfromthisstudy are dlscussed an audit of skin biopsy specimens from general practitioners in grampian region: changes in requesting practice and specimen type assessmen ofresaction marginsofsurgicalspecimens~s becming more important m manyfieldsofpathology. we wwe interested in developing a means of assessing surgical margins dermatopatholqy in both conventionally removed skin ellipses and m skin ellipses removed during m o~s chemasurgery technique many of the conventionally used markers, such as indian ink, alcian blue and tipp-ex correction fluid are difficult to use i" that they are messytoapply. slow todry and show insufficient contrast wiih oneanother both lnthegrass specimen and microscopically weused"superman'paint,apaintusedforresinandplastermodels,wh,chcomesin awlderange of colours. the paint was easyto apply. did not run and dried qwckiy becauseof the variety of colours avaciablewe were able to apply contrasting c~l o u n to the vertical and horizontal axes of b x c~s i o~ af skm elhpses removed by mohschemosurgery.thepaint proyidedagaodmarkergrasslyand wasnot affected byfreezinqthetissue model paint provides another marker lor surgical bxcij~ii margins and s particularly useful for moh's chemasurgery where horizontal and vertical axes aremarked inorderto assess theadequacyoftheexcision.the model paint may also be usetul in other branches of surgical pathology where resection margins are important. in psoriasis there is altered bpidermal dmwentiattan and increased epidermal turnover both which involve changesin intercellularadhesion aquantnative imm~noh~st chernicaicompa~~sonof the expression of the integrin staining of integrin p subunlts - and e subunlts were disclosed usmg an andin-blatln peraxidase techn!que. large epidermal dendritic cells (antigen presenting cells) expressed p- the psoriatic skm.showed increased - a- . - . and p- expression. buta- and~- showednosignificantdifferencefromnormal thelncreasedlntegrin expression by keratinocytes seems to be a reflection of awered epidermal differentiation rather than increased keratinoc ?turnover themcrease in ~- pasitivedendriticceilscauidbeareflectianofalteredanfigen handkngin psonatic gin. interleukm- (il- ). used m the treatment of patients with metastabc disease fallmg to respond to conventional treatment, can induce regression oftumwrbuik in certain patients however, the systemlc administration ofil- is associated wlth a number of toxic effects, including dermatological compl#catlons. these have been poorly documented we have prospectivehl studied the dermatological reactions patlents treated wiih il- for metastats cdorectal ~arconoma. pre-and past-treatment biopsies were obtwied where possible, arid sect~ons stained with h e. giemsaand pas: hesh tissues weresubjected to immunophenotyplng.there were femaleand malepatients.with treatmentrour~sranginginnumbwfrom - onlyonepatlenthadapastmedlcal hlstoryof any skm complaint (eczema]. four patlenk suffered a dlffilse eryihematous reaction with mild desquamatm and dryness the other patient developed genwalised erythroderma and an additlonal photosensitivity type reaction. histology, anerthe initialcourse. revealed patchyspmgios s. exocylosis and basal layer epidermaldamagewnh a mild perivascular chronic inflammatory cell mfinrate. with suhsequent treatment there wasthlckenmg ofthe epiderrnls, plgrnent incontinence. dermal oedema and more marked chronic perivascular cell tnfiltrate. immunohistcchemistry revealed markedchanges inthe expression of cdt , hladr, cam- and cd m the derm . these changes were greatly heightsnedwith subsequent liealmsnts. with addnional changes tn other t cetl markers. clearly il- enhances the parenchymal expression of antibody-dependent and antigen-mdependent accessory molecules which are important in focusing the immune response. claire m. thornton. maureen y. waish weexaminedalfprimarycutaneaus malignant melanomas seen rn thmdepartmmtover a fweyear permd from to . the total number of tumoum was the number of cases of malignant melanoma both invasive and m m u , increased from cases ~n to cases in . superfmal spreading melanoma was the most common type of melanoma, accounting for % of the total cases this was followed by the nodular melanoma whch accountedfor %. wlthlentigamallgnamelanomaandacral lent~g~nousmelanomabe~ngtheleast commontypes. therewasanincreasmg numberoftumourspresentlngwlth abreslow'sdepthoflessthan t mm. thefigurermng from % of cases in lo % of cases m most cases were stdl of ciar*e kud ivanhough mcreasing numbersaftumour presented at clarke levels i and i wlth a corresponding reduction ~n cases presenting at clarke level v more lesions presented with a flat cross sectlonal profile ~n the later years the figures lncreaslng from % in % in i w o he numbor of i c s~s showlng surface ulceratan at predentatlon d-reased from % in t to % in themltotlcact~vlty. thedegreeofp~gmentalion. theintensiiyofthe~nnammatorycell infiltrate. the predominant cell type and the mcldence of vascular lnvaslon showed no change over the study penod classification of benign vascular tumours is notoriousiy difliwlt and ciinicopathological correlation is often imprecise. this almost certainly reflects the tendency of pathologists to lump together different lesions under the broad heading 'haemangioma', sometimes with capillarylcavernous subtyping. twelve cases of a distinctive subset of cavernous haemangiomas, to be known as sinusoidai haemangioma, are presented. these presented in adws ( female. male. mean age years. range & ). five arose in the upper limb and five an the trunk (of which two developed in mammary subcutis). all were solitary and presented as a bluish cutanmus swelling up to . cm in diameter of variable duration. one case was associated with ipsilateral gynaecomastia. average foiiow-up of . years has revealed no tendency far local recurrence or metastasis. histologically these were subcutaneous/deep dermal lesions with a lobular, sieve-like appearance and focally ill-defined margins. they were composed of dilated. thin-walled interwmmunicating vascular channels with a pseudopapillary architecture. thrombi were common and two cases showed central infarction. vascular spaces were lined by monolayered endothelium which was often plump and hyperchromatic but n d mitotic. distinction from wnventional cavernous haemangioma and angiosarcoma (particularly in breast lesions) is discussed. current methods forthe identification of herpes simplex virus (hsvj may fail to identity the presence ofthe virus in biopsy or autopsy material. we have investigated autopsy cases and neurosurgical biopsy cases of clinically suspected herpes simplex encephalitis by a nested polymerase chain reaction (pcr). dna was extracted from routineiyprocessedand paraffin embedded material byproteinase k incubation, phenol chloroform extractionand ethanol precipitation. nested pcr was performed using known oligonmlaotide primers designed from the hsvtype giywprotein d gene from an area with the lowest homology with hsv type . two of autopsy c a w and of neurosurgical biopsy cases ware pcr positive for hsv. the third neurosurgical biopsy case was not confirmed by pcr as being due to hsv. such primers lo hsv allow the rapid retraspeaive diagnosis of herpes simplex encephaliis and should be of value to neurapathologists. tissueembedded at lowtemperaturein lowicryl-k m resin hasbeenshownto besuitableforimmunogoldlabellng of cellular antigens, and to be capable of withstanding the processing required for hybridization of nucleic acid probas.theaimofthisstudywastoestablish basic wnditionssuitabieforhybridirationof digoxigenin labelled dna probes to lowicryl embedded material, both at the light and electron microscope levels. cultured haemopoietii cells wereembedded afler brief aldehyde fixation. digaxigenin labelled whole human dna or plasmid per (negative control) probeswereappliedtothinandsem-thinsectionsafterproteolytictilgestion and/or denaturation by heat or alkali. hybrids were detected in semi-thin sections by standard wlourimetric methods. and in thin sections by immunogold techniques. elaborate blocking procedures and prolonged washes were found to be unnecessary. specificnu~l~earsignaiwasseenatbdhthelightandemisv~iwiththewhalednaprobe,revealingdetailsof nuclear dna distribution not evident in paraffin sections or cytospun preparations. non-specific binding and background were minimal. signal was greatly reduced if the denaturing step was mined, and was slightly increased by protwlytic digestion, though at the expense of cytoplasmic morphological integrity. while the sensitivity of this system is limited by the fact that hybridization occurs only at the suriace of the section, it is a rapid and specific means of nudaic acid detection, and offers the dossibilitv of accurate iocalization of intracellular human and viral nucleic acid sequences at a fundamental level. of prostatic carcinoma, mouse liver, kidney and gut were used. acp was demonstrated using an kc-dye cwpling method. the acp was unaffected by the addition of mm tartrate. although the acp was known to be 'tartrate sensitive'. the addition of mm tartrate or mm sodium fluoride weakened but did not eliminate the reaction. mouse tissues, prostatic carcinoma and leiomyosarcoma tissues were processed using variausfixatives and embedding procedures. these were tested for acp and trap. the acp in the muilinucleated giant calk of the leiomyosarcoma survived standard formalin fixation and paraffin wax processing and was tartrate resistant. as expected mouse liver, kidney and gut acp and prostatic acp did nd survive most fixatives and embedding procedures. however acp could be demonstrated in those tissues processed bythe 'amex' method. i.e. fixed in acetoneat - o' c. processed throughmethyi benzoateand xyianeto paraffin wax. theacp.thus imalised in paraffin blocks was eliminated by the addition of mm tartrate to the incubating medium. if needed the amex pracedure prrsenes some tartrate sensitive acp in paraffin blocks. acp that survives standard fixation and embedding procedures is iikeiy to be tarfrate resistant. showed acp only in osteoclasts. various elements ot tissue processing procedures were examined to find the wnditionsnecessarytoachievemaximum acp localisation. tissue blockswere pr-ssbdintowaxusingstandard embeddingprocedures. threefixativeswereused. % neutral bufferedfmalin, formalcalcium and ethanol allat + 'cfor hours.twodecalcifyingfluids wereemployed, %ethylenediamine-tetraaceticacid(edta)ph . at + %, zo' c and 'c.andformicacidisodium citrateat+ %forw hats. formalinfixed edtatreatedtissuaat + % produced maximum acp activity. acp was shown in osteociasts, some ostwcytes, chandrocytes. cement lines., tide mark, periosteaicellsand large macrophage likecells inbona matraw. formalinfixed tissues decalcified in edtaatzo'cand 'c werenegativeforacpaswerealltissuesfixedinethana . lnformalcalciumfixedtissueand formic acidlsodium citrate decalcified tissue the acp reaction was weaker. with some elements, e.g. chondrocvtic acp missing. all the acp preserved through paraffin processing was tartrate resistant. it is well recognised that morphology and optical resoiution are vastly improved with resin as opposed to paraffin embedding of tissue. however, difficulties in producing consistent immunopetoxidase reactions on resin sections have caused many departments to abandon thetechnique. although antigenicity is preserved the low optical density of diaminobenzidine (dae) means that the reaction product is barely visible in thin resin sections. the aim of this study was to develop a method whereby antibodies wmmonly used on paraffin sections wuld be successfully applied to pm resin sections. tissues fixed for - hrs in % formol saline were partially dehydrated and infinratedwithlr whiteresinat °cfollowed bypolymerisationat cusingacatalyticmethod. pmsectionswere reacted with polyclonai and monocionai antibodms using a standard indirect immunoparoxidase technique. visualisationwas with a silveramplification systemfordae (amenham) appiiedasthetinalstage. excellent resuns have been obtained with a rangeof antibodies including s , von wiilibrand factor. immunoglobulins, uchll and l (dakoltd.). usingthistechniqueitisnowpossibletocombinehighresoiutioniightmicroscopywithaprecise immunocytochemicai reaction. the advantages are obvious and are particularly relevant in the field of lymph node pathology. however, at and gythe number in muscle dropped significantly . and hoursfoilowing treatment, but hadrecoverad to w n t m levels byfivedays. in the lamina propria, agnor numbersincreasedinitiailyaflerthe . and gy treatments but returned to control values by five days. with gy the agnor numbers showed a significant fall hours after irradiation: this decline continued up to five days. it is evident that &nor numbers within the small intestine are affected following irradiation. the variation in counts is dependent on dose, cell type and time since irradiation. karen m. britten, w. r. roche oepartment of pathology, southampton unwewty general hosp~lai, southampton, so xy in response to ever-increasing demands for immunophendyping in inflammatory disorders, we have developed anernatwefixation and embeddingtechniquesforsmall biopsy specimens. bronchial biopsieswerefixed in buttered formaiin and processed lor embedding in araidite or were fixed in acetone containing protease inhibitors and embedded in the water-soiubk resin glycol methacrylate (gma). gma allowed for the investigation of a full phenotypic profile akin to that which may be wfwmed in frozen section while yielding far superior morphology and greater numbers of d a n s from small biopsies. the phenotypic markers included those for t-ceiis (cd . cd , cd ,cd andlfal),macrophages(cdllc, cd ),mastcelis~b b andaat)andeosinophils(mbp, egl and eg ). wehaveaisodemonstratednautrophiieiastase,cytokinesandtheceiiadhesionmolecuiesicaml .elamand vcam. similar high qualty sections were obtained with araidne but the repertoire of antibodies was restricted to thoseantibodieswhichcannarmailybeappiiedin paraffin. wesuggestthatforsmaii biopsies whichrequiredetailed immunohistochemistry, such as in the areas of transplantation and mucosa immunology, fixation in acetone at - % with the inclusion of protease inhibrors and processing into glvcolmethacwiate with careful temderature control gives optimum results. these included % formaiin at vanous temperatures, microwave treatments. bouin's fluid and a wmmercialiy avaiiabieproduct "rapid fix".thetissueswereroutinelyprocessed. embedded in paranin wax andstained wilhthe haematoxylinandeosin method. an eval"ation~fthefi~alion methodswasaisocarriedoutforimmunocytachemicai stains. from microscopic examination of the results t was evident that for the haematoxyiin an+eosm stains. % formaiinat o'cwa~ wnsistentiytheoplimum methodof choice. farimmunocytochamistryali methodsrasuited in a poor performance using a standard trypsinisetion time. however, an acceptable result was achieved from % formalinat °canda microwave fixative method. byreducingthetrypsinisation time. this. however, required strict wntroi. thus it is possible to fix tissue within one hour. by a method which is cost effective and which can be used within limitations for immunacytochemistry. we set out to study the extent and time dependency of storage related artefacts in cytospin fluid (shandon). we assessedtheeffectof stwage in cytospin fluidon the nuciearmarphoiqlyof breast fnac. fnaspecimsns were coiiected on day and slides were made from each specimen on day . day and day . these were fixed with a spray fixative. between each sampk preparation, the cytospin colleaion fluid was kept at %. the slides were fauigen stained and nuclear morphological parameters (area, perimeter, form ar. farm pe. and convexity-concavity) weremeasured using aseescansoiitaire plus imageanalysis system. there wasasignlticantchange in ail the parametem betwwn day and day , and between day and day (p < . ). the measurements were repeatableon differentoccasionswithoutsignificant difference in theresuns. these results indicate thatstorage in cytospin fluid significantly aners nuclear morphology. the nuclei become progressively larger and more irregular in shape. il is therefore important to standardize the storage conditions of fna specimens. if accurate objective comparisons are required. the identification of macromolecular components of hydrated patholqlicai tissues revealed by low temperature scanningeiectron microscopy (ltsem) isan emergingfieldof enquiry. in orderthatthetechnicaidetaiisforltsem labeilingmaybeestablishedanexperimentaiprotocoi has beendrawninwhichiabeiparticiesofhighatomicnumber are visualized. the experiment comprises a mcdei system in which bovine serum albumen as a known amigen is dissolved in phosphatebutteredsalineand adsorbedontonitroceiiuiosemembtane. theantigen onthemembrane is subsequently reacted mth rabbt anti-cow antibody and then elther protein a-goid or goat anti-rabbn-gold. aner treatmentwithadeveiopertoaddaiayerofsiivertothegoidparticies,thepreparationsareanachedtostubs,rapidiy frozen in nitrogen slush at - ooc, coated with aluminium and observed on the ltsem stage at approximately - °c. backscaneredeiectronimagingathighacceleratingvoltages( kv)isusbdtodetectsilver-enhancedgoid partic es.these areciearlyvisuaiized. thefrozen hydrated preparationsarestabieunderthe described wndtions of ltsem operation. in contrast dry, wnventionai. sem preparations are beam sensitive; their initially observed delicate uitrastncture quickly dearades an exeosure to even the moderate i kw electron beams emdioved in secondary electron imaging. a new approach to systematic storage of pathological specimens for low temperature scanning electron microscopy as greater numbers of pathological specimens are stored for wnvenient future imaging by iow temperature scanning electron microscopy (ltsem). it is increasingly important to maximise expensive cryostare capacity. a cassenesystem hasthereforebeendeveiopedwhichincreasesthe hoidingcapacityof cryostoresbyafactord . over conventional bee storage. each c-he consists of an aluminium disc . mm in diameter wilh cylindrical wells mmindiametereveniyspacedand . mmapart. specimens. mountedan jeolstubs,areretainedinthe wells by set screws. five cassettes fit into each x mm glass storage jar, gwmg a capacny for a canister cryastore(w th @rs percanister)of oostub-mounted specimens an addtionai benefit ofthecassettesystem is that each specimen is afforded a mechanically and thermally protected environment. specimen collection for ltsem may take place in a distant operating theatre or laboratory. lmmobiiiration of specimens whilst they are in transit excludes any possibility of their being damaged during normal handling and improves the prospects for survivalof accidents.afurther benefitisthataparticuiar mountedspecimencannow bemorespeedily identlledand removed for ltsem from rs well. many pathological condtions are charactensed by the presence of cellular degeneration accompanied by cytoskeietai abnormalities. in many such diswders it is not clear whether cytoskeietai abnormalities are pftmary process or are part of a secondary response to cellular insun by other agents or mechanisms. we have used a fibroblast ceii culture mcdei to study the effects of physical distension on the cellular cyioskeielon. inert beads of and pm were introduced intoceils byallowingendocytosisor bymicroinjection.thecytoskeietai response tothese beads was studied using immunofiuorescence microscopy. beads introduced by endocytosis migrated to the prinuciear region and over hours became enmeshed in intermediate filament and mlcrotubuiar aggregates. actln microfilament organiselionwas not alfected. incontrast. microinjectionof beadsproduced an immediatecollapseof morofilaments,wiulvimenbnandtubuiin distribution being presened.the immediateresponseto microlnlectlon is similar to the collapse of actin filaments s w n upon thermal stress. this experimental model has shown !hat aggregates of cytoskeietai proteins may be produced within cells as a secondary responseto intraceiiuiar debris, and that microinjection may induce cytoskeietal abnormalnies similar to those seen in thermal stress. thedetection of numerical chromosome aberrations m interphasetumourceiis bynonisotopic insitu hybridisation has been previously described but the application d t h s technique to paraffin embedded material has been wmpiicatsd bythe requirementfottissuesectioning with the production of partial nuciei. inthisstudy, the analysis - pm thickparaffin sectionsolconventionally processed caski ceilsusing both human papliiomavlrus(hpv type l e a d achromosome specificalphoid probe wascompared with resunsomained usingintactceils.the use of sectioned material did not give signal distributions comparable to those obtained using whale cells. this is consistent wnh a mathematical model derived for the relationship between section thickness. nuclear size and nuclear retention in paranln sections. a method was therefore developed for the extraction and analysis of nuclei fromthick( pm)paraltinsectionsandappiiedtotheanaiysisof squamouscelicarcinomasofthecervix(n = ). the number of copies of chromosome varied from to and this variation was apparent both between lesions and between tumour ceiis within the same ieaon. this is comparable to resuns obtained wth cervical carcinoma derived csll lines. there was. however, no clear relationship between the presence of hpv sequences and chromosome number. these preliminary results suggest that the postulated loss of host suppression of hpv gene function by deletion of genes on chromosome does not occur through gross chromosomal abnormalities. membranous giomeruionephritls (mgn). an immune-mediated disease, is a frequent caused renal morbidity in man. cationic bovine serum albumin (cbsa), given to nzw rabbits in a chronic serum sickness-type protocol. is knowntoinduceglomerularchangessimilartothe humandisease. we~ssedtheenectofashortcourseofthe immunosuppressivedrug cyaon thedevelopmentof early stage.cbsa-induced mgn. fourteen male nzwrabbts received an iv immunismg dose of mg cbsa and p g e. coliendotoxin. one week later they commenced daily iy inlectionsaf mgcbsafor ~onsec~t~vedays. three rabbtsweresacftficed atthistime. sixofthe remaining rabbits were commenced on a short cou~sb of o m cya. whilst continuing to recewe daily doses of cbsa. the remainingrabbitswsregivencbsaonly.after consecutivedosesofcbsathesell anlmaisweresacnficed.ali rabbns given doses cbsa showed early stage mgn and those given doses of the cationic protein showed a more mature, established disease (thickened glomerular capillary wails wth diffuse. global. granular deposition of igg/c and subepithelial electmn dense deposits). four of the cya-treated cbsa rabbns showed a marked reduction in giomerular capillary wail c deposition. three of these rabbns had considerably less severe disease ultrastructuraiiy. these results suggest that cya may aker the course of cbsa-lnduced mgn. 'oroartment of pathology university of edmburgh m&~aischooi: late renal aliogran loss s due to arterial intimai proliferation and iumenai narrowing. there are few studies ofthe phenotypes of the intimai cells. we analysed these lesions by light microscopy and immunmyiochemistry using antisera against t-lymphocytes. b-iymphocytes. macmphages, smooth muscle cells, class ii hla or molecules and the proideration antigen pclo vessels were studied from gran nephrectomies resected between and months post-transplantation. we identified an arterial endolheliaiilis, r-rted in cardiac aliografts, but not emphaszed in renal graftrejection. panernsolarterial pathoiogywwerewgnisd: ( ) endotheiialitis in nnteriobuiar arteries without intimal proliferation, ( ) endothelialiis in larger arteries accompanied by intimal prdtferat!on of smooth muscle. ( ) '"inactive" lesions with thickened intima (i foam cells) but no endotheliaiitis, and ( ) 'natural" atherosclerosis of larger arteries. the endothellaidis tended to m u r in shmw sumving grafts. the pedominant cell was the macrophage. with fewer t-lymphocytes. pclo was expressed in mononuclear ceiis, smwth muscle cells and endothalid ceiis. pmicuiariy. but not exclusively in younger grafls. we proposb these ledonsevolve, variably. from an early endotheiiaiitis to late chronic vascular rejmlon or gralt athwmierosis. the predomlnance of the macrophage at ail stages, suggests it plays a significant roia in the evolution of these lesions. the rat is used to study the response of the renin-angiotensm system m diseases such as hypertension there are structural differences in the jga butthere are few comparisons of its response to stimulalion between species we used renin antisera and an lmmunoperoxidase technique to stain renin-containing cells (rcc) ~n rat (n = i t ) and human kldneys(l nephrectomy and autopsycases). westmulated therenin-angintensin system experimentally by clipping one renal artery ( rats) and inducing sodium depletion ( rats) and studied the analogous human diseases-renal artery stenosts ( cases) andaddison'sdissase( untreated and treated eases). wecounted the rcc and plotted their distribution on scatter diagrams. there were some dinerences in distribution between the species but in boththerewasagradient in distributionof rcc which predominated m the superficial renal cortex. in sodium depleted rats. recruitment of rcc in the iuxtamedullary jgas abolished this gradient. while in some of the animals with renal artery clip hypeitension the normal gradient was reversed, with most rcc in the deep cortex. in bdhu~nreatedaddison' diseaseand~nrenalanerystanosistherewasanoveralincrease(xs)m rcc butthenormal gradientoftheirdistnbution withintherenalcortexwasmainlained.theseresults haveimpl,cationsfortheroleolthe intrarenal renin-anglotensln system in the control of renal haemodynamcs. we have developed a model of adult human prostatic epdhelium that allows arch$tectuml and cytologioai features to be maintained. epithelial organolds produced by enzymic digestion of human benlgh prostatic hyperplasla tissue weresuspendedm type collagengelandsubcutaneouslyxenogranedintointact malenudemice.thexenogranis progressively invaded by mouse stromal cells thesesurround theepithelial organoids and supportthe reformation of epithelial structures with a lumen, lined by a mixed epithelial layer. as the lumen forms tall columnar epithelial cells begin to be seen, these progressively express the prostate specific epithelial markers. psa and psap further the xenograflsexpress appropriatecyiokeratin markers in boththeluminaland basaleplthellal cells. gelsplaced within a . pm millipore chamber, which do not undergo stromal invaston. lose all epithelial organisation with disorganised sheetsand ballsofcellsbeingfound.these cellsdonot express thesecretory markers. in the absence lanandr genic~tim~i~sep,th~l~~l structures wsth a lumen are formed but there are no tall columnar secretory cells and noexpression ofthesecretory markers. thismodel hasfurther been investigated todetermine theresponseof human prostatic cells growing in vivo to the antiandrogen flutamide and to a -aza-steroid e-reductase inhibitor these observations indicate the essential role of both stromal cells and androgens m dictating functional dlfferentlatnon this model will allow the dissection of the regulatory processes involved in prostatic differentiation. hepatocyte growth factor (hgf) is the most potent known mitogen for adult rat hepatocytes in pimary cuiture and isthoughtto haveanimportant rolelnlivergrowihandrepalr linle~sknownaboutthemechanismofactionof hgf on hepatocytes. since the adenylate cyclase system has been implicated in hepalocyie growth control. we examined the role of adenylate cyclase and cyclic amp wmp) in hgf-stimulated dna synthesis. human recombinant hgf (hrhgf. numl) had no elfect on baa or stimulated adenylate cyclase activity in membranes prepared from freshly isolated rat hepatocytes. similarly, hrhgf had noeffect on intracellular camplevels in cultured hepatocytes. furthermore, agents which increase camp inhibited hrhgf-stimulated dna synthesis m primary hepatocyte cui ures glucagon had an i . c. , of lo-'; fotskolin ( . ibmx ( "m). -broma camp ( pm) and dibutyryl camp ( pm) completely inhiblfed hrhgf-stimulated dna synthesvs. from thts. we conciude that adenylate cyclase and camp do not have a role m hgf-stimulated dna synthesis m pnmarycultures oladult rat hepatocytes. the receptor for hgf has recently been identified in tissues other than the liver, as c-met a protooncogenewith intrinsictyrosinekinaseactivitv. whether or notc-metactsasthsrecedtorfor hgf ~n heoatocvtes s currently under investigation a novel in wvomodel of intestinal differentiation is descnbed. fourteen-day, undifferentiatedfetal rat small intestine. stripped of the major part of s mesenchyme. then suspended in a type i collagen gel and renografted ~n a nude mouse. undergoessmall mntestbnal morphogenesis and cytod$ffeienbat$on. all tourmapr epithelial imeages. namely paneth. goblet. columnar and endocrine. are present. double labelling m sit" hybndlzatlon, employing biotinylated and digoxigenin labelled dna probes to whole rat dna and whole mouse dna, reveals an unusual ]uxtaposition of species specific stroma. the outer longitudinal smooth muscle layer, and the major part of the lamina propria. p isthemost commdnlyalteredgene~n humanturnours. mutati~nleadstothep~oducfionolanabnormalprotein which can be detected by immunohistology. such abnormalales are seen in a wide variety of turnours, including colon cancer. abnormal p protein levelshavebeen detewed in & % of sporadiccolorectaltumours. in order to determine i p mutations occur in carcinomas arising from dyspiasla. we have investigated the prevalence of such mutations m colorectal carcinomas from patients wr)h long-standing ulceratwe colitis %). lmmunocytochemicalstalnlng waspwformedon freshand paralflnembeddeduccarclnomasand sporadlc carcinomacontrols matched forsite, stageand grade six areas ofdysplasla, (fourassoclatedwlth uccancers)and sporadic adenomas were also stained. p protein was detected by immunohistochem~stry m the fresh uc cancers and / ( %) ofthe paraffin embedded uc cancers usmg the antibodies cmi, pab , pab and pab . slmllarresuitswere obtained in thesporadic carcinomasw,th fresh cancers positlvefor p and / ( %) of the paraffin embedded cancers. two areas of dysplasla which were associated with p postlve uc cancer~also showed positive p staining, alongwith an adenoma. our results indicatethatunlike k-ras mutations, p proteinabnormalities occurat a similarfrequencyln sporadic colorectal carcmomas, carcinomasar sing !n uc as well as being present in uc dysptasasla. this work suggests that p mutations play a mle m the dysplastacarcinoma sequence. threepanernsofstainingareloundin humancolonusingatechniquetodemonstrateo-acetylationofsialomucins (mpasj. % afindivdualsshowunifwmmpas-positivity. theremaindwareeitherent~~ympas-negativewmpasnegativewith occasional positive crypts. we suggestthatthisrepresentspalymorphismof an autosomal gene ( sa) controlling -acetylation of sballc acid. isolated crypt-restricted mpas-posltivky in otherwise negative indivtduais representing somatic mutation of the ma* gene in crypt stem cells of osa'/osa-individuals. to test this we have studied colons from patients with rectal carcinoma, half of whom had received cgy radiation days preoperataely. radialiond~dnotaffecttheprevalenceofthethreepnenotypesbutincreasedthefrequencyofmpasposltlvecryptsinanegativebackground(t ~ ~~~ . ~ to*, p < . ), largelyduetocryptsshowingseclorial mpas-positivlty ( . x lo* ys . x to , p < o.c€ql). consistent with incomplete crypt colonisation by a recently mutated phenotype. the prevalenceofthisosa*/osa~phenot~e(radiated %, non-radiated %) isveqcloseto the predicted heterorygosily rate ( . %. hardy-welnberg law). thew resuns suggest that human colonic crypts are monoclonal with a longer stem cell cycle than the mouse and that mpas staining provides a method for measuring human stem cell mutattonal load. pouchrris m leo-anal resarvoirs is a frequent complication of restorative practocolectamy which is associated wtih constderable morbidity. long term complications of pouchitis are unknown, however patient follow-up wlth sigmoidoscopic surveillanceis mandatory to assessdysplamc or neoplastic changes in resldual rectal mucosa. we examined epithelial cell proliferative activdy in the ileal mucosa of pouch biopsies using the antibody pclo which detects nuclear expression of the kd nuclear protein proliferating cell nuclear antigen (pcna). ten patienls with functtoning ,lea-anal pouch reservoirs of at least one year duration were biopsied and assessed histologically for evldence of pouchitis formalin fixed paraffin embedded biopsies from anterior and posterior pouch wall were examlned with pclo using the pap technique. ten terminal ileum sctions from right hemicokctomy specimens werenormalcontrols. cryptsin pouch biopsiesand normalcantralshadasimilarpciolabelling indexmeanof k and % (mean values - and w ) respectiveiy the sides and tips of villi had a significantly greater pclo labellmg indexinthepouch biopstes( mean. range - ). compared withnormaicontrols(t % mean, range - ) these resulk demmstiate an expanded proliferatwe epnhelial compartment in ileal pouch mucwhich in contrast to normal terminal ileum involves villous surfaces. considering the frequency and long term natllrs pouchitis. thesefindingssupportthe need for continued pathalogical and clinical assessmentof the ileal mucosa in the neo-rectum of these patients. we, cui, i c talbot, j. m. a. northover signiticant alterations in structure. function and gene expression of mltochondna have been reported in cobrectal turnours. but it $ not known rf these abnormalities are due to mitochondria genetic alteration. in this study, total cellular dna was isolated from l lo rectal carcinomas, adenomas and their adlacent histologically normal mucosa. these dna samples were digested separately with different restriction endonucleases. and then analysed by southern blotting using apurified mtdnaprobe. the restriction fragment pattern oftumourmtdnawas comparedta thatofeorrespondingnormalmucosalmtdna.theseresultsshowedthattherearenalargedeletions. insert~ons, or rearrangements $n turnour mtdna. and no single base changes m the delectable regions in spite of some polymorphic vanations. our results suggest that mtdna changes are unlikely to have a malor role in human colorectal tumoungenesis. hence, alterations in colorectal turnour mitochondria must be dependent upon other mechanisms crescentic colitis: the clinicopathological spectrum of a distinctive endoscopic feature in the sigmoid colon n. a. shepherd, s. gore, s. p. wilkmson oepariments ofnsfopafholcgyand gastreoterol~y, gloucestershre royal hospml, great western road, gloucesfw, crescentic coibs describes an endoscopic appearanc of the sigmoid colon charactwised by mucosal swellmg, eqhema and haemorrhagestrictly localised tothecrescentic mucosal folds. in alive yearpericd thisdiagnosiswas made m patrents, representvlg .a% of ail fbeopta endoscopies. there was a male predominance and most plients were middle-aged or elderly. dwerticulosis waspresent m most ( %) but theabnomallf~es wareconfined to the crescentic mucosal folds with sparing of the divenicular onfices. the malorlty of patients presented with a history of bleeding perano. histologically there wasaspectrum of changesvaryingfrommlnorvascularcongestian to florid active inflammatory disease with crypt architectural abnormalities mimicking ulcerative colitis. three patients presenting with crescentc colitis later developed the clinical, endoscopic and histopathological features of distal ulcerative coliitls: two other patients with a history of distal ulcerative colitis were found to have the charaderlstlc changes crescentic colitis only at endoscopy. three cases showed the histological features of mucosai prolapse the findings in thisstudy demonstratethat arelativelyspecificendoscopicfeaturemayexhibit a wide spectrum of pathological changes whilst luminal mucosal inflammation of the sigmoid colon, usually in a sociat on wiih diverticuioss. may mimic the pathology of chronic inflammatory bowel disease. asmall propartion of these cases may represenl a strictly locallsed form of chronic ulcerative colitis. of genome and antigen a n various anatomtcal sees the resuns have imolications lor the mechanism of e n t~ of mv into neurons and for mechanisms of transynaptic viral spread neuiopathology, fnstitute of pathology j chow, j tobias.' k co ston. t j chambers estrogen is generally considered to maintain bone mass through suppression of bone resorption we have previously demonstrated that administration of pharmacologic doses of estrogen increased bone formation in ovary-intact rats to assess theeffectsof physiological concentrationsol estrogen on boneformatian. estrogen was administered to ovariectamised rats fi n which bone resorption was suppressed by ahprbp. animals receiving exogenous p-estradtal (ed ( wglkg. t o wgikg and w pgkg daily for days) showed a dose-dependent increasemtrabecularbonevolumeaft %. . % and . % respectively, compared wnhthoseratstreated with ahprbp alone. the increase in bone volume was due to bane formation in e,-treated animals, in which bone resorption had been almost completely suppressed by ahprbp neither ovanectomy. ahprbp nor e,-treatment had asignificanteffecton thevolumeorrateoflormationofcortical bone thus. theiocreasedbonerasorptionwhichis aconsequence estrogen-dellclency entralns lncreased bone formation, whlch masks a slmunaneous reduction in estrogen-dependent bone formation. it thus appears that estrogen maintains bone volume not only through inhibition of bone resorption. but also through stimulation of bone formation. pgs m bone formation m vmo. which may represent a pathway common to bone anabolism that s observed !n response to many e""lio"mental t m"ll pgf , was wlthoul effect we found that nodule mductton by pgs occutred early the cunures, belore nodules lntegrln expression e n human bone was examkned by immunohistolqlical stalnmg of edta-decalcrfec and undecalcllned cryostat sections of fracture-and tumour-associated callus dbtalned at surgery and neonatal costochondral )unctions obtained at autopsy. cases were stained wlth a panel of well-charactsnsed monwlonai antibodies against p - . el- and uvp integrios using abc peraxidase and indirect immunofluorescence techniques. osteoclasts stained lor pl , p . u and ovp mtegnns. indicating that they express u p nlp- ) and uvp (classm vttranectin receptor) ostmblasts stained lor pl, and u , and osteocytes stained for p l and . indicating that ostmblasts express n pl nlp- ) and m gl nlp- . classical fibronectin receotorl and that vlp- expression s reduced or lost during differentiation to astwcytes j qulnn. n a. athanasau nufield depanment of paihology and bactenotogy, level , john radcoffe hospital, oxford, ox du osteoclasts are known to effect bone resorption in inflammation and malignancy but whether other cells of the mononuclear phagocyte system. particularly macrophages and macrophage palykaryons, are similarly capable of pathological bone destruction is uncertain macrophages derived from tumours (human lung camnomas. murine mmw-assmated mammary carcinomas) and inflammatory lesions (munne foreign body granubmas) were cuituredonboneslicesbothin thepresenceandabsenceaf stz slromalcells.theseneoplasticand inflammatory iesions contained a heavy macrophage infiltrate but no giant cells and no calcified tissue. there was superficial roughening ofthebonesurfacebymacrophagesboth inthepresenceandabsenceofst cellsand.aher t days. scattered areas of lacunar resorption n co-cultures of macrophages and st cells. normal pulmonary tissue macrophagesdid not produce resorption lacunaeunder these conditions. the results show that macrophages alone are capable of atype of low-grade boneresorption and that a subpopuwion of turnour inflammation-assaoated macrophages following specific interaction with slromal cells, can differentiate intocellscapable of the special sed function of high-grade lacunar bone resorpt on macrophages may thus directly contribute to the mteolysis associated with metastatic turnours and inflammatory lesms in bone differences in the grade of osteolysis may also account for clinical differences tn the degrees and rate at which pathological bane resorption occurs the george washmgton university medtcal center, washnqton, c , u s a for decades, new technological advances have been hailedor condemnedas representing the extinclion cla~s~cal diagnostic surgical pathology, but so far the reports of the death of our specialty have been grossly exaggerated. indeed, the newtechnologies of the sare seenas aids torathet than replacementsfor careful and intelligent gross and micmscopic examination and interpretation, which havealways remained the 'gold standard" against wnichnewtechniquesaremeasuted thedecade will bemarked by anexplosionof pos ibilitiesfort ueas well a non-tmue diagnosis, balanced by a shrinkage in both specimen sizes (already evident in breast pathology) and healthcare budgets.thus. thesurgical pathalogist will haveto becometoanevengreaterextentthecomplete physician. in order to beable to choose wiselyand economically from thediagnostic"menu"availab e. thesurgical pathologist w d also have a greater need than heretofore to be a competent cytopathologist as well, as many of his or her cases will also have fine needle aspiration material and a nuclear grading will assume greater significance o n tumor pathology. finally, the roles of the surgical pathologist ~n both research and patient care (including direct patteoi contact) will require emphasis m order to attract more medical students into our specialhl. womenorthosewithlowgradedisease.this hasledto theviewthathpv s~nielatedtocer~ c~l cancer asimple pcr protocol was developed using standards containing . - fg of hpv dna in ng of normal human placental ona toestimate levelsaf hpv dnain smearsor biopsies. the quantltativedistribution of hpv dna tn normal and abnormal ~ervlcal epithelium was mapped thmughout loop biopsy and hysterectomy specimens using micro-dissectionand histologyof alternate millimeter slices levels were measured in parallel c~n~c~i smears. preliminaryresuits show a low lwel of hpv dna(equiva ent to one copy per cells) was usual in women with normal smears or low grade abnormalities replication of hpv dna to a level of one copy or more per cell was limited to areas of cin ~ which may be very small and to atypical immature metaplasia. the level was reflected ~n the smear aswitch to a high level of hpv dna s a biological and potential diagnostic maherfor high grade precancer. cerv cal biopsies were stained by the cracker technique to demonstrate agnors. for the purposes of the study, b~opsiesweredividedlntaflvegroups-normal, kallocytosis.cini.cin iiandciniii.usinganiemps/ hastingan a m s photon framestore a customised image analysls system was created. the image s dllated and eroded to close any cavities an outlining procedure locates the agnors and records their mid-pants. from this st of midpants themeandistanceofeach agnor'slhreenearest neighboursiscalculated.thlsprocedurescanledoutfor several lelds of view. this study attempts to relate the shape of the associated "mean distance" histograms to the histologlca diagnoses in flry cases. primary adenocarcinoma of the cervix: a retrospective clinicopathological study of cases r ananoos, kamta nahar.' alison b,grigg.'s. roberts? sergin m. sma the patientfen healthy with a tendency togain weight being heronlycomplaint. gynaecological referral revealeda week gestation abdominal mass and bilateral varicose veins. laparotomy, lee oapharedomy, total abdominal hysterectomy and right salpingo-oophorectomy were carried out the len ovarian turnour was cyst~c, smoath surfaced. unilocular,cantainedturbid brownnon-greasycontentsand was cmln diameter.al cmsquarearea of lhe lining showed papillary tutmg. and the remainder was smooth. hiatology of these papillary areas revealed a papillomatow proliferative squamous neoplasm with keratinization and mild acute inflammatm. wthout evidence of cytologicalatypia.stromal invasionora benigncysticteratoma.theremainderofthecystshowednat swamous. cuboidal, or low-coiumnar type epithelium with an underlying vaguely endometrial type stroma in minute focl, with occasional small secondary cysts thls unusual squamous neoplasm was diagnosed as a proliferative epbdermoid meglomerularperipolarcell isan epithelial cell snuted atthe vascularpole ofthe glomerulus recently, theexistence of the penpolar cell has been doubted the aims of this study were first, to establish whether the peripolar cell is a m q v e cell type ~n the mammalian kidney. and second, to compare their numbers and morphology in different speces we used scanning electron microscopy to study - kidneys from each of mammalian species including man. we removed the glomeruli by microdissection and examined a minimum of vascular poles from each kidney penpolar cells were largest and most numerous m goat and sheep kidneys ( w% and % of glomerul~) meywerescant~estandsmallest humanand bovinekidneys( % and %).whileaspectsotsome per~polarcells resembled podocytesand otherperipolar cells had features reminiscent of parietal epithelialcells. in each ~pe~ie~wewereabletadistinguishperipolarcellsasaspeciflccellhlpe. weconcludefirst. that theglomerular peripolar cell s a unique cell type ~n the mammalian glomerulus, and second. that their morphology and number cystlc renal disease occurs in various forms characterised by dilatation of different pans of the nephron. the morphologoal, clinical and genetic features of these diseases is variable but some animal models have been developed m an attempt to understand the mechanism of cyst formation within the nephron. we describe a polycybtic . .ofthe kidneyinthscea-n mousew~hanx-linkedrecessive~mmunodetic,entsyndrome there is progressive cystlc dilatation affecting all parts of the nephron. the cyst lining is composed of a single l a y m c epithelium with focal nuclear crowding and the formation of micropapillary structures. the cystlc epithelial cells show subnuclear vacuolation focal basement membrane thickening is also a feature. there is no signdicant inflammatory infiltrate present within these ktdneys. election rni~roscoptc examination w e a l s that the subnuclear vacdation s due to loss of the membrane infoldings at the basal pole of the eplthelial cell wlth flud accumulatlon within the extracellular space. the basement membrane thickening is due to expansion of the lamina dense. the finding of a plycystic kidney lesion on these mice offers an opportunity to investigate the relationship beween the immune system and renal cyst formation. eqthropomtin. a circulating glywprotem, is the principal humoral regulator of elythropoiesis. it s produced in the kidney butthe precisecell oforigin iscontroversial. erythropoietin participates in a~ ~ssicaif dbackcontroi system whch attemots to restore oxvaen deiivew lo the tissues. normallv erdhrowiatin is resent in the sbnm at picomolar c mntrations bui revels may h e up to oid durilg ?&erehkpoxic ;tress. the mechanism linking renal oxygen sensing with erythropoietin synthesis is p o~l y understood but there s evidencethat cells of the innsr cortex respond to tissue hypoxia by producing elythropoietln. eryihropoietin geneexpression in the kidney can be detected by northern blot anaiysis within one hour of exposure to hypoxie stimulation. the cellular location of erythropaietin m-nger rnawasdetected inamurine model byinsdu hybridizationemploying radioiabeiled dna probes and autoradiography techniques. tubular cells of the inner renal cortex wbi b identifled as the main site of ervfhropoietin gene transcription. the location oterythropaetm production was confirmed by immunohistochemistry with anti-sera ra sed to pure recombinam dna-denvea human elythropoetin. the specific antibodies bound oniy lo tubular cell cytoplasm, confirming the tubular location of erythropoiet,n-producing cells. severe reversible acute renal failure and iga nephropathy r jackson, k l. c. mclay unrvemty diws~lon of pathology, giasgow ropl infirmary, castle sireel. glasgow, g osf whilst progressive, irreverable renel failure is well known in the evolution of a stgnificant proportion of cases of iga nephropathy, disease-related. reuerstbie, severe deteiioretion in renei function s a less well recognized phenomenon. relatively few cases aredocumented in the literatureand these have invariably been associated with m-iw haematuna. renal failure has been largely attributed to a combination of tubular obstruction by red - cast~andacutetubularn%crosisthaughttabetheresunof adirecttubulo-toxiceffectof released haemoglobin. in a review of cases of i@ nephropathy we have encountered two cases of this type. the clinical. histological. immunohistochemical and ultrastructural data relating to these cases are presented. on the basis of this evidence and an analysisof the relevant literature. isconcludedthat the accepted pathogenetic hypothesisdnot provide an entirelysatisfactaryexpianationof the phenomenon. itisfurlhersuggested that immunologically mediated injury dfrected at the glameruiar vascular pole and possibly the extraglamerular mesangium could compromise thetubular blood supply and might also lead to haemorrhage into the distal tubule at the macuia densa. scanning electron microscopy (semi when combined with electron microprobe analysis (mpa) s an expeditious and accurate method of identitying substances deposited in tissues. we report a case in which these techniques were used to study crystals in a transplanted kidney. the patient presented at age three with vitamin d resistant rickets and fanconi syndrome on the basis of cystlnosis. he gradually went into renal failure and received a kidney transplantfrom hismother. itfunctionedwsiifarafewmonths butthen deterioratedandwas removedone year after transpiantation.afewcrystalswereob~ed by light microscopy ma biopsytakenattwomonths. arepeat biopsy at four months was examined by sem and contained the typical hexagonal cryystais of cystine. the s o i i i sulphur peak obtained with mpa confirmed their elemental compojltion. the hydrophilic nature of the cystlne prevented their identificetion within routine sectionsfor transmission electron microscopy (tem). the hexagonal o~tline of aystaiswas confined to interstiitat macrophages rather than tubular arglomeruiar ceiis. pm frozen sections mounted on carbn pianchets diowed vlsualisation and analysis of the crystal deposits by sem. the use of appropriate processing methods may be crucial m identifying crystal dspasition within pathological specimens. during the year period - . new cases of dense deposit disease (m~~b,a"op,olif~=ti"~ giomerulonephritis type ill were diagnow in nonhern ireland. two further known patients developed recurrent disease in renal transplants. the mean age of onset was years (range eight- years) with five males and eight females affected. renal function was impaired in % patients at presentation which ranged from nephrotic syndrome ( %). nephrilidnephrotc syndrome ( %) and macroscopic haematura with mild protelnuria ( %) lo mild protemuria f microsopic haematuna ( %). serum c was universally low. haw of initial biopsies showed cr-entsusuailyaiatedwith rapidansefrenaifailure. % ofpatientsrequired diaiysisatorwithin twomonths of presentation. a further % d patmnts developed renal failure within three pars of disease onset of the six patientstranaplanted haw havedaveioped rmrrent diseasin their grafts six patients havenot required dialysisat ameanfoilaw upoffour yearr(ranse neloeightyears). mhaugh reialweiyuncommon, dense deposit disease isan important causeof renal failure in children and adolescents. we have confirmed that this disease has a poor outlook in terms of renal function with a tendency towards recurrence in grafts a review of renal biopsies from patients with diabetes mellitus. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . showed with diabetic giomeruionephropathyalone, with other renal diseaseand with both diabetic giomerulonephropathyand other renal disease. eight with the histologiwl appearances of diabetic nephropathy without known diabetes were alsn identified. clinical details were available on ofthe patients. these showed that in type (insuiin dependent) diabetes ( ). most ( ) showed diabetic giomeruionephropathy, showed both diabetic glome~lon~phropathy and another diagoaas, and showed aniy another diagnosis in type (non insuiin dependent) diabetes ( ). showed diabetic glomerulonephropathy with showing another renal diagnosis without diabetic giombrulonephropathy, and with both diabetic glomeruionephropathy and another diagnosis. patients investigated with nodular glomerulosclerosis resembling that seen in diabetes but not known to be diabetic at the time of biopsy, showed abnormal glucose metabolism on investigation and t showed a paraprotein. this review showsthatinpatientswithciinicairenaldiseaseanddiabetesmeliitus,therenaldiseasecannotalways beassumed to be diabetic nephropathy especially ~n type diabetes where a wide range of other renal disease can occur histological findings of diabetic nephropathy may occur in patients before diabetes is diagnosed cllnlwiiy. a j. hawie, r. l. bryan in a biopsyof arenaltransplant. we notedan artmalabnormalitythat wasunrecorded in standard texts. in an arcuate artery. theiewastheappearanceafformation ofanewartery inside theoid, with layersof muscleandelastic fibres that in places resembled an internal elastic lamina. separated from the original internal elastic lamina by loose connective tissue. in a systematic study of consecutive transpiant biopsies and consecut~ve transplant nephrectomies. another six examples of this abnormality were found. these six and the index specimen showed changes cons~stent with chronic vascular rejection. in a systematic study of consecutive nan-transplant renal biopsies showing interstitial nephritis. another example ofthis lesion was found, in a kidney showing chronic interstitial nephntis. thischange isprobablyavmantfom of muscularisation ofthsarteriai intima, isseen in chronlc renal damage, and occurs in transplanted and native kidneys. localised myloidasis ofthe lower genito-unnarytractisararedisease. few studleshaveanempted tocharacterise theamyloidtypeusing immunohistochemicalstains. wereportaseriesofnin~casssinvoivingthebladder( ), lower ureter ( )and penile urethra( ). thesecomprised malesand pfemaies with an age ranged & years. nonsof the patients had evidence of systemic amyioidoss. three patients had a past history of lower genito-urinq tract infection and of these, one had repeated instrumentation. one patient had prostatic carcmma the cmmonast presenting wmpiaint washaematuriaand the mostfavouredsurgicaldiagnosiswasa neoplasm. fowpat#ents had repeat biapsiesfor persistent orrecurtentamyloidosiswith atimeintmal ofupto t yearsfram initial presentation. bght microscopy showed amyloid deposmon throughoutthebiopsies, in lamina propria. muscle. adiposetissueand vessel wails. with a variable giant cell reaction and lymphoplasmacytic infillrate. an abc-immunaperoxidase techniquewas usedwith antibodmsto pcamponent, serum amyioidaprote n prealbummandkappaand lambda light cham in an attempt to classify the amyloid type. this was found to b e d non-pa. non-prealbumm type. a negative or equivocal reaction was seen for kappa and lambda light chains however, such antibodies may not necessarily be immunoreactive with light chains or fragments of light chains m amyloid deposits. features of bile reflux type gastntis: glandular distmion (branching, "carkscrewmg"). nuclear regeneratwn, intramucosal smooth musclefibresandpaucityof inflammation. helicobacterpylanarganiams werenoted bileacid concentration #n gastrc aspirates collected by endoscopy at the time of biopsy was estimated using an optical densitymethod.gi casesshowingglandulardistorti~ hadraisedgasttic bileacidconcentration.m cases with mahednuclearregeneration hadraised bileacidsandof case~withinbamucosalsmoothmuxlefibres had raised bile acids nine of cases with raised bile acids showed a chronic inflammatory response the histological features of bile reflux type gastritis which correlate best with estimation of bile m gastric juice are glandular branching, "corkscrewing" and intramucosal smooth muscle fibres whereas pauclty of chronlc inflammation does not h pylon was identified in of cases with raised bile acid. aims to compare the bacterial flora of normal and inflamed appendices. and to conelate this with various histological features. to establish the incidence of yeninla infection ~n acute appendicihs !n southampton. methods resectedappendicesweresentheshta histopathologyandal cm portionwasremovedandcultured for aerobic and anaerobic organisma. the remainder was fixed ~n % buffered formalin and pocessed for rwtine histology results: appendices showed acute ap+endicitis, and were normal. histology there were statistically significant differences between the two groups for the p s e n c e o f faecollfhs (p < . ). fibrosis (p < . ) and prominent follicles (p < ) -all were more common ~n the normal appendices. microbiology no yer~inia was cultured in enher group. there were statistically significant differences in the number in each group which grew anaerobes and streptococci. anaerobes were more common in the normal group (p < . ). and streptococci morecommon in inflamedappendaes(p< ) conclusions. .thereisanaiiwed bactenalflora in acute appendicitis. yersinia does not contnbute to acute appendicitis in southampton. faecoliths. fibrosis and prominent follicles are significantly more common in normal appendces le antigen (using cat - ) a i d type h antigen using a specific moncbnal antiserum. we f w n d a widespread distribution oftype structures in both benign and malignant epnhelium oftheextrahepatic biliarytract and ampulla of vater. type h antigen expression was seen in benign epnhelium and in non-papillary tumoun and ampullary carinomas. lmmunohistochemical detection of tyw blood qrou antlqens does not adpear to be ofprwnostlc the majority of cases bearclose relationship to human ulcerativecolitisclinically, endoscopcallyarid i" re-nseto treatment one hundred postmortem iiyerswerestudiedfromconontoptamanns with totalseversul~ataecolitis who were pathogen free and had a histological picture resembling human uicerallve cdhs only iivwere normal in there was a mild penportal chronic inflammation. had extens~ve steatosts, had an appearance resembling chronic active hepatitis and ~n the histology resembled that of sclerosng cholangitja. other hepatic pathologies were s e n in smaller numbers. these changes parallel the itver disease seen in association wlfh "icbrativbcolitisinman. webelievetheconon toptamannprovidesthefirst modelof l i~e r d i~~~~e m u l~e r a t i v e~~l n to allow sturty of the pathogenesis of the enralntestinal manifestations of ulcerative colitis. r-rvolr and ilm-anal anastomosis. pre-and pan-surgical specimens were studied and compared using routine histological and histochemicaltechniqm themalontyofcasssexhibitedan lncreasem chronc lnflammatlon wlth n or noacute inflammation.the levelsof chronic innammation were found to bemost severe i" those cases which ~~fferedhompouchiti~. morphomstncalanalysisrevealedan~ncreaseincrypt depthtovillousheightration(cdvh) ~n % of cases. the cdvh in the pouchitis cases was greater than n the other specimens studied the resuns of m u m histochemical analysis did not show any charactenstic changes occumng. in the cases studied. lectm histochemistry demonstrated an increase an supranuclear staining of the pouches with dba. sea, wfa and wa. which is similar to that fwnd in poximal colon. staining with psa, lca. u f and lta revealed charactenstic changes in binding panem th-may mdjcale changes m tucosylation of certain cellular canponents. certain changes in lectin binding were found to be more sjgnificant in the pouchitis group of cases. the changes in the ra~ewoir mucosa are most irkely to be an adaptive response to the new mtra-lummal environment wlth the acqmdfon of cmain mime chaiacteristics. thereare alsosp~ciftc changes which occur to a greatsrdegree m the cases with wuchits, these may occur as a result of or lead to the occunence of puchms. these resuns may be cryostat sctionshomten patientswith ileal pouches lor extensive colitis, ten distal ulcerative ~olitics and five normal mall intestines were assessed immunohistochemically for macrophages (leu m antibody). rfdl gnterdigitating antigen presenting cells). and rfdt (resting macrophages)antigencantainingcellsin lminapropna. result~areexpressbdasape~=~~t=g~~f thetotal. normal small intestine contained a mean ? % and % (ranges - . - , and - ) leu m . rfdl and rfdt positive ~ell~re~pecl~~elywh~chcontrastswiththoseh~ghervaluesinpouchbiapsies- %, %and %(ranges - , - and )respectively. distal ulcerative coilticscontalned increased macrophagenumbers( . and % for the three antibodies) but have a predominance of the rfdi positive population in contrast to pouchitis patients in whomthe rfd positive cells predominate thesefindings demonswate remarkably high macrophagenumbersin pouchitis, howeverthe rfd positive sub-populations predominance suggests an aetiologv other than ulcerative colitis for muchitis. margaret balsltis. yah mahida the wide family of cell adhesion molecules (including the subgroups of the immunoglobulin supergene family. the integnn receptors andtheselectins) are involved in controlling interactions between endothella cells and leucacytes !n inflammatory states. this is partly by their influence on adhesion and migration of leucacytes to and through endothellurn. using monoclonal antibodies, we have compared expr~ss~on of the three cell adhesion molecules icam-t (interceiiular adhesion molecule- ). elam-t lendothelial leucocyte adhesion molecule-t and vcam- (vascular cell adhesion molecule-t) ~n normal colonic mucosa with muwsa from cases of inflammatory bowel disease. wehavefoundexpressianafthese threemoleculesto beincreased~ninflammatarybaweldiseaseandthis change involvesendothelial cells as well asleucocytes we will dixussthbrelationship between adhesion molecule expression and dlsease actlvlty and posslble therapeutic implications granulomatous enterocolitis associated with therapeutic irradiation . . mangham. k m newbold, s dover the universty of bmmgham department of palhology, sci ool of m&mi science the medrcal schwl, edggbaslon. chronic radiation-induced entero~olitis s a weli recognised complication of radiotherapy for mtra-abdominal or pelvbcmalignancy. the pathological features includepwforatlon. fistula formation. segmental necros~s. and stncture formation. the histologicalfeaturesareessentially lxhaamic in nature. consequent upon the characteristic vascular damage. two case of inadtation-induced granulomatous enterocolltis are described ~n which non-caseating epithelcoid granulomas were present ~n the bowel well. to our knowledge. granulomas have not been previously &="bed in radiation coldisor any other formof ixhaemic bowel disease thsgranulomaswere largely confined to the mmosz-associated lymphoid tissue and the draining lymph nodes. naked submucosal granulomas were, however atso present. this distribution is similar to that seen m crohn's disease and tuberculosis. no evidence of these, or any othersystem!c ~r a n " l~r n a t~~~~~n d i t l~n~ was present these cases suppon theview that granulomas in bowel disease are secondary to an tmpaired muwsal bamer to antigens and highlight the non-specifiaty of granulomas m the diaynoscs of inflammatozy bowel diseases between and % of crohn's disease patients have involvement of the upper gastrointestlna tract the identification of granulomata is normally required to make a specific diagnosts but this may be dinicult in small endoscopic biopsies. we present t o cases where there was a strong suggestion of upper gastrolntestinal crohn's disease with a definitive diagnosis established on more distal invoivbment. four of the biopsies contained granulomata. the remaindershowedeitherpatchy inflammation, local ulceration orvillo~s dtjtmtlon only in aneffort to establish whether crohn's disease biopsies contained specific macrophage or lymphoid populations we applied the~onoclonal antibodies. mt (cd ). uchll (cd ro)). muramidass. mac , alpha- antltwsin and kpt (cdg ) using immunohistochemistry. theresuns were compared with casesot "on-specific duodenitis orlelunltis crypt-restricted loss of g pd activity has been used to quantify carcinogen-induced somatic mutation of the x- samples of peripheral nerve (sciatic nerve) of mice from varylng age groups were examlned over the past years. development olage~related penpheral neivefibredegeneraflon was observed among mlce over months age. the spontaneous peripheral nauropathy was characterized by walierlan type degeneratlon. teased nerve flbres from mice of years old showed evidence of swelling of the myelin sheath. fragmentation d myelin into myelin balls or ovoids, with areas of segmental demyellnatlon. light microscopic examination of h e stained sections revealed axonal degeneration, and myelin fragmentation. with vawolation of nerve fibres. on ultrastructural studies. there was evidence of axa-myelin degeneration. axxoplasm showing dark stained bodles suggemng of degenerate mitochondria the myelin sheath a l~o showed disorganization and fragmentation myelin, sometimes with whorl lormatron rithcm the cyroplasm of schwann cetk, wtth evidence of autophagocytosis by schwann ceiis. in some inslancestherawere sgnsofproliferation at schwann cells. nervefibredegeneration affected both non-myelinated and myelmated fibres generally, therewereapparentlygreaternumbers fdegeneratenan-myelinated nerrefibres than myelmated nerve fibres. furthermore the numben of degenerate myelinat& nerve fibres were less than in seontaneous peripheral neuopathy in ageing rats (personal obsenat on) swntaneous peripheral neuropathy of sciat,cnenesmainlyappearedin miceaged manlhsormore, s eenonlvvelyr rely in mlceof months. younger mice did not show any evidence of this change age-related penpheral nelimpathy is not usually accornpan~~d by any clln,cal ,gns neur~. fhology. uhversrty ofiowa, iowa. u s a v~scuiarendothellalcclls(rn)conslitufet~elntertace betweenthe bloodstreamand thetcsske and pertoormsevetal key roles ~n the development immune and inflammatory responses endothellal cell from the braln display slgnlflcantly different momhoiogy from other en. having ilght ]unctlons between them and pauclty at micropinacytolic ves~cles dunng inflammatory conditions of the cns interaction between mlammataly cells and rbb en s an initial important event. it s known that expression of several adhesion molecules on b r m en is upregulated !n intlammatorycondtti~ns and cytokine-induction ofthese molecules has been investigated however an ~mparlanl mechanism of adheson molecule induction and hence modulation of tmflammatq cell/en binding might beviral infect onofbrainen wehaveexamined theabilityofmeaslesvirus(human )andherpes simplex virus to infect cerebral endothelial cells. adhesion of syngenelc splenocytes to both vlrally-infected and mock infected cells was determined using a chromiums assay. by h of infection with measles yins, cytopathic effect was eadent. and splenocyte adherence was increased to a mean of % of the control mock-infected aner h mock-infecled expression of the adhesion molecules meca- and mala-lon virally-infected cells was determined possible mechanisms for enhancement of adhesion will be discussed and subsequent implications of virus inlectiun of cerebral en ~n relation to homing of inflammatory cells into the brain. ultrastructulal dbsenamns were made on the mechanism and route of innanmatory cell diapedesis through cerebral vessel walls in an experimental model of can~ned~stempervlrusencephalamyel~t~s ~n the hamster migrating monocyies and lymphacyiesextended pseudopodiawhich contacted, indented and adhwedtoendothelwm. they lhen invaded the endnthel l cells. becoming enveloped by endothelial cytoplasmic processes which re-establishec the continuity of the vascular lining as the migrating cell passed through although mlgratlng cells were frequently seen close to intei endotreiml junctions. they were ever seen wlthln iprctlons. or between adlacent endothelial cellsand therewas noev!denceof opening of mterendothelial tlghtpnmons aherpasslng through the endothel~al layer. cells squeezed through small pores (migration porest in the t h r sub-endothellal basal lamina me present study confirms and extends previous observations on the operation u;#thr the cns of a trans-endothel~al. paraiunclianal route for d apedesis of mflammatary cells. ireland falbology royal viclona hospital, bcl/asl hi bl lrrlnrld a sewice for the blochemtcal dlagnosts of lysosamd torpge diseases has now been o n operalaon m betlast forslx y n a r~ it currently lids assays available for the measurement lysasamal acid hydrolases in plasma. serum. lkwcocvtes. cultured sk nfibroblasts. amnioticfluld and cultured amniatlccellstordlagnos~s of lysosomalstorage diaurders solar patients have been referred from throughout ireland wtth having a positive diagnosis of these. were diagnosed as being hamozygous for a specific lysosomal enzyme deficiency, were identified as having multiple enzyme acl~ciencies (mucolipidosis type -cell disease) and had heterozygote (carrier) enzyme ibvpis t t v latier. w~i e either parents (obligate hetemrygotes) or siblings of homozygotes and one was a heterozyquti' lor x~lirlhrll recessively inherited fabry's disease. in addttlon. prenatal diagnosis has been performed on motherswithafam,iy hi~toryofi-celldiseaseand/orhurler'ssyndmme.oneofthesepmvedto bepositivefor huller tht' resuiic the biochemical investigations of these cases are presented. high levels of circulating immunoglobulins are common ~n liver dlsbase. in alcohol c hver disease depos s of mmunaglobullna(lga) have been found in the liver and therenal mesanglum, and werepart acaseof a oyearoldfemale with a year history of cardiomyopathy and progressivemuscle weakness anerthe birth of her baby shewas subsequentlyflnedwith apacemaker. clin~cally, therewas sevweweaknessof neck flexors with proximodistal weakness tn both arms and mild weakness of htp flexors. the most stnking weaknesswasin herbreathmg muscles therewas no ptosisorfaaal weakness rellexeswere symmetrical and her plantarswere flexor herckwas normal. aquadriceps musclebiopsy revealed abnormal vanatcon o n fibre diameters affecting bothflbrelypes. occasional pink hyalme ~nclus~ons which stainedforacid phosphataseand wlth pas wwe seen i~i both fibre types electron microscopy showed these i n~l~s i o n~ to cwtsist of aggregates nm diameter filamentaenmeshedwithinacentral coreofdenseamorphousmatenal. inotherareastheamorphausmatsnal layas inegutar patches within !he sarcoplasrn mainly at the level of the "z" tine causing disintegration of the sacomere immunoelection microscopy wing collmdai gold showed that the denseamorphous material reacted slrongiyw<th desmiri antisera and calld therefore represent a phosphorylated form of the pratein. tubular aggrcgatesitas] hayebeendescr,bedinassociat,on witha widevarietyofdisorders wherethey most onen occurin type %fibres recentlytwolam~i~eshavebeenrepored with muscularweaknessandtasasthedom~nant biopsylinding wereporla thtrdfamilyol whatseemsla beadistinctcondition twopatients afalherandda-ohter presented with slowly progressive p.oxima weakness. limitation of eye movements and aciiilles t e o d a r.antractufes creatine kina-was s t times normal by light mcroscopy, basophllic accmulations were ~e e n in both fibre types clecson m!croscopy revealed that these conslsted tightly packed parallel tubular a m p (tas) these varied somewhat m theiiulliast,wct~ral appearance and were therefore clawfled accordingly thisreport lhrows furner bwdexe m support of a otstmct cllntcopathologicai enttry. the c-myconcaprotein (p ) is a dna-bmdmg protem. increased expression of which is assmated wth shartenmg of the gog, phaseof the cell cycle. quantitahon of p m breast cancers may have prognoshc and therapeutc implicatrons. flow cyiometnc quantitatton (fcmo) of p has been shown to correlate wxth certatn parameters m breast carcinoma. however, thsre are factors which we felt could potenttally confound this fcmq m foimalln flxed paraffinembeded(ffpe)tissues. bothnuclear(n)andcyt~plsmic(c)express~oncanoccur. dependlngonthetlssue fixative used. cytoplasmic stripping and exposure of nuclei to the digestwe enzyme, trypsin, occw dwng the preparation of the nuclear suspension from ffpe tissue. this study documents c-myc oncoprotein stainlog i" * tissue senion of p m a r y breast carcinomas: turnour cells, adlacent benign epithelium and stromal cells were assessed. the monccional antibody om- - (crb) was employed using the abc immunocytochemical technique thi~antibodyrecognisesp andacyiaplasm ~ kd protein the resultsoftrla trypsmsatm of tissue sbctionspriortostsiningwerealsonoted. positivbstainingwasseenin y~oftumaun. both nandcstainmg belng present in % benign epithelium, many endotheiial, fibroblast and lymphocyte nuclel showed posltlvlty trypsinisatwn ledto lossof staining ntensity.thusin ffpe breastcawnomas. ~, " g l e p~~~m = t =~f c m q~f om- - anti-c-mycantiobody isconfounded by facts((i) much extratumoural c-mycexpresslon cccurs, ( ) cytoplasmic stripping may iead to oncoprotrin loss and ( ) breast oancercells,iisinducedaver -fald by phorbolssten.amphiregulin bindstotheegfreceptotandcan be stimulatoryorinhibitorydependingan theceil line. ltal~oblndsto~tsown receptor, possiblyerbb- .weevaluated expression of amphiregulin in primary breast cancers using immunocyiochemistry. % of turnours showed staining using amonoclonal antibody toamphireguiin which wasdlffuseorfocal in tumourcells.amphiregulin was not expressed in stroma and much less often in normal breast ep!thellum than m tumours. immunohistochemistry waspositiveincases withthe highest mrnaexprsssion(concordance %)asmeasuredusingacdnaprobeand qualitativenortherblotsanddat blotsanabplatecounter all caseswereassayedforegfraod er. er'egfr-. er* egfr*. er-egfr-. er-egfr'. there was no association of amphtregulln expresston wlth other receptors. these results suggest amphiregulm may be an independant factor regulating growth of a substantla proportion of breast cancers. in h a s t cancer, overexpression of the c-erbb- oncogene is associated with poorer prognosis, and in a prwious study we found it to be associated with lack of response to endocrine therapy bolus doxorubicin with infmons of ifosfamiddmesna appears to be a very active chemotherapy combination for advanced breast cancer in young women. thirty-nine women (median age = yrs) with rapid recurrence of breast cancer (median disease free interval = mth) were treated with doxarubicin and ifosfamlde/mesna. and c-erbb- expression in their pnmary tumours (determined immunohistochsmically) related to treatment response. the tumours of patlents strongly expressed c-erbb- protein and of these patients had objective responses to chemotherapy in patlents with turnourseitherweakly expressingc-erbb- (n = )otshowingnostaining(n = )therewereobject,veresponses,n . these data indicate that c-erbb- expression ~n primary breast carclnoma, while associated with resistance to hormonetherapy. daesnot preclude responsetosystemic chemotherapy. and sugge~taposs~blerolefarc-erbb- status in directing treatment strategies for individual breast cancer patients. pmicularly adpvant therapies ~n early stage disease. transforming growth factor is a muitifuncyional regulatory protein which can either stimulate or inhibit cell proliferation. can inhibit immune responses and can enhance both angiogenesis and the formation of connective tlssue.tgfpmrnahas beendetectedin breastcancercelliines, andtheprotein hasbe~nfoundinthemediafrom several such lines. in view of the prominent stromal reaction seen in many breast cancen. tgfp may have a srgnificant role in tumour development and subsequent behawour. tgfb, protein expression has been examined ~mm~noh~stdchemically using a specific polyclonal antiserum in formalin fixed. paraffin embedded invaswe and in s,fu breast carcinomas occasional staining of normal acinar cells was seen. % of invawe cancers had prominent staining of cells for tgfb. often with associated stromal staining. % showed occasional cellular and stromal staining. and staining of stroma only wasobserved in %. theremainder werenegative staining of ins it^ carcinomas was less with only % having prominent staining and % being negative. there was no conelatkon with type or grade of tumour. or oestrogen receptor status but all carcinomas with prominent reactivity had metastasised to nodes. this along with the differences between lnvasive and m srtu carcinomas suggests that tgfp may be a determining factor for invasion and metastasis. depanment ofpathoiwy, univers,ty ofshemeld, poboxsq , shefiela s ul chromatin changes ~n neoplasticcells are well recagnised quantitation of nuclear chromatin distcibuhon is dcfficult becauseaf thevanetyof parameterswhich couldpossibly bemeasured in thisstmiyamethodof quantttatmg the distribution of dna was developed for a seescan solitaire plus image analysts system. the parameters homogeneity, condensation and clumping were calculated by segmenting the nucleus grey scale intensity into three levels, black, grey and white, byamethod of adaptivethresholding.theseleveis represent the darkwand lighter regions of chromatin distribution. the relative pr~portlons and positions of each of these regions were calculated by viewing the nucleus through a pixel wide computer generated "mesh" the system was used to compare benign and malignant feulgen stained breast fnac specimens. the homogeneily and clumping parameters were significantly different between the two groups (p < ). but complete discrimination was not possible in order to test the valtdity of the system. spicimens were graded subp wely ( to ) and compared with the image analysis results. there was % correlation for homogeneity and % lor clumping. indicating the difference in chromatin distribution s real and not a result of system error the role of nuclear dna analysis. oestrogen receptor and thymidine kinase (tk) activity as prognostic indicators in human breast cancer was assessed. these parameten were investigated in relation to traditional pmgnostlcators. theoveralloblectivewastodeveiopaprognosticindexthat canpredlctforsunlval.tumourmatenalfmm breast cancer patients was anaiysed the nuclear dna content and oestrogen receptor status were determined by static cylophotometry and a multipoint titration assay respectively thymldlne klnase actlvlty was assessed using atp or ctp as a phosphate donor. patients were followed-up for a median of months. the cox proprtlonal hazards model wasused todetermine which parameterscould predict survival.pathologicalgrade, tumours e. totaltkand ctpjatpratiowerefo~ndto beindependent predictonof survival.aprognosticscore wasderivedforeachpatlent and these were assigned to one of three prognostic groups. using these prognostic groups the rlsk of reduced sunwpi could be predicted in . % of patients. inconcluslon, the assesment of pathological grade, turnow s m and thymidine kinase activity provides a reliable means of predicting prognosis year periodarereviewed.the median follow-up was months. on the basiso margln contour, degreeofstmmal overgrowth and atypla, mitotic activity. nuclear pleomorphism and the presence of speclallsed stroma, benlgn and malignant phyllodestumounwereidentified turnoursoccurredin womenof msdlanage years whomost often presented with a palpable, occasionally painful mass with a medlan diameter of cm exclson ( ) or smple mastectomy(l ) werethemorefrequentformsof therapy. localrecurrenceswereexperlenced by gpatlentswhose pnmary tumours although classified as benign contained areas of stromal overgrowth and specnaltsed stroma: all had been primarily treated by a histologically inadequate local e~~i~i~n~r~~b c~t a n e o~~ mastectomy (l).these local recunenceswere successfully controlled by local surgery ( )and mastectomy( ) mf~vemal~gnant tumours treated by mastectomy, none has recurred locally or developed metastases. flve mcompletely emsed turnours whlch showed neither evidence of stromal overgrowth nor speciallsed stroma remaln disease free at years. it s concluded that histologically malignant phyllodes tumoun are unllkely to develop distant dlsease i treated appropriately andthatstromal overgrowth may indicatethe possibilityof recurrence ~n incompietelyexcisedbenign lesions. several recent studies have evaluated the incidence in primary breast carcinoma of "micrornetastases" ~n multiple sections of axillary lymph nodes. howbyb~, the significance of these findings ~n terms of prognosis and sunw remainsunclear. inanattempt toelaborateon this, wereviewedthecasesof patlentsdlagnosedas havlng'bodenegative'' breast carcinomaon routine histologyand in whom therewasafollow-upperladof at least fwe years. the anginal diagnostic h e slides were reviewed in conjunction with eptthellal immunohistochemical pr-aratlons usmgcea,ema. hmfg, andkeratin, andappropriateclinicalfollow-updatawasaccumulated.thestudycasesfell into two categories, those which retained a 'node-negatlve' status after revew, including immunohistachemical appraisal ( %). and those which demonstrated individual or grouped malignant epithelial cells ( %) ~n the peripheral or deeperainuses of the lymph node. of the former group. all pattents but one werealive with noevidence of recurrent breast carcinoma five yean after mastectomy. of the second cohort. posnttve far "micrometastases". % showed nodiseaserecurrence. while % had gross metatatic d i s e a s e o r h a d w c c~m b t o thecarcmoma within hefiveyearpenod.thefindingsdescnbed.aithough basedon low patient numbenln thisprelimmarysludy. indicate the appropriateness of a large multicentre sunw to evaluate completely the prognostic significance of these '"msrometastases". sadhna bhatnagar. j. mcclure. d a. kalbhen the injection of sodium iodoacetate ( . mg) into the avian knee joint on two consecutive days induces changes which macmscopically and radiologically resemble osteoarthrosis. knee-pnts from groups of six hens were studied atregularinterval~upt weeksalterinjection by histological and histochemlcel methods. characteristlcles~ons were identified and assessadon asemi-quantiative basisand indwdual ision profilesacrossthetime course of the expenment were constructed. during the first five weeksthe obvious changes wwe chondrocyte loss and depleted matrix staining. chondrone formation was also noted. fmm six weeks there was also tibravascular proliferation at the chondro-asseousiunction wim modelling activity and new bone fwmation. in addition near the insenion of ligaments there was chondrocyte differentiation and proliferation. the amount of cartilage fibrillation and flsswng increase during the expenment aithough there were so areas of apparent resurfacing. the decreased matrlx staining noted early in the expenmen stabilised and many lacunar spaces are occupied by chondmcytes towards the end. subchondral bone hyperplasia and osteaphyte formation are prominent persisting features. such sequentialcharacterisation may be usefulin a-ssinglhee(fectsofput=~~vetherapeuticcompounds onthe model. rodentachilles tendonsweiesubiected to midpointtenotomy and allowed to recover for various timesin sllu before the operaled tissue was mmved, placed into a millipore dinusion chamber and insened inhaprntoneally into syngeneic hosts. diffusion chambers were removed at weekly intervals such that the tom time post-operation :i.e. time sru plus time wnhin the diffusion chamber) was up to weeks. histological examination demonstrated that ectopiccartilage wasprodwed withinthediffusion chamber byatotal of weeks but only if thefirst weeks were recovery in situ. wnh increasing time. calcified cartilage, mtteoid and pnmary spongmsa bone was obseived evtdence suggests that the camlage forms via direct tendon metaplasia and that the bone may form as a result of dinerentialive changes within hypertrophic chondrocytes. tlbla and fibula appearto be directly exposed to tha surface of the medial femoral condyle and inner ridge of the lateral femoral condyle. the exposed load-bearing area of the femur and tibia contain demonstrable amyloid around chondrocytes and in the matrix below the surface. these areas are also covered with the densest hyalmd fibro-cartilage. amyloid p r e e u~r s from h e synovlum may penehatethe surface ofthe cartilage under pressure or precursm from cartilage matrix or chondrocytes may be converted into amyloid and are napped below the surface. the inpction of sodium todoacetate into avian knee iolnts is followed by the development of an osteoarthrotic pmess to studytheeftectal sodium lodoacetateon chondrocytemetabalism m wire, chickstmal chondrocytes grown i~th~ee-dimensionalcollagen gels wereexpmedtothe compound. sterna from chick embryos(l - days in om) were digested with collagenase releasing the chondrocytes which were then cultured in collagen gels. w i u m iodoacetate was added to -day old cultures at concentrations of . . and wg/rnl. ( , . and .o y lodm)tor hours. theeftectswere assessed lor up to days by: ) staining wnhfluorescein diacetate which pmducesfiuorescenceiniive. metabolisingceilsonly. )thelevelofproteoglycansinthemedium wasestimated by complexing with dimethylmethylene blue and measuring the increase in absorbance at nm. ) histologycultures were fixed in glutaraldehyde, embedded in paraffin, sectioned. and starned wm alcoholic toluidine blue. alcaan blueandpicmsinusred.treatmentwlthiodoacetateproducedadoserelateddecreaseincellnumbersand intheamount of proteoglycmsreleased into themedium (lessthan % at pg/ml).the histolagyshawed thatthe treated cells were smaller and produced less matrix. these results show that sadium iodoacetate reduces cell number and interferes wqth the manufacture of matrix components. instftute of pathology, ro i vcfona hosprtal, belfast. l. irelend theoblectivesof this study were tocorrelatecertain clmcal features of ependymoma. (i e. age, sex, anatomcai slte. turnour size. meningeal dissemination and raised intracranial pressure with certain pathological features. . e dna content, mnotic rate. immunocytochsmistry and ultrastwctural features) with prognoss a total cases was available for clinical pathological assessment univewty of liverpool, p box , liverpooi bx and 'sari andres unrvemty it is not surprising that wmal exposure to high altitude causes increased activity of the hypothalamic-pltultaryadrenocortical axis. but fflidence suggests that continued exposure to hypoxla leads it to perstst long after homeostas sis restored and acclimatization achieved their pltuatary glands were not sgnlflcantly different insizefromthoseof thesea-levelsubpcts, but c~ntainedlargerpopulat~onsofcort~cotrophs~n termsof the total population of their anterior lobes ( . vs . %: p < ) these fmdlngs are in keeping with those of previous studies of the hypothalamic-pituitary-=d~=~~cottical axis in man and animais exposed to prolonged hypoxia at natural high altitude or in the laboratory. they suggest that greater amounts of acth might be necessary to sus ain normal adrenocoltid function under such circumstances and that adrenocortical hyperplasla bone marrow wnsists of a haamopolstic system whch is nn intimate association with aftbrous stramal network.adun mammalian bonemanowstromais knownto have both chondmgenicand ostecgeniccapabilitiesinvlvoand stromal tissue has been shown to tom fibroblastic wlonies capable of giving nse to bone in vitro. however, the mqbility of manow stroma to form cartilage in who has not been addressed when grown in identical conditions to mammalian stroma. mechanically dissociated single cell suspensions of chick embryo bane manow hom > days in ovo and from day hatchlings readily lorm bath fibroblastic and cartilage colonies in vitro. cells within the cartilage colonies are polygonai in morphology, are separated by a relradile exhaceiiuiar matrix and svnthesise catilage specific pmeogiycans and collagens. as shown histochemicelly, biochemically and immunocytochemically. in contrast, adult chicken bone manow does not form cartilage. instead, the cells appear osteoblast-like and synthesise coilagens typical of bone. a. m. fianagan t. j. chambers &wtmment of h~slopatholosy. sl george's hosptal medlcel sch-i, london. sw ore osteaclastshavebeensuccessfullygeneratedinculturesofmurinehaemopoisticcells. itwouldbeuseful ifasmila, model were available to analyse the mechanisms of regulatlm of human mteaclast formaim ir normal and pathological states. although the osteoclast is present in reverai neoplastic conditions. it is not known whether it forms part of other haemowietic malignancies, lor example, polycythaemia rubra "era or chrmic lymphatic leuhemia. we used strategies based on our experience with murine osteoclastogenesis. however, we have been unabletogeneratefunctional human osteoclastscapableoiresorbingbone m vrtro. large numbersofmultinucleate cells developed in these cultures mese cells did not show a typical panem of reactivity with asteociast-specific manoclonal antibodies, nor did they bind ' sct but rath@r possessed an antigenic profile characteristic of macmphage polykaryons. it is pecullarthat human tissue fails to support osteoclast generation stnce cells of the other haemopoietic lineages were consistently generated in our cuitures. in murine cunuras it is known that a particular shomal cell type is requtred for osteoclastagen@sis; it is possible therefore that this cell population is spamin adult humantissue.miswouldreflecttheiownumberofosteoc asts presentinhuman adults comparedto mice elucidation of conditionssuitableforthe generation mthehuman osleoclastm wtrow~ll heldus understand the mechanisms by which it is regulated in health and disease spaceof one year. the other case s b middie-agedwoman who &=being affected bythe disease far sevaral years developed separate sarcomas in each lower limb dunng a nine month pwiod. each tumour was treated bv pamal amputation but she died from widespread metastases within one year of the first amputationthe distribution of proline- -hydroxylase in a range of human tissues shown by a monoclonal antibody, fib s. smlth, p. revell aepariment of mobd anatomy and bone and h m f research unrt, the london hospw medcal cdl€ge, london. e l fib . a commercially available monoclonal antibady to the b subunit at the enzyme proiine- -hydroxyla~ invalved~ntheproductionofcallagen, wasappl'edtaacetonebxedcryostat sect~onsofawiderangeoftissues. in hver, hspatocytesshowedvarying degrsesof labelling with the antibody, along with spindle shapedcells(sscs) in associated connective tissue in both tonsil and lymph node. sscs in the connective tissue were melled with the antibody along with a number of other cells (lymphocytes) within the germinal centres. in addition to this. perivascularcellsand cells intheconnectivetissue iayerimmediatelybelowtheepithelium also labeliedin sections of tonsil. in skin, very few cells in the dermis were positive for pm ne- -hydroxylase. as were also only a small numbetof epidermal cells. afew chondrocyteswere marked bythisantibody in artlcularcartilageand intervertebral disc,along withswneostwcytesin bone.manycellsinthepleuraandtheepimysiumof normal~keletalmusclew~e posotive. there was no staining at all in specimens of kidney. in addition to these 'normar tissues, examples of a seminoma, a breast carcinoma and a thymoma were examined. each of the turnours showed fib labeliiw of ssc~withinthestromalt~ssue.theseresultsshowthat fib b sele~tivelylabelssscs.whlcharapresumedto be f brobiasts. in a wide ranged both normal and pathological tissues. which produce and maintaln the collagen matnx. proime- -hydoxylase is an enzyme involved in the pmduction of collagen and demonshahle in the rer of fibroblasts. it may therefore be cmsldered as a possible matker for fibroblasts in tissue sections. we have applied the mouse moncclonal antlbdy fib , raised against the $ subunn of prol ne- -hydroxylase. to alcohol fixed paramn embeddedsections and acetonefixedcryostat sect~onsolnormal (n = ) and meumatoid (n = ) synovia. synoviocytes labelled very strongly, but the distribution vaned between samples. insome, mostsynoviocytes weremahed, whilein others ~nlythedeepiayerofcells wasmitive. it isnot known whether thts dillerence ~n prdine- -hydroxylase expression is due to the severity and duration of disease. drug treatment or other factors. it is of interest, however, that fib labelling was not wnflned to the type b (fibroblast-llke) synoviocytes and that the more supficlal type a synoviocytes also contained proline- hydroxylase. spindleshapsdeells i" thesublntlmaleonnectiva tissue were labelledma vanablemanner. inaddltlm to these findings for fibroblastic cells. a small cap of fib posnive cells was seen around lymphoidfollicles. sometimes polansed towards the synovial surface. this enzyme expression by lymphocytes is under further invsstlgatlo". vitronectin s an adhesive glycoprotein which shares several functional similarities with fibronectin. it is a major component of extracellular matnx and plays a role in cell-matrix mteradians, monocyte function. and the coagulation and wmplement systems. we have employed a monoclonal antibody to assess the distribution of vironectin in frozen synovial biopsies from patients with rheumatoid arthntis, ostwnhntis. ankylosing spmdyiitis andtraumatic non-inflammato~ control^ ( cases). lmmunoreactive vltronectin was identified in the synovium. similarto the panem of immunoreactiyefibronectin, itwas located in adensefibrillarpanem surrounding cellsof the synovial lining layer. vitronectin was also associated with fibres throughoutthe sub-intimal connective tissues. being most prolific in fibrotic areas. vltronectin was identified in the sub-endothellal layen of blood vessels. but unlike fibronecttn was not associated with basement membranes. similar distributlon patterns wme observed in all biopsies studied thelocalisation ofvitronectitin in synovial tissues suggestsapossibleroie inanachmmtofcellsto the extracellular matrix and that may be important in the pathophysiology of inflammation and repair manolayercultured articularchondrocytesare known to rapidly losethelrexpressionof collagen typeil.the purpose of this study was to compare the expression of various collagen types in twoand three-dimensional cultures articular chandracytes. in addition. the expression of s- w protein and its alpha and beta subunits was studied in monolayer cui ures. bavine articular chondrocytes were isolated by collagenase digestion from ankle joints and cuhured in monolayerandspheroid culturesin dmemwim % fcs lmmunocytochemical studieswere performed using the indirect peroxidase technique an monolayers affer methanollethanol ( vlv) fixation and on frozen sections of spheroids. the reaction was scored semi-quantitatively as negative (-), weakly (+) moderately (++) ontha basisofthenumbersafcasesrefsiredtoustottheidentificalionofjointcry~talsintissuesectians, webelieve that calcium pyraphosphatedihydrate(cppd) deposnion disease isbeing underdiagnosed byhistopathologists. a crltical light microscopic reviewof cases. all with thediagnosisconfimed bymicroanalysis(energydispersivexrayspectroscopyln thescanning electron microscope, infrared spectroscapywboth) revealed adistinctwefeathery or br"shlikeappearanceinal suchdeposits.thisfeaturewasapparentat lowpower, whileconvincingvisualisation of crystalswithinthedepositswasdiff~cuneven withanoil immersionobiective.thesignof birehingeneeof cppd crystals is more dificult to demonnrate i" tissue sections than synoviai fluids. due to iactois such as stain. fragmentation and heaping up of crystals, buf it can be more readily assessed in unstained sections or following microincineration of the section. in six of our c a m the deposits were exclusively within bone: demonstration depended on relatiyeunderdecalcification: deposits in this position have not previously been recognised. this study thus provides new information an the histological identdication of cppd deposns in tisues and implies heterogeneity in the pathogenesis of such deposits. in human knee osteoarthrosis (oa) overt unicornpartmental disease is frequently accompanied by a macroscopically normal second cornpartmsm. however. there are a number of light micmscopical changes within the latter which might amount to an "early osteoarthrosis". these are chondrocyte pmliferation and chondrone formation. decreased proteoglycan staining and disruption of the chandra-osseous interface with duplication of the tidemark and resorption ofthe cartilage by chondmclasts. tidemark duplication is also a feature of established oa re~resenti~gadistu~ancedfcabif~cat~oninrone ofarticularcartilage.slnceslwp~~t~i~~~~ knowncalciumion transponmoleculs.altsrationm~~sstainingpanernand~ntens~tym~grn beanlndexotearlyo~. sl~~protemstamng was studied in the tiblai plateau cartdage of cases: with unicornpartmental oa. with bicompartmental oa and controls. lntheianerall chondrocytesgavepositivemembrana and cytoplasmic staining (especially in zones e n d ) , d~u s e w e a k m a t~~c~l s t~~~~~g~~~~~e l , s t~o~g p a r i c e l l~i a~a~d~~t~~-t~~t o~~l s t~i~~~g~~~o~~s a~d . in overt and early oa the orderly malrlcal panem was disrupted with prominent patchy staining around chondrocyte clusters. the significance of the change in the matncal distribution ~n oa is unknown and unlikely lo be related to a disturbance of calcification in zone . road, london. wcl m e histogenesis at alveolar son part sarcoma (asps) is still unsenled: nonchromaffin paraganglioma. malignant granular cell myoblastoma, neuml tumour, myogenic tumour. habdomyosarcoma and renin-producing tumoui theories have been proposed lmmunohistochemical studies have yielded discrepant results we recentlyobsewed that the diastase resistant crystalline material in asps was strikingly reactive for vimentin by the immunoperoxidase method on paraffh sections one case. we then studied paraffin sections of seven other cases from our files between and ~ by routine light microscopy, immunohistochemistry and electron microscopy all eight cases were reactlve for vimentin. which highlighted the crystalline materlal as in the first case. vanabie numbers of immunoreactlve cells for desmin were found in three. and smooth muscle specific actin in four. all cases showed some reactivity for neuron specific enolase. and seven of the eight cases reacted for s- protein. the clinical and pathological lea lure^ of cases of synoviai sarcoma have been reviewed. these tumours are aggressive with a poor prognosis. typically affecting young aduns with a long history before presentation and detinltive diagnosis. a high index of suspicion is required if the diagnosis is not to be missed several features, including age, size. site. histological type (monophasicibiphasic), mitotic actlvity and necms~s have been investigated withaview toestablishing possible prognosticindicators. the bestguideto prognosisis assessment mitotic actlvity. size and histological type dld not affen the outcome. in this study the effecl of subcapsular orchidectomy on skeletal metastases from prostatic carcinoma was studied using bone histomorphomatnc paramsten. twenty-eight patients with bone metastases were studied immediately before and for seven months after orchidectomy. tetracycline labelled bone biopsies were taken from metastases and turnour free areas at the beginning and end of study. sixteen of the patients also underwent o~teocla~t inhibition tor six months using disodium pamidronate ( mg i.v. weekly for weeks then anemale weekly for five months) to enable differentiation of the skeletal response to castration. histamarphametnc anslysi~ tumour frse bonerevealed adrapin overall bonevolume. histological analysisofmetastasesshawedadecrease m osteoblastic activity but widespread osteoclast mediated ostealysis. tumour regression and manow recolonisation were present in most c~s b s but malignant foci remained in % of repeat metastatic biopsies, inducing a typical. lacalised disruption of bone metabolism. it is concluded that orchidectomy causes ostmblastic regression blrt induces increased ostboclast mediated bone destruction, which is most pronounced within metastases. although tumour regression and marrow recolonisation usually occur within metastases, active turnour foci anen perstsf. ( ) chairman s. g. silverberg, washington theeffectsafoncogeneson thedeveloping nelvoussystem havebeenstudiedinaneuraltransplant system ~ntats, taking advantage of the extraordinary capacity of fetal cns to differentiate in and fully integrate with, the adult host brain. gene transfer tnto tetal brain cells was mediated by in viiro infection with replication-defsctwe retroviral vectors. fetal rat brain suspensions were then stermtaxically injected into the caudoputamen of adult f rats.animals carrying transplants exposed in vim lo the polyoma medium tantigen developed endothellal hemangiomas in the graft which often led to fatal cerebral hemorrhage within - days alter transplantatm lntroductm of the viral sic gene caused astrocytic and mesenchymai tumors affer latency periods of months.following infection of fetal donor cells with a vector encoding the v-myc oncogene led to the development of only a single embryonal cns tumor whereas exposure to v-ha-ras and v-myc resulted in the rapbd induction of multiple malignant neoplasms. when injected intracerebrallyinto newborn ratsin vivo, complementation of theseongogenes led the development of malignant hemangioendotheliomas, undinerentiated neural tumors andlor leukmia.oncogene transferthus constitutes a challenging new model to assess the effects of translorm~ng genes on the developing nervous system. since the presentahon of molecular genetic evidence supporting the knudson hypothesis m the eariy s there hasbeen increasing interestinthe ideathatasimilartwo-hit mechanism mayaperateln sporadadlccancers therens now evidencbthatlassofonealleleandretentionottheother~nmutated/wildfarmaccuninsparad~ccalono. lung. renal and breast cancers in breast cancer lossof heterorygoslty(l h) has been demanstratedfarseveral alleleson chromosomes l p q and an the short arm of chromosome . loh has also been demonstrated on other chromosomes thegeneral viewisthat loh indicatesthatasuppressorgene may bepresentattheseloct. hawevet in interpreting lohthere sdifficulty indetermining when loh becomessignificant because itsincidencevariesfrom % / depending on the locus examined. in this presentation, knowledge about loh on chromosome p (and q where a new deletion has been recently discovered), and p will be presented and the pathqlenetic significanceand ciinicsl relevanceexamined. lnthe lattercontext anewrapid methodfordetemlnmg alleljc dosage will a l x be described which does not requlre rflp analysis. expelt systems - .e. computer software that can function as cansuhant, decision support system, or process contr~llerhas undergone a rapid development durlng the past decade. applications to histopathology have gnaw some ,ocqdiffuse maiignantmesotheliomas( mm) occurannuailymn. americaat thepresenttime.cumulat~~ely, substantial numbersot lesser-knownfarms of mesothelia turnourare also seen, including serous paptllary tumoun of the peritoneum, well differentiatedpapillarymesothellomas and benign cystic mesotheliomas, but incidencedata are not available.the relativeiyfrequent atypical reactive hyperplmasof w o s a membrane add further vanstytothe range of mesothellai lesions which may present diagnostic problems. the expenence of a u.s.-canadian mesotheliomapanel suggests that dmm vs. metastatic carcinomacontinues to bethe commonest mesatheliamarelateddiagnostieehaliengeandthatthedifficunyandalwayssolved bytheappiicationofspecialstains almostas common are le~ions in which the ditferential diagnosiscenters on dmm us. a reactive process (atypicai mesotheliai hyperplasia, fibrous pleurisy) within the laner group it is particulady difflcun lo obtain a strong consensus opinion, andfoilaw-uphasconfirmedthemaiorityopinioninonly %ofcases. sarcomatousdmmvs.sarcomanosisaiso a notable area of difficuny. most mesothelial lesions tn north amenca. including dmm. are reviewed within a short time of biopsy by a pathologist in a major teaching centre, only a small number ( - %) being subpct to further scrutiny by a panel or ~n the coune of epidemiological or clinical studies however, many cases resurface later ~n a legal sening when the material and clinl~al information s often available. the original diagnosis of the hosoital pathologist s usually endaned by the courts. sharon w. weiss llposarcoma is one of the most common forms of son tissue sarcoma and may be divided into several subwes well-differentiated. myxoid. round cali, phmwpha, and dedifferentiated. this presentation will discuss the ) diagnosticcnteriaof lipblasts ) d agnosisand behaviorof myxoidround cell iiposarcoma andthe ) behavior and incidence of "dedifferentiation" dwell-differentiated ihposarcoma. the diagnosis of lipsarcoma depends in part on the identdication of lipoblasts or pnmitive fat ceils. since iipoblast-l!ke cdls may be seen m a variety of conditions apart from iiposarcoma, strict diagnostic c!i tma must be applied in identlfving these ceiis. these cells contain a hyperchromatic. indented or scalloped, ecmtnc nucleus set in a cytoplasm containing one or more lipid-nch vacudes. these cells must also occur in an appropriate histaiqic background. vanous lesions which may contain ihpoblast-like cells and which may. therefore, mimic liposarcoma include fat necrosis, fat atrophy, silicone granuloma, signet nng carcinomas or melanomas, and a variety of malignant tumors with fixation artifact. myxold androundcell lipsarcomarepresentthemost commanfarmofsarcomaaccuningmearlytamidadunlifeandare commoniy located #n the region of the mlgh and popitteal fossa. although the designation of "myxold and "raund cely suggest two separateturnortypes. they represnt ends d a common h~stologlcspbctnm. myxaid liposeicoma represents the well differentiated end of the spenrum. whereas round cell represents the pcarly differentiated end.however, transitional or mixed forms ex~st. accounting for confusion as to how such tumors should be classlfled mymd liposarcomas are characterized by nodules of bland stellate or rounded cells set n hyaiuronic aod-rich strama. an intricate plexiform vasculature and numerous lipblasts are easily vdentaed. wlth progression to round celliiposarcomathecellsbecomelarger,moreatypicalandthestromalessmyxaid. lipoblastsaremoredlfticultto find and a plexiform vasculature s less apparent. recent work by evans suggests that evaluatlon of myxoidlfound cell ihpasarcomashould includethe percentage of round cell component wdhin atumorsincethisdirectclh/ mtluences prognosis. our policy is to carefully sampk such tmors, submining t section for each centimeter in greatest diameter of the tumor. a rough estimate of the percentage of a round cell component s made. we w a r d tumors havinglessthan faroundcellcomponentasgradei.thosehav,ngbetween & % roundcelicomdonent are considered grade . whereas those with more than % are considered grade . well dmerentiated liposarcoma is one of the most common sarcomas accuning m late adult life. characteristically affecting the deep musclesaftheextremities, theretropentonealspace. and the groin, this tumor ischaractenzed by varying amounts of mature fat interspersed with fibrous bands, atypical hyperchromaric spmdledceiis, and lipoblasts. these lesions are considered low grade sarcomas having a high rate of local recurrence but no abilny to metastaslze. recently evans and ammi eial suggested that well-differentiated liposarcomas occurring m the wbcutaneous tissue and muxles of the extremity cause so hnle morbidity that the use of the term "atypical lipma" should be used rather than ihposarcoma. in contrast. well-differentiated liposarcomas in the retropentoneum cause significant morbidihl and pose a significant risk of death from local disease. thus. nearly all pathologists agree that the term "weii differentiated lipasarcoma" should be retained for these tumors ~n the retroperitoneum. however. none of these recent studies have followed a large number of these tumors for a proianged period at time m order to assess the long term behavior and specihcalty risk that such es ons may progresswith time to a higher grade lesions ( . dedifferentiate). we have recently completed a follow-up study of cases of well ditferentiated iiposarcoma accurringmthemusc~oftheextremity,retropntoneum,andgroin. inalllmatlonsthenskof local recurrence was high. however,dedifferentiationoccurred~n % of casesandwasnotrestnnedtotumors~nanyparticularlacatlon. butcouldbe bestconelatedwiththedurationofthetumor. withmostcasesoccun~ngaflerl mmoreyean thus. dedifferentiation is not a site-dependent phenomenon, as has previously been suggested. but rather a time dependentphenomenon.anhough thesedatado notneee~~arily indicatethatwe should abandon theterm"atwca itpoma" they do indicate the need tor proianged foilow-up of patients wlth wei&-dlfferent,ated ddosarcoma and the small but deflnne risk of dedifferentiation as a long term complication of the disease. key: cord- - jy zoz authors: edens, fw; parkhurst, cr; qureshi, ma; casas, ia; havenstein, gb title: atypical escherichia coli strains and their association with poult enteritis and mortality syndrome date: - - journal: poultry science doi: . /ps/ . . sha: doc_id: cord_uid: jy zoz abstract to date, no definitive etiology has been described for poult enteritis and mortality syndrome (pems). however, two atypical escherichia coli colony types are isolated consistently from moribund and dead poults afflicted with pems. to test the infectivity of these e. coli strains, poults were placed into floor pens in three isolation treatment rooms: ) control: no bacterial challenge, ) e. coli colony types or posthatch oral challenge: ( ) cfu/per poult at d, and ) e. coli colony types or posthatch oral challenge: ( ) cfu/per poult at d. daily intramuscular injections of cyclophosphamide ( micrograms per poult) from to d posthatch were given to half of the poults in each treatment. atypical e. coli challenge caused bw depression, and cyclophosphamide treatment exacerbated the response. all e. coli-challenged poults developed diarrhea similar to pems. mortality was increased by both atypical e. coli colony types, but at d e. coli colony type caused greater mortality than colony type . with cyclophosphamide treatment, mortality was exacerbated with both colony types, but colony type at d caused the greatest mortality. ultrastructural damage to ileum epithelium cell microvilli and subcellular organelles indicated that part of the bw depression could be attributed to malabsorption of nutrients. it was concluded that the atypical e. coli colony types and play a significant role in the pems disease. since , the number of verified cases of poult enteritis and mortality syndrome (pems) has increased significantly each year. until , pems was a problem localized in the carolinas and georgia (barnes and guy, ; barnes et al., ; brown, ) , but in pems was verified in at least six additional states and was suspected in outbreaks of severe enteritis with diarrhea and high mortality in three other countries (brazil, canada, and portugal; anonymous communication) . these reports signaled the possibility of a turkey disease outbreak in epidemic proportions on a national and international scale. poults between and d of age appear to be at the greatest risk for pems (brown, ; barnes and guy, ; barnes et al., ) . symptomatic poults display signs of irritability (walking and high pitched vocaliza-tion) initially, followed by anorexia, diarrhea of increasing severity, dehydration, growth depression in excess of %, near total morbidity, and finally mortality in excess of %/d for or more consecutive d. survivors are stunted severely and never reach target market weights. recently, qureshi et al. ( ) reported that pems afflicted poults are severely immunosuppressed. our observations indicated that t cell-mediated immune response and b cell-mediated humoral immunity both are suppressed significantly. furthermore, the bursa of fabricius, spleen, and thymus are atrophied as a result of pems. whether the dysfunction is a direct result of a viral or bacterial infection or whether it is indirectly associated with a severe stress response due to an infection from an unidentified etiology has not been ascertained. the pems problem is complicated by the fact that no etiological agent(s) have been identified (brown, ; barnes and guy, ; barnes et al., ; qureshi et al., ) . numerous potential viral agents have been investigated, including adenovirus, coronavirus, coronavirus-like particles, enterovirus, astrovirus, birnavirus (serotype ), rotavirus (type d), reovirus, bursa epithelial virus, and others. however, alone these viruses have not been shown to induce pems (brown, ; barnes and guy, ; barnes et al., ) . nevertheless, brown ( ) reported that a coronaviruslike particle and a serotype birnavirus could both reduce growth in -wk-old poults. co-challenge with these two agents depressed growth and feed conversion significantly and caused % mortality, resulting in a condition analogous to pems. it also has been observed that cryptosporidiosis complicates the pems problem on some farms, but cryptosporidium infections alone do not cause pems (brown, ; barnes and guy, ; barnes et al., ) . on the other hand, barnes et al. ( ) suggest that after an unidentified virus infects the turkey poults, opportunistic enteric bacteria (such as salmonella, escherichia coli, or clostridium) further complicate the pems condition and cause mortality in the immunocompromised poult. research on pems has been very slow to yield insight that could lead to a solution to the problem. however, we have consistently isolated from moribund pemsinfected poults two pure cultures of atypical e. coli along with several other atypical e. coli strains. although the isolation of these strains was fortuitous, it appears that their presence in nearly every set of pems-infected poults sent to one of the north carolina department of agriculture (ncda) regional diagnostic laboratories suggests that they may be very important. indeed, these e. coli strains may represent a significant breakthrough in the diagnosis and control of pems. this report describes the effects of single oral inoculations of these two atypical strains of e. coli on growth and livability of turkey poults. this project was approved and was conducted under the supervision of the north carolina state university animal care and use committee, which has adopted animal care and use guidelines governing all animal use in experimental procedures. british united turkey poults from a commercial hatchery were obtained within h after hatching. the poults received neither vaccinations nor hatchery services such as beak, snood, or toe trimming. they were transported to north carolina state university's dearstyne avian research center disease isolation facility. for experiment , they were segregated randomly into treatment groups, wing-banded, and placed into electrically heated battery brooders, where they were maintained through d of age. in experiment , they were placed in floor pens with pine wood shavings litter and were maintained through d of age. a turkey starter ration, containing . % crude protein and a metabolizable energy level of , kcal/kg (table ) , was provided. water was available ad libitum in zinc galvanized drinkers. ambient temperature in the two experiments was regulated at c for d after placement, when it was reduced to c until d. from through d, the temperature was set at c. in experiment , two radiant heaters were suspended in above each floor pen to provide a supplementary heat source, if required by the poults. the poults were provided continuous light throughout the experimental periods. air exchange rate in the isolation rooms was designed to provide a minimum of four changes per hour to prevent ammonia build-up. nevertheless, environmental conditions were altered by reducing air flow in the isolation rooms to allow moisture build-up in the litter under the poults. by the end of d, the litter moisture was elevated to more than %, a condition not unlike that found in commercial turkey production areas in the southeast during periods of hot humid weather when there are severe outbreaks of pems. in experiment , moribund pems-infected poults were obtained from a commercial turkey producer, killed by carbon dioxide asphyxiation, and dissected to remove the ceca. precautions were taken to prevent contact between the excised ceca and either the body surface or necropsy table. the isolated ceca were placed into sterile plastic packets before stomaching. the contents were streaked onto macconkey agar plates and incubated h at c. escherichia coli colonies were isolated from these plates and propagated anaerobically h at c in brain heart infusion (bhi) broth and were used in three separate trials. in experiment , e. coli, identified by colony morphology (smooth, raised, mucoid, and slow-growing colony as type ; rough, flat, congo red-positive fast-growing colony as type ) were isolated consistently from pemsinfected poults at a ncda regional diagnostic laboratory and propagated on bhi broth. the type and type colonies were designated as atypical forms based upon their bbl biochemical profiles (edens et al., unpublished data) . in experiment , day-old healthy, mixed-sex poults from a commercial hatchery were given a single oral challenge with ml of the anaerobic e. coli culture ( cfu/ml per poult) derived from the ceca of pems-infected poults from a commercial source. control poults were given orally an equal volume of bhi broth. in trial , there were control poults, poults challenged at d of age, and poults challenged at d of age. in trials and , there were control poults, challenged at d with a live bacterial culture, challenged at d with a killed bacterial culture (autoclaved culture) followed at d with a live bacterial culture, challenged with killed organisms at d, and challenged with a live bacterial culture at d followed by killed organisms at d. no attempt was made to identify the serotypes of this mixture of e. coli colonies used in this oral inoculum. the birds were maintained in heated metal brooding batteries from hatching until wk of age. in experiment , either atypical e. coli colony type or type was given orally to male poults at either or d of age. the challenge dose of ml for each poult contained cfu of a single colony type. an equal volume of bhi broth was given to the control poults. there were four replicate groups of poults for each of the colony types administered, and two replicates of each for the controls. thus, a total of poults were involved in experiment . in experiment , two replicate groups of the poults were given either atypical e. coli colony type or atypical e. coli colony type at either or d of age and one replicate of the control group were given cyclophosphamide (cpd, mg per poult) intramuscularly for consecutive d beginning at day of hatch. cyclophosphamide is an alkylating agent that binds readily to b cell dna, rendering those cells inactive (lerman and weidanz, ; glick, glick, , , and it was administered to mimic the immunodepression or immunodysfunction found in field cases of pems. the cpd treatment was administered in sterile avian saline ( . % sodium chloride) in a volume of ml. an equal volume of avian saline was given to all other poults. a total of poults were given cpd. body weights were determined on a weekly basis, and mortality was determined on a daily basis. at d of age, surviving poults were killed by carbon dioxide asphyxiation and each poult was examined and rated for its level of enteritis based upon the presence of distended, gaseous intestines and ceca each containing yellowish, liquid fecal material with excessive mucous content. the bursa of fabricius from each poult was examined macroscopically for the presence of bursa cores consisting of hard caseous material. at the time of necropsy, representative samples of the ileum from five poults from each treatment were collected for transmission electron microscopic examination of the effects of e. coli infection on the morphology of the epithelial lining of the ileum. tissues ( to mm ) were fixed in ice cold glutaraldehyde and . m sodium cacodylate buffer, ph . . the samples were washed · min each in ice cold . m sodium cacodylate buffer, then postfixed for h in % osmium tetroxide in sodium cacodylate buffer. the tissue samples were then washed again · min each in ice cold sodium cacodylate buffer, trimmed to -mm cubes, and dehydrated in % graded series of ethanol solutions ( to % for storage at c). samples were then dehydrated to · % ice cold ethanol for min each, and finally the tissues were dehydrated to · % ethanol for min each, with gradual return to room temperature ( c). the tissues were infiltrated with a : ethanol:spurr's (firm mixture) for h at room temperature followed by overnight infiltration in : ethanol:spurr's at room temperature. the samples were then placed in % spurr's mixture for · h changes before embedding in flat molds overnight at c. both thick ( m) and thin ( Å ) sections were cut using an ultramicrotome. thick sections were mounted on glass slides and stained with toluidine blue. thin sections were mounted on -mesh grids and stained with % aqueous uranyl acetate for h at room temperature, rinsed in three changes of filtered distilled water, then stained min at room temperature with reynold's lead acetate and rinsed. grids were viewed with a joel s transmission electron microscope at kv. weekly bw, total weekly mortality, and the percentage of survivor poults found to have bursa cores were analyzed statistically using the general linear models procedure of sas (sas institute, ) . the bw and mortality data were subjected to analysis of variance and the bursa core data were analyzed by t test. percentage data were converted to arc sine square root percentages before analysis. means were separated using least significant difference. statements of significance were based onp £ . or less. the effect of anaerobic-isolated and cultured e. coli from pems-afflicted poults on bw and mortality in experiment are presented in table . oral challenge with the e. coli isolates at d caused a significant decrease in bw by and d postchallenge in trials , , and , respectively. this depression in bw persisted through d. challenge at d of age also caused a significant depression in bw at d. presentation of killed cultures at d provided some protection from the challenge with live organisms at d, based on intermediate bw gain compared to groups given live organisms alone at d and to control poults. mortality was increased significantly in trial when live organisms were inoculated at d. mortality was not recorded in trials and because poults were taken daily for determination of other parameters (data not reported here). table are the bw data for experiment . during the first week after oral challenge with atypical e. coli colony types or , poults inoculated at d had significantly reduced bw in comparison to the controls, whereas those inoculated at d had bw intermediate to the controls and those inoculated at d. during week , poults given the cpd experienced a small but nonsignificant decrease in bw. however, this decrease in bw due to cpd reduced the difference between controls and the e. coli-treated groups resulting in no group differences. at d, there was a clear difference between control poults and poults given the two colony types of e. coli. among the e. coli treatment groups, poults given colony type at d had significantly smaller bw than all other treatment groups. in the groups given both cpd and one or the other of the two different atypical e. coli colony types at either or d, the treatment groups given atypical e. coli colony . transmission electron micrographs of microvilli associated with epithelium cells in the ileum of -d-old turkey poults from a) control, b) atypical escherichia coli colony type and c) atypical e. coli colony type . the electron micrographs indicate that the microvilli in the poults given atypical e. coli colony type at d of age were beginning to degenerate with membrane separation and were thinner, taller, less numerous than control, and that there was disruption of organelles within the cells. the microvilli in the poults given atypical e. coli colony type at d of age were degenerating with membrane separation, decreased numbers and disruption of the organelles within the cells. (mv: microvilli; tw: terminal web; g: glycocalyx; jc: junctional complex; bar = hm). type at d or e. coli colony type at d were not different from the treatment group given only atypical e. coli colony type at d. mortality data for experiment are presented in table . treatment groups given the cpd in combination with atypical e. coli colony types or given at d and atypical e. coli colony type given at d had significantly elevated mortality during week . during week , mortality of control poults was significantly lower than all other groups given atypical e. coli colony types or with or without cpd treatment. poults given atypical e. coli colony types or at d experienced the greatest mortality rates among these groups not given cpd. however, the addition of cpd to the treatment regimen further increased mortality rates of those groups given atypical e. coli colony types or at d. nevertheless, by d those poults given cpd and inoculated with atypical e. coli colony types or at d were also showing significant increases in mortality compared to their respective controls. at d, there was significantly elevated mortality in the poults given atypical e. coli colony type at d in comparison to all other groups not given cpd. there was no difference between groups given atypical e. coli colony type at either or d. mortality in the group of poults given the atypical e. coli colony type at d was significantly higher than in their controls but less than in all other treatment groups given the atypical e. coli colony types and not in combination with cpd. the cpd treatment, due to its immunosuppressive effects on b cell function, allowed relatively high rates of mortality during the first d posthatch, and this presumably was due to reduced ability to resist naturally occurring, opportunistic bacterial pathogens. the cpd treatment regimen had an additive effect on mortality in all groups given the atypical e. coli colony types at either or d in comparison to control + cpd and groups not given cpd. the atypical e. coli colony type given at d in combination with cd had the highest mortality rate among all the treatment groups whereas the group given atypical e. coli colony type at d in combination with cpd had the lowest mortality rate. presence of caseous cores in the bursa of fabricius of survivor poults at d is indicated in table . control poults with and without cpd treatment did not show any bursa cores. however, cores of various sizes were found in each of the treatments in which the two atypical e. coli colony types were given. the greatest number of cores was found in poults given atypical e. coli colony type in combination with cpd treatment, and this greater core number coincided with the greatest mortality rate. furthermore, the frequency of bursa core presence in survivor poults appeared to be related to poult age at the time of e. coli exposure and to the cotreatment with cpd, which induced immunosuppression due to its rapid alkylating action on dna in b cell populations. presented in figure are transmission electron micrographs of epithelial cells from the lumen of the ileum in -d-old poults in control and atypical e. coli colony types and treatments. control epithelial cells appeared to be normal in every detail. in figure a , the microvilli in a control poult are highlighted, and it can be seen that they are erect, in large numbers, and have glycocalyx-covered tips that appeared to be functional. the central core of the microvilli extend into the apical portion of the cell to form a well-defined terminal web. membranes on the microvilli were intact and were continuous with the cell membrane. membrane tight and intermediate junctions and desmosome between epithelial cells appeared normal. organelles below the terminal web of the epithelial cells appeared to be normal and contained rough and smooth endoplasmic reticulum with large numbers of ribosomes, mitochondria, lysosome-like bodies, and some small, smooth membrane-enclosed dense granules that appeared to be similar to recently absorbed lipids reflecting the reduced lipid content of the lower small intestine. in figure b , the microvilli on a commonly appearing epithelial cell from the ileum of a -d-old poult given atypical e. coli colony type at d are emphasized. the microvilli are more slender, more uneven, and fewer in number than those found in control poults. the membranes of these microvilli also appeared to be degenerating, and the glycocalyx on the tips of the microvilli were absent or appeared to be losing their integrity. the multiple filaments forming the central core of these microvilli appeared to be reduced in number, reflecting the absence or degenerative appearance of the glycocalyx, and this continued to be evidenced by the degenerative appearance of the terminal web. the junctional complex also had a degenerative appearance, as evidenced by a breakdown in the tight junction, loss of integrity of the intermediate junction, and desmosome. organelles within the cytoplasm appeared to be abnormal, especially the mitochondria, which were enlarged and appeared to be nonfunctional. however, there was little evidence to suggest that absorption of nutrient from the lumen was occurring because no lipid containing smooth granules were observed. the rough endoplasmic reticulum was disrupted with few attached ribosomes along with other disrupted membrane structures in the cytoplasm. in figure c , the microvilli on epithelial cells from a -d-old poult given atypical e. coli colony type at d have lost their integrity. the microvilli were fewer in number, had lost rigor, had degenerating membranes, and the glycocalyx structures were largely absent or abnormal in appearance. the multiple filaments forming the central core of these microvilli appear to be reduced in number, reflecting the absence or degenerative appearance of the glycocalyx, and this continues to be evidenced by the degenerative appearance of the terminal web, which is irregular and not well formed almost to the point of being nonexistent. the junctional complex also appeared to be degenerating, as evidenced by a breakdown in the tight junction, loss of integrity of the intermediate junction, and desmosome. organelles within the cytoplasm appeared to be abnormal, especially the mitochondria, which were enlarged and appeared to be nonfunctional. the rough endoplasmic reticulum was also disrupted and appeared to be degenerating based upon the lack of attached ribosomes and an apparent increase in free cytoplasmic ribosomes. shown in figure are goblet cells in the ileum of poults from a -d-old control (figure a ) and from a -d-old poult exposed to the atypical e. coli colony type (given at d; figure b ). in control poults, the secretory vesicles in the apical portion of the goblet cells appeared to be filled with mucous ready for secretion. the reticular area beneath the secretory apical region was well developed, and the endoplasmic reticulum was recognized easily. however, a drastic contrast was presented when goblet cells from poults given the atypical e. coli colony types were examined ( figure b ). these goblet cells were depleted of secretory material and the reticular area within the cytoplasm was not distinct. additionally, the nuclei of many of the goblet cells appeared to be degenerating. shown in figure are sections of a goblet cell and adjoining epithelial cells from the ileum of a poult that had been given atypical e. coli colony type at d. however, the third cell type shown in this figure was a macrophage that had migrated through an epithelial cell tight junction or a goblet-epithelial cell junction and was feeding on bacteria in the ileum lumen. this appearance was not an isolated condition but was seen in many sections from poults given one or the other of the atypical e. coli colony types at d. although several viral agents have been isolated from pems-afflicted poults, barnes and guy ( ) and barnes et al. ( ) have indicated that no single virus or combination of viruses has been shown to induce the disease. they suggest that the mortality is caused by one or more specific or novel infectious agents that may act singly or in combination. however, brown ( ) did indicate that a combination of a coronavirus and a serotype birnavirus (infectious bursal disease virus) created a condition analogous to pems. newberry et al. ( ) demonstrated that virulent hemorrhagic enteritis virus (hev) can interact with e. coli strains (o :k and an atypical/untypable strain isolated from a dead hevinfected poult), causing turkey poult colibacillosis and a high rate of mortality shortly after e. coli challenge. presumably, the hev had already damaged the intestinal tract allowing rapid bacterial translocation. this observation was consistent with earlier findings by larsen et al. ( ) and sponenberg et al. ( ) , who also found increased incidence of poor performance and increased mortality as a result of e. coli infections after hev infection. this work was confirmed and extended by van den hurk et al. ( ) , who observed a synergistic effect between hev and e. coli o (via an intratracheal route of administration) co-infections in which there was higher mortality ( %) than with e. coli alone ( %), and survivors had decreased bw gain ( % less per day) with the hev and e. coli co-infection. the induction of colibacillosis with high mortality and decreased performance as a result of co-infection with hev and e. coli untypable, o , and o serotypes points out the potential for a virus etiology, which may potentiate the development of pems. the presence of the atypical e. coli colony types and in moribund pems-afflicted turkey poults and the fact that these strains of e. coli cause severe diarrhea, body weight gain depression, bursa cores similar to the pems condition, and high rates of mortality in both infected and infected/cpd-immunodepressed poults, represents the first clear evidence that there may be specific virulent organisms involved in the pems disease condition. whether the atypical e. coli infections in association with pems is a primary or secondary infection remains to be determined. nevertheless, anecdotal evidence from field reports during the summer of indicated that a new fluoroquinalone antibiotic, sarafloxacin , which has efficacy against gramnegative bacteria such as e. coli and salmonella ssp., stopped mortality in pems-afflicted flocks of turkey poults. however, after the antibiotic had been terminated there was a recurrence of a pems-like condition. abbott laboratories, north chicago, il - . as these atypical e. coli strains are sensitive to sarafloxacin and resistant to all other commonly used antibiotics that have been tested (edens et al., unpublished data) , this field observation supported the concept that pems can be due to bacterial agents acting singly or in combination with an unknown viral etiology, which may induce immunodysfunction allowing these atypical e. coli strains to penetrate the gut epithelial barrier. after these e. coli strains become systemic, high rates of mortality and unthriftiness in the survivors can be observed. one of the signs of pems is inhibited or reduced growth accompanied by wasting of the muscle mass (brown, ; barnes and guy, ; barnes et al., ) . bacterial infections are known to result in whole body nitrogen loss proportional to the duration and severity of the disease (beisel, ) . in fact, wasting of muscle in the pems condition is a classic example of catabolic losses of amino acid nitrogen from skeletal muscle (beisel, ; rennie, ) that is used in noncarbohydrate gluconeogenesis. in this study with the atypical e. coli strains, wasting of muscle tissue was not always apparent in every poult, but in many of the survivor poults, there was very little muscle mass remaining at d of age, similar to the condition in field cases of pems. it has been noted that virulent e. coli strains in both chickens (tian and baracos, ; leitner and heller, ) and turkeys (leitner and heller, ) can cause diarrhea, wasting, and mortality. however, it was noted by both groups that stressors, such as inanition after virulent e. coli infection, exacerbated the disease and subsequent mortality. indeed, feed consumption was depressed within h postinfection with virulent e. coli (tian and baracos, ) and remained depressed for at least d after infection similar to the pems problem associated with field outbreaks. leitner and heller ( ) noted that a brief period of inanition at d of age resulted in rapid penetration of the intestine by virulent e. coli causing bacteremia and colonization of liver and spleen of poults. thus, this suggests that the atypical e. coli strains, which are known to have binding and penetrating ability for avian epithelial cells, have an opportunity to translocate from the intestine to the viscera causing septicemia during the time when pemsafflicted poults exhibit feed refusal but are eating litter that can be heavily ladened with atypical e. coli colony types and (edens et al., unpublished data). however, it is still not clear whether the atypical e. coli strains may be the primary or secondary cause of the feed refusal behavior in pems-afflicted poults, but as an enteroinvasive organism, the atypical e. coli have the potential to cause septicemia quickly. on the other hand, the disruption of the cellular integrity of the intestinal epithelium in response to infections by the atypical e. coli colony types and suggests that there may be another problem associated with pems. simply taken, the loss of functional microvilli and ultrastructural damage to the organelles within the epithelium cells suggests that there is a significant malabsorption problem associated with pems. this conclusion is based upon observations of elevated feed conversion ratios in pems poults (barnes and guy, ; brown, ; barnes et al., ; edens, unpublished data) . it appears clear that part of the reduction in weight gain can be attributed largely to the inability of the infected poults to absorb nutrient from the chymal content of the intestine. furthermore, the observation that there are large numbers of macrophages that migrate through the intestinal epithelium cells to phagocytize bacteria in the lumen of the ileum suggests that in cases of secretory diarrhea both transcellular and paracellular exudation of body fluids might occur which would exacerbate the diarrhea and dehydration associated with pems. additionally, the breakdown of the epithelial cell junctional complex integrity would also aid in the translocation of the atypical e. coli strains and other potentially pathogenic bacteria as well. protection against the infectivity of anaerobic bacteria from the ceca of pems-infected poults was observed in our first studies when killed bacteria were given orally at d followed at d with an oral challenge by a live culture of the anaerobes. the resistance to infection by the d administration of live organisms is not clearly understood. perhaps, the presentation of the killed organisms at d, before the intestinal epithelial cells seal and form a barrier to invasion of microorganisms, allowed the immature immune system in the intestinal tract to rapidly process antigenic information presented by the heat-killed bacterial cells. this would be consistent with the observations that even in ovo administration of antigen can stimulate early immune response in chickens (sharma, ; williams et al., ) . this finding suggests that antigenic sites on the killed bacterial cell membranes or in the cytoplasm can induce an immune response that can be protective to the poults regardless of the level of and kinds of maternal antibodies passed to the poult. on the other hand, this observation may indicate that there are insufficient maternal antibodies in the newly hatched poult to protect it from the ravages of these and other atypical and pathogenic bacteria and that a specific immune stimulant may be required to develop immunocompetence against specific pathogens such as these atypical e. coli strains. it appears that the dominance of atypical e. coli colony types or in a majority of the pems and flushing cases examined by us to date argues for their involvement in the etiology of pems. the fact that these atypical e. coli strains, colony types and and an additional unique and atypical strains, are resistant to most of the commonly used antibiotics (edens et al., unpublished data) also argues for their involvement in the pems disorder. the fact that these atypical e. coli colony types can cause significant depression in weight gain, cause development of bursa cores similar to the earlier observations of barnes and guy ( ) , brown ( ) , and barnes et al. ( ) , and cause high mortality rates, especially in immunocompromised poults, also argues for their involvement in the pems disease. the fact that these atypical e. coli colony types and are resistant to nearly all commonly-used antibiotics but sensitive to sarafloxacin and that sarafloxacin therapy completely stops pems-associated mortality argues for the inclusion of these bacteria as potential etiologies for pems. therefore, we conclude that the atypical e. coli colony types and are involved in the etiology of the pems disease, but we can not make a definitive statement at this time about whether these e. coli strains are involved in primary or secondary infections associated with pems. furthermore, there are numerous other atypical strains of e. coli that have been isolated from pems and flushing poults that are also antibiotic resistant, and these can not be ruled out as possible etiologies for pems. spiking mortality of turkeys (smt) and related disorders: an update poult enteritis and mortality syndrome ("spiking mortality of turkeys") and related disorders-an update and overview. newsletter, college of veterinary medicine and college of agriculture and life sciences metabolic effect of infection spiking mortality: pathology, performance, and prevention morphological changes and humoral immunity in cyclophosphamide-treated chicks the bursa of fabricius and immunoglobulin synthesis colibacillosis of turkeys exacerbated by hemorrhagic enteritis virus. laboratory studies colonization of escherichia coli in young turkeys and chickens selective suppression of humoral immunity. the effect of cyclophosphamide on the ontogeny of the humoral immune response in chickens use of virulent hemorrhagic enteritis virus for the induction of colibacillosis in turkeys immune system dysfunction during exposure to poult enteritis and mortality syndrome agents muscle protein turnover and wasting due to injury and disease sas user's guide: statistics. version edition embryo vaccination of specific pathogen free chickens with infectious bursal disease virus and tissue distribution of the vaccine virus and protection of hatched chicks against the disease field outbreaks of colibacillosis of turkeys associated with hemorrhagic enteritis virus effect of escherichia coli infection on growth and protein metabolism in broiler chicks (gallus domesticus) commercial broiler studies of marek's disease vaccination in ovo key: cord- -fk um nw authors: farver, carol f.; zander, dani s. title: molecular basis of pulmonary disease date: - - journal: molecular pathology doi: . /b - - - - . - sha: doc_id: cord_uid: fk um nw pulmonary pathology includes a large spectrum of both neoplastic and non-neoplastic diseases that affect the lung. many of these are a result of the unusual relationship of the lung with the outside world. every breath that a human takes brings the outside world into the body in the form of infectious agents, organic and inorganic particles, and noxious agents of all types. although the lung has many defense mechanisms to protect itself from these insults, these are not infallible; therefore, lung pathology arises. damage to the lung is particularly important given the role of the lung in the survival of the organism. any impairment of lung function has widespread effects throughout the body, since all organs depend on the lungs for the oxygen they need. pulmonary pathology catalogs the changes in the lung tissues and the mechanisms through which these occur. this chapter presents a review of lung pathology and the current state of knowledge about the pathogenesis of each disease. it suggests that a clear understanding of both morphology and mechanism is required for the development of new therapies and preventive measures. pulmonary pathology includes a large spectrum of both neoplastic and non-neoplastic diseases that affect the lung. many of these are a result of the unusual relationship of the lung with the outside world. every breath that a human takes brings the outside world into the body in the form of infectious agents, organic and inorganic particles, and noxious agents of all types. although the lung has many defense mechanisms to protect itself from these insults, these are not infallible and so lung pathology arises. damage to the lung is particularly important given the role of the lung in the survival of the organism. any impairment of lung function has widespread effects throughout the body, since all organs depend on the lungs for the oxygen they need. pulmonary pathology catalogs the changes in the lung tissues and the mechanisms through which these occur. what follows is a review of lung pathology and the current state of knowledge about the pathogenesis of each disease. we believe that a clear understanding of both morphology and mechanism is required for the development of new therapies and preventive measures. lung cancer is a major cause of morbidity and mortality throughout the world. the most recent estimates available from the surveillance, epidemiology, and end results (seer) program of the national cancer institute are that in over , people in the united states were diagnosed with cancer of the lung and bronchus, and over , will have died due to this disease [ ] . however, in the past decade incidence and mortality rates have begun to move in a more positive direction, particularly in men. overall, men show a decline in lung cancer incidence, while in women, although lung cancer rates grew from through , they stabilized from through [ ] . similarly, cancer death rates due to lung cancer have declined for men and have slowed for women. although, for women, lung cancer death rates have increased since , the rate of increase has slowed to . % annually from to [ ] . these trends parallel changes in the prevalence of tobacco smoking, the most important risk factor for development of lung cancer. given the tremendous societal and individual impacts of this disease, it is not surprising that the molecular biology of lung cancer is a major focus of investigation. elucidation of the molecular pathogenesis of these neoplasms has progressed significantly, offering insights into new, targeted therapies, and predictors of prognosis and therapeutic responsiveness. recognition of precursor lesions for some types of lung cancers has been facilitated by our expanded understanding of early molecular changes involved in carcinogenesis. the world health organization (who) classification scheme is the most widely used system for classification of these neoplasms (table . ) [ ] . although there are numerous histologic types and subtypes of lung cancers, most of the common malignant epithelial tumors can be grouped into the categories of nonsmall cell lung cancers (nsclcs) and small cell carcinomas (sclcs). nsclcs include adenocarcinomas (acs), squamous cell carcinomas (sqccs), large cell carcinomas, adenosquamous carcinomas, and sarcomatoid carcinomas. sclcs include cases of pure and combined small cell carcinoma. common pulmonary symptoms associated with these tumors include cough, shortness of breath, chest pain or tightness, and hemoptysis (coughing up blood). since some tumors cause airway obstruction, they predispose to pneumonia, which can be an important clue to the existence of a tumor in some patients. constitutional symptoms can include fever, weight loss, and malaise. some neoplasms will declare themselves with symptoms related to local invasion of adjacent structures such as chest wall, nerves, superior vena cava, esophagus, or heart. sclcs are known for early and widespread metastasis and are therefore particularly prone to being discovered through presentations as metastases in distant sites. some tumors are discovered due to pathophysiologic changes triggered by the release of soluble substances from tumor cells. endocrine syndromes due to elaboration of hormones are well recognized, and include cushing syndrome, syndrome of inappropriate antidiuretic hormone, hypercalcemia, carcinoid syndrome, gynecomastia, and others. hypercoagulability commonly occurs with lung cancers, leading to manifestations of venous thrombosis, nonbacterial thrombotic endocarditis, and disseminated intravascular coagulation. hematologic changes can include anemia, granulocytosis, eosinophilia, and other abnormalities. other paraneoplastic syndromes such as clubbing of the fingers, myasthenic syndromes, dermatomyositis/polymyositis, and transverse myelitis are noted in subsets of patients. when lung cancer is suspected, evaluation of the patient includes a thorough clinical, radiologic, and laboratory assessment, with collection of tissue or cytology samples to establish a pathologic diagnosis of malignancy and to classify the tumor type. fiberoptic bronchoscopy is often performed to collect samples for diagnosis. sample types can include transbronchial and endobronchial biopsies, bronchial brushings, bronchial washings, bronchoalveolar lavage samples, and transbronchial needle aspirates. submission of sputum samples for cytologic malignant epithelial tumors examination can provide a diagnosis in some cases, particularly for centrally located tumors such as sqcc and sclc. tumors arising in a peripheral location can also be sampled, in many cases, by fine needle aspiration or core needle biopsy performed under radiologic guidance. if a pleural effusion is present in combination with a lung parenchymal tumor, analysis of the pleural fluid cytology often allows one to establish a diagnosis. pleural biopsy, mediastinoscopy with biopsy, and wedge biopsy can also be performed, depending on the clinical and radiologic findings. for tumors with apparent distant metastasis, biopsy of the metastasis focus can both establish a pathologic diagnosis and determine the stage of the tumor. the prognosis of lung cancers is closely related to tumor stage. for nsclcs, the american joint commission on cancer tnm staging system is widely used (table . ) [ ] , and for sclcs, disease is classified as limited (restricted to one hemithorax) or extensive. overall, for lung cancers, the -year survival is . % for men and . % for women [ ] . an important factor leading to this relatively poor survival is the late stage at which many lung cancers are diagnosed. information from the seer database, from - , indicates that %, %, %, and % of patients were diagnosed with localized, regional, distant, or unstaged disease, respectively [ ] . the corresponding -year survival rates are . %, . %, . %, and . %, and year survival rates are . %, . %, . %, and . % [ ] . for patients with nsclcs, treatment depends on stage and comorbid conditions [ ] . surgical resection is the preferred approach to treatment of localized nsclcs, provided there is no medical contraindication to operative intervention. lobectomy or more extensive resection (depending on tumor extent) is usually recommended rather than lesser surgeries, unless other comorbid conditions preclude these procedures. tumor cm in greatest dimension, surrounded by lung or visceral pleura, without bronchoscopic evidence of invasion more proximal than the lobar bronchus t tumor with any of the following features of size or extent: > cm in greatest dimension, involves main bronchus ! cm distal to the carina, invades visceral pleura, associated with atelectasis or obstructive pneumonitis that extends to the hilar region but does not involve the entire lung t tumor of any size that directly invades the chest wall, diaphragm, mediastinal pleura, parietal pericardium; or lies < cm distal to the carina but without involvement of the carina; or is associated with atelectasis or obstructive pneumonitis of the entire lung t tumor of any size that invades the mediastinum, heart, great vessels, trachea, esophagus, vertebral body, carina; or has separate tumor nodule(s) in same lobe; or is associated with a malignant pleural effusion. regional lymph nodes (n) nx regional lymph nodes cannot be assessed n no regional lymph node metastasis n metastasis in ipsilateral peribronchial and/or ipsilateral hilar lymph nodes, including intrapulmonary nodes involved by direct extension of the primary tumor n metastasis in ipsilateral mediastinal and/or subcarinal lymph node(s) n metastasis in contralateral mediastinal, contralateral hilar, ipsilateral or contralateral scalene or supraclavicular lymph node(s). mx distant metastasis cannot be assessed m no distant metastasis m distant metastasis; includes separate tumor nodule(s) in a different lobe. occult t n m stage tis n m stage ia t n m stage ib t n m stage iia t n m stage iib t n m t n m stage iiia t n m t n m t n m t n m stage iiib any t n m t any intraoperative mediastinal lymph node sampling or dissection is also recommended for accurate pathologic staging and determination of therapy. subsets of patients also benefit from chemotherapy and radiotherapy. for more advanced nsclc and for sclc, chemotherapy and radiotherapy are the primary treatment modalities [ ] . rare patients with limited-stage sclcs can be considered for surgical resection with curative intent. development of lung cancer occurs with multiple, complex, stepwise genetic and epigenetic changes involving allelic losses, chromosomal instability and imbalance, mutations in tumor suppressor genes (tsgs) and dominant oncogenes, epigenetic gene silencing through promoter hypermethylation, and aberrant expression of genes participating in control of cell proliferation and apoptosis [ ] . there are similarities as well as type-specific differences in the molecular alterations between nsclcs and sclcs, and between sqccs and acs [ ] [ ] [ ] . oncogenes that play a part in the pathogenesis of lung cancer include myc, k-ras (predominantly acs), cyclin d , bcl , and erbb family genes such as egfr (epidermal growth factor receptor) (predominantly acs) and her /neu (predominantly acs) [ , ] . also, lung cancers often display abnormalities involving tsgs including tp , rb, p ink a , and new candidate tsgs on the short arm of chromosome (dutt , fhit, rasff a, fus- , bap- ) [ , ] . as research advances, these lists continue to grow, and as knowledge has expanded about the roles of these genes in carcinogenesis and tumor behavior, new targeted therapeutic agents have been designed to treat this disease ( figure . and table . ) [ ] . many other agents are under investigation. in cancers, chromosomal regions harboring tsgs and oncogenes are often deleted or amplified. allele loss involving loci in p - is a consistent feature of lung cancer pathogenesis [ , ] . wistuba et al. reported allelic losses of p, often multiple and discontinuous, in % of the lung cancers studied and in % of the precursor lesions [ ] . larger segments of allelic loss were noted in most sclcs ( %) and sqccs ( %) than in acs ( %) and preneoplastic/preinvasive lesions [ ] . there was allelic loss in the -kb p . deletion region in % of the lung cancers; % of the normal or reneoplastic/preinvasive lesions associated with lung cancers; and % of the normal, mildly abnormal, or preneoplastic/ preinvasive lesions found in smokers without lung cancer, but no loss was seen in the samples from people who had never smoked [ ] . p - deletions are also frequent and early events in the pathogenesis of lung carcinomas [ ] , and other common alterations include loh at q, q, q, and p [ ] . allelic losses that are more frequent in sqccs than acs include deletions at p (tp ), q (rb), p (p ink a ), p - , and several regions of p [ , , , ] . a recent study utilizing a bacterial artificial chromosome array to perform high-resolution whole genome profiling of sqcc and ac cell lines showed that regions of frequent amplification shared by both types of tumors included p; chromosome , q, q , q, and q; and common regions of deletion included p, q, p, p, q; chromosome ; and chromosome [ ] . however, acs appeared to have higher frequencies of deletion of chromosome ; p, q, q; and chromosome than sqccs, and possess small regions of amplification on chromosomes and not seen in sqccs. chromosome arms q and q were frequently deleted in ac but amplified in sqcc cell lines. both types of tumors showed deletion of chromosome arm p, but it was more frequent in the sqcc cell lines, while amplification of chromosome p was more frequent in acs. amplification of chromosome q was common to both types of tumors but showed frequent alteration at q - q in the sqcc lines and at q in the ac lines. inactivation of recessive oncogenes is believed to occur through a two-stage process. it has been suggested that the first allelic inactivation occurs, often via a point mutation, and the second allele is later inactivated by a chromosomal deletion, translocation or other alteration such as methylation of the gene promoter region [ ] . inactivating mutations in the tsg tp , which encodes the p protein, are the most frequent mutations in lung cancers. these mutations are found in up to % of nsclcs and over % of sclcs, and are largely attributable to direct dna damage from cigarette smoke carcinogens [ ] . tp mutational patterns show a prevalence of g to t transversions in % of smokers' lung cancers versus only % of lung cancers in nonsmokers [ ] . p protein is a transcription factor and a key regulator of cell cycle progression; cellular signals induced by dna damage, oncogene expression, or other stimuli trigger p dependent responses including initiating cell cycle arrest, apoptosis, differentiation, and dna repair [ ] . loss of p function in tumor cells can result in inappropriate progression through the dysregulated cell cycle checkpoints and permits the inappropriate survival of genetically damaged cells [ ] . the p ink a -cyclin d -cdk -rb pathway, which plays a central role in controlling the g to s phase transition of the cell cycle, is another important tumor suppressor pathway that is often disrupted in lung cancers. it interfaces with the p pathway through p arf and p waf/cip . thirty percent to % of nsclcs contain mutations of p ink a , including homozygous deletion or point mutations and epigenetic alterations, leading to p ink a inactivation [ ] . almost % of sclcs and smaller numbers of nsclcs, on the other hand, display loss of rb expression [ ] , and mutational mechanisms usually responsible include deletion, nonsense mutations, and splicing abnormalities that lead to truncated rb protein [ ] . p ink a leads to hypophosphorylation of the rb protein, which causes arrest of cells in the g phase. the active, hypophosphorylated form of rb regulates other cellular proteins including the transcription factors e f , e f , and e f , which are essential for progression through the g /s phase transition. loss of p ink a protein or increased complexes of cyclin d-cdk - or cyclin e-cdk lead to hyperphosphorylation of rb with resultant evasion of cell cycle arrest and progression into s phase [ , ] . cell cycle progression is inhibited by p waf/cip through its inhibition of the cyclin complexes. the %- % of nsclcs lacking detectable alterations in p ink a and rb may have abnormalities of cyclin d and cdk , which cause inactivation of the rb pathway [ ] . figure . provides an overview of the p and retinoblastoma (rb) pathways, showing the complex interactions between the components [ ] . epigenetic alterations (hypermethylation of the cpg island) of tsgs are also frequent occurrences during pulmonary carcinogenesis, and methylation profiles of nsclcs show relationships to smoke exposure, histologic type, and geography. methylation rates of p ink a and apc and the mean methylation index (mi) (a reflection of the overall methylation status) in current or former smokers were significantly higher than in never smokers; the mean mi of tumors was highest in current smokers; methylation rates of apc, cdh , and rarbeta were significantly higher in acs than in sqccs; methylation rates of mgmt and gstp in cases from the united states and australia significantly exceeded those from japanese and taiwanese cases; and no significant gender-related differences in methylation patterns were found [ ] . proto-oncogene activation and growth factor signaling are important in pulmonary carcinogenesis. the tyrosine kinase epidermal growth factor receptor (egfr) is frequently mutated in nsclcs, particularly in acs, and the mutational status is important in determining response to tyrosine kinase inhibitors. a related pathway, the phosphoinositide -kinase (pi k)/akt/mammalian target of rapamycin (mtor) pathway, is frequently deregulated in pulmonary carcinogenesis. as reviewed by marinov et al., this pathway has been reported to mediate the effects of several tyrosine kinase receptors, including egfr, c-met, c-kit, and igf-ir, on proliferation and survival in nsclc and sclc [ ] . clinical trials are ongoing, investigating the efficacy of the mtor inhibitor rapamycin and its analogues on lung cancer [ ] . her /neu is another related receptor tyrosine kinase that is upregulated in approximately %- % of nsclcs [ , ] , but unlike the situation with her /neu-positive breast cancers, treatment with anti-her /neu antibody (trastuzumab) does not seem to yield comparable benefits for nsclc when used alone or in combination with chemotherapy [ , ] . point mutations of ras family proto-oncogenes (most often at k-ras codons , , or ) are detected in %- % of lung acs and %- % of all nsclcs [ ] . although farnesyl transferase inhibitors prevent ras signaling, these agents have not shown significant activity as single-agent therapy in untreated nsclc or relapsed sclc [ ] . myc family genes (myc, mycn, and mycl), which play roles in cell cycle regulation, proliferation, and dna synthesis, are more frequently activated in sclcs than in nsclcs, either by gene amplification or by transcriptional dysregulation [ ] . vascular endothelial growth factor (vegf) is a homodimeric glycoprotein that is overexpressed in many lung cancers and directly stimulates endothelial cell proliferation, promotes endothelial cell survival in newly formed vessels, and induces proteases involved in the degradation of the extracellular matrix needed for endothelial cell migration [ ] . its angiogenic effects are mediated by three receptors: vegfr- , vegfr- , and vegfr- ; ligand binding leads to tyrosine kinase activation and activation of the signaling pathways required for angiogenesis [ ] . monoclonal antibodies to vegf (bevacizumab) and tyrosine kinase inhibitors to vegfrs have been developed and show promise for treatment of nsclc. a phase iii trial of bevacizumab showed significantly improved overall and progression-free survival when this agent was used in combination with standard first-line chemotherapy in patients with advanced nsclc, and several smallmolecule vegfr tyrosine kinase inhibitors have yielded favorable results in phase i and ii trials in nsclc [ ] . micrornas are a recently discovered class of nonprotein-coding, endogenous, small rnas which regulate gene expression by translational repression, mrna cleavage, and mrna decay initiated by mirna-guided rapid deadenylation [ ] . some micrornas such as let- have been suggested to play roles in carcinogenesis by functioning as oncogenes or tumor suppressors, negatively regulating tsgs and/or genes that control cell differentiation or apoptosis [ ] . investigations of the therapeutic potential of micrornas are also under way. in the version of the who classification scheme, ac is defined as "a malignant epithelial tumour with glandular differentiation or mucin production, showing acinar, papillary, bronchioloalveolar or solid with mucin growth patterns or a mixture of these patterns" [ ] . ac has become the most frequent histologic type of lung cancer in parts of the world. it occurs primarily in smokers, but represents the most common type of lung cancer in people who have never smoked and in women. a small subset of these tumors arise in patients with localized scars or diffuse fibrosing lung diseases such as asbestosis and interstitial pneumonia associated with scleroderma [ ] . these neoplasms usually arise in the periphery of the lung, and are more likely to invade the pleura and chest wall than other histologic types of lung cancers. radiologic studies can show one or more nodules, ground-glass opacities, or mixed solid and ground-glass lesions. on gross examination, the neoplasms are often solitary gray-white nodules or masses, sometimes with necrosis or cavitation, which pucker the overlying pleura. mucin-producing tumors can have a glistening, gelatinous appearance. other presentations include a pattern of consolidation resembling pneumonia (usually bronchioloalveolar carcinoma) ( figure . ), multiple nodules, diffuse interstitial widening due to lymphangitic spread, endobronchial lesions with submucosal infiltration, and diffuse visceral pleural infiltration and thickening resembling mesothelioma. common histologic patterns displayed by acs include acinar ( figure chapter molecular basis of pulmonary disease mixtures of these patterns are very frequent. less common histologic subtypes include fetal ac, mucinous (colloid) ac, mucinous cystadenocarcinoma, signet ring ac, and clear cell ac [ ] . acs usually exhibit differentiation toward clara cells or type ii pneumocytes or, less often, goblet cells. they manifest a range of differentiation extending from very well-differentiated tumors with extensive gland formation and little cytoatypia, to poorly differentiated, solid tumors that cannot be categorized as acs unless one orders a mucin stain (figure . ) . however, most examples include readily identifiable glands. invasiveness is reflected by the presence of neoplastic glands that infiltrate through stroma or pleura, stimulating a fibroblastic (desmoplastic) response ( figure . ) , or by cells in the lumens of blood vessels or lymphatics. in recent years, atypical adenomatous hyperplasia (aah) has been recognized as a precursor lesion for peripheral pulmonary acs. this lesion is defined as "a localized proliferation of mild to moderately atypical cells lining involved alveoli and, sometimes, respiratory bronchioles, resulting in focal lesions in peripheral part iv molecular pathology of human disease alveolated lung, usually less than mm in diameter and generally in the absence of underlying interstitial inflammation and fibrosis" (figure . ) [ ] . aah exists on a histologic continuum with bronchioloalveolar carcinoma (bac), which is defined as an in situ (noninvasive) form of ac, in which the neoplastic cells grow along alveolar septa (lepidic growth) without invasion of stroma or vasculature ( figure . , figure . ) [ ] . most bacs exceed cm in diameter and consist of cells with greater degrees of cytoatypia than aah. although aah is found in approximately % of patients without lung cancer at autopsy [ ] , it has been reported in %- % of lung resection specimens with all types of primary lung cancer and %- % of lung resection specimens with ac [ ] . the progenitor cell for bac and aah is believed to be an epithelial cell located at the junction between the terminal bronchiole and alveolus, termed the bronchioalveolar stem cell [ ] . a recently published large-scale study of primary lung acs, using dense single nucleotide polymorphism arrays, described significantly recurrent copy-number alterations in these tumors (table . ) [ ] . twenty-six of autosomal chromosome arms showed consistent large-scale copy-number gain or loss, and recurrent focal events, including amplifications and homozygous deletions, were found. although some of the alterations involved regions known to harbor a proto-oncogene or tsg, these genes remain to be identified in some of the other regions affected. amplification of chromosome q . was the most common event noted, found in % of samples. this region includes nkx - , which encodes a lineage-specific transcription factor (thyroid transcription factor- [ttf- ]) that activates transcription of target genes including the surfactant proteins, and may be an important proto-oncogene involved in a significant fraction of lung acs. immunohistochemical staining for ttf- can be performed to detect expression of this factor in most lung adenocarcinomas, aiding in the determination of the lung as the site of origin of the tumor (figure . ). additional work using small interfering rna (sirna)mediated knockdown of this gene in lung cancer cell lines with amplification led to reductions in tumor cell proliferation, through both decreased cell cycle progression and increased apoptosis, suggesting that gene amplification and overexpression contribute to lung cancer cell proliferation rates and survival [ ] . egfr and k-ras mutations are mutually exclusive mutational events in ac of the lung, which suggests the existence of two independent oncogenic pathways [ , ] . egfr is a receptor tyrosine kinase whose activation by ligand binding leads to activation of cell signaling pathways such as ras/mitogen-activated protein kinase (mapk) and phosphatidylinositol- -kinase, which in turn propagates signals for proliferation, blocking of apoptosis, differentiation, motility, invasion, and adhesion [ ] . tumor-acquired mutations in the tyrosine kinase domain of egfr, often associated with gene amplification, have been found in approximately %- % of nsclcs in the united states, and are associated with ac histology, never-smoker status, east asian ethnicity, and female gender [ , , ] . egfr mutations are frequently in-frame deletions in exon , single missense mutations in exon , or in-frame duplications/insertions in exon , and occasional missense mutations and double mutations can also be detected [ , ] . egfr mutation has an inverse correlation with methylation of the p ink a gene and sparc (secreted protein acidic and rich in cysteine), an extracellular ca þ-binding glycoprotein associated with the regulation of cell adhesion and growth [ ] . egfr status is an important predictor of response to egfr kinase inhibitors: patients with egfr mutations are most likely to have a significant response to egfr tyrosine kinase inhibitor therapy, and egfr amplification and protein overexpression have been reported to correlate with survival after egfr tyrosine kinase inhibitor therapy [ , ] . k-ras is a member of the ras family of proteins, which function as signal transducers between cell membrane-based growth factor signaling and the mapk pathways [ ] . k-ras mutations are associated with smoking, male gender, and poorly differentiated tumors [ ] . her (also known as egfr or erbb ), a member of the egfr family of receptor tyrosine kinases, is mutated in less than % of nsclc, and does not occur in tumors with egfr or k-ras mutation [ ] . the her mutations are in-frame insertions in exon and are significantly more frequent in acs ( . %), never smokers ( . %), asian ethnicity ( . %), and women ( . %), similar to egfr mutations [ ] . alterations in dna methylation appear to be important epigenetic changes in cancer, contributing to chromosomal instability through global hypomethylation, and aberrant gene expression through alterations in the methylation levels at promoter cpg islands [ ] . this lesion, which has been defined as a precursor lesion for peripheral pulmonary adenocarcinomas, consists of a wellcircumscribed nodule measuring several millimeters in diameter, in which alveolar septa are lined by mildly moderate atypical cells. epigenetic differences exist between egfr-mediated and k-ras-mediated tumorigenesis, and may interact with the genetic changes. a recent study showed that the probability of having egfr mutation was significantly lower among those with p ink a and cdh methylation than in those without, and the methylation index was significantly lower in egfr mutant cases than in wild-type. in contrast, k-ras mutation was significantly higher in p ink a methylated cases than in unmethylated cases, and the methylation index was higher in k-ras mutant cases than in wild-type [ ] . sqcc is defined as "a malignant epithelial tumour showing keratinization and/or intercellular bridges that arises from bronchial epithelium," in the who classification scheme [ ] . it is a common histologic type of nsclc that is closely linked to cigarette smoking. in most patients, this tumor arises in a mainstem, lobar, or segmental bronchus, producing a central mass on imaging known tumor suppressor genes and proto-oncogenes defined as found in either cosmic , cgp census , or other evidence; if there is more than one known proto-oncogene in the region, only one is listed (priority for listing is, in order: known lung adenocarcinoma mutation; known lung cancer mutation; other known mutation (by cosmic frequency); listing in cgp census). @myc is near, but not within, the peak region. ksingle gene deletions previously seen, this study provides new mutations as well. part iv molecular pathology of human disease studies. many of these tumors have an endobronchial component that can cause airway obstruction, leading to postobstructive pneumonia, atelectasis, or bronchiectasis. not infrequently, it is the pneumonia that prompts evaluation of the patient and leads to discovery of the tumor. less often, sqccs develop in the periphery of the lung. gross examination reveals a tan or gray mass that usually arises in a large bronchus and often includes an endobronchial component (figure . , figure . ). partial or complete airway obstruction can be associated with changes of pneumonia, bronchitis, abscess, bronchiectasis, or atelectasis. necrosis and cavitation are very common in these tumors. involvement of hilar lymph nodes by tan-gray tumor can be visible in some resected specimens. microscopically, the key features of this tumor are its keratinization, sometimes with formation of keratin pearls, and intercellular bridges ( figure . ). as is true of acs, the degree of differentiation of this tumor varies from very well differentiated cases, in which there are abundant keratinization and intercellular bridges and little cytoatypia, to very poorly differentiated cases, in which keratinization and intercellular bridges can be quite inconspicuous and the tumor consists of sheets of large atypical cells with marked cytoatypia and frequent mitoses. however, most cases fall more toward the middle of the spectrum. invasiveness is reflected by the presence of irregular nests and sheets of cells that infiltrate through tissues, stimulating a fibroblastic response, or by cells inside vascular or lymphatic spaces. invasive sqccs are often accompanied by sqcc in situ and dysplasia, their precursor lesions. these lesions arise in the bronchi and may be contiguous with the invasive tumor or exist as one or more separate foci. these precursor lesions can also be observed without coexisting invasive carcinoma. like sqcc, tobacco smoking is the main predisposing factor for sqcc in situ and dysplasia. unlike invasive sqcc, however, these lesions are not invasive-they do not extend through the basement membrane of the bronchial epithelium. grossly, they may be invisible or appear as flat, tan or red discolorations of the bronchial mucosa, or tan wart-like excrescences. microscopically, these lesions encompass a chapter molecular basis of pulmonary disease range of squamous changes that include alterations in the thickness of the bronchial epithelium, the maturational progress of squamous differentiation, cell size, and nuclear characteristics ( figure . , figure . ) [ , ] . as dysplasia increases from mild to moderate to severe, the epithelium thickens, and maturation is increasingly impaired. the basilar zone expands with epithelial cell crowding, the intermediate zone shrinks, and there is reduced flattening of the superficial squamous cells. cell size, pleomorphism, and anisocytosis usually increase, and there is coarsening of the chromatin and appearance of nucleoli, nuclear angulations, and folding. in carcinoma in situ, although the epithelium may or may not be thickened and the cell size may be small, medium, or large, there is minimal or no maturation from the base to the superficial aspect, and the atypical nuclear features are present throughout the entire thickness of the epithelium. mitoses appear in the lower third (mild or moderate dysplasia), lower two-thirds (severe dysplasia), or throughout the full thickness of the epithelium (carcinoma in situ). basal cells in the bronchial epithelium are believed to represent the progenitor cells for invasive sqcc, and the sequence of events leading to sqcc is believed to include basal cell hyperplasia, squamous metaplasia, squamous dysplasia, carcinoma in situ, and invasive sqcc (figure . ) [ , [ ] [ ] [ ] . regression of lesions preceding invasive sqcc can occur, particularly the earlier lesions [ ] . however, severe dysplasia and carcinoma in situ are associated with a significantly increased probability of developing invasive sqcc in patients followed over time with surveillance bronchoscopy [ ] . wistuba and colleagues evaluated sqccs and precursor lesions for loss of heterozygosity (loh) at chromosomal regions ( p , p . , p . - . , p , p - , p , q , p , q rb, and p tp ) part iv molecular pathology of human disease frequently deleted in lung cancer and found multiple, sequentially occurring allele-specific molecular changes in separate, apparently clonally independent foci, early in the pathogenesis of sqccs of the lung, suggesting a field cancerization effect [ , ] . they observed clones of cells with allelic loss at one or more regions in % percent of histologically normal epithelium and % of specimens with hyperplasia or metaplasia; increasing frequency of loh within clones with increasing histopathologic lesional severity; the most frequent and earliest regions of allelic loss at p , p - , p , and p ; increasing size of the p deletions with progressive histologic changes; and tp allelic loss in many histologically advanced lesions (dysplasia and cis) [ ] . an overview of the sequential molecular events leading to invasive sqcc is shown in figure . [ ] . large cell carcinoma is an undifferentiated nsclc without light microscopic evidence of squamous or glandular differentiation, although squamous or glandular features may be detectable by ultrastructural examination (figure . ) [ ] . histologic subtypes of large cell carcinoma include large cell neuroendocrine carcinoma (lcnec), combined lcnec, basaloid carcinoma, lymphoepithelioma-like carcinoma, clear cell carcinoma, and large cell carcinoma with rhabdoid phenotype [ ] . clinical signs and symptoms resemble those of other types of nsclc. most tumors develop as peripheral lung masses, except for basaloid carcinomas, which usually form centrally located masses. histologically, large cell carcinomas consist of sheets and nests of large cells with vesicular nuclei, prominent nucleoli, and moderate or abundant amounts of cytoplasm. lcnecs demonstrate neuroendocrine architectural features and immunohistochemical or ultrastructural evidence of neuroendocrine differentiation. basaloid carcinomas display nests of small, monomorphic, rounded or fusiform tumor cells with little cytoplasm, numerous mitoses, comedo-type necrosis, and hyaline or mucoid stromal degeneration. clear cell carcinoma consists of large tumor cells with clear cytoplasm. precursor lesions are not currently recognized for any of the subtypes of large cell carcinoma. however, basaloid carcinoma is associated with squamous dysplasia in about one-third of cases [ ] . large cell carcinomas are poorly differentiated carcinomas that can demonstrate features of ac (most frequent), sqcc, or neuroendocrine differentiation when examined by immunohistochemistry, electron microscopy, or molecular methods [ ] . these tumors often demonstrate losses of p, q, p, q, q, and p, and gains of q and p, more closely resembling acs than other histologic types of lung cancer [ ] . common molecular abnormalities include tp mutation, c-myc amplification, and p promoter hypermethylation, while k-ras mutation is less common [ ] . egfr tyrosine kinase domain mutation is not characteristic of large cell carcinomas, and egfrviii (deletion mutations in the extracellular domain of egfr) is uncommon [ , ] . the major categories of pulmonary neuroendocrine (ne) neoplasms include small cell carcinoma (sclc), large cell neuroendocrine carcinoma (lcnec), typical carcinoid, and atypical carcinoid. sclc and lcnec are high-grade carcinomas, typical carcinoid is a low-grade malignant neoplasm, and atypical carcinoid occupies an intermediate position in the spectrum of biologic aggressiveness. in one large series, the -year and -year survival rates for typical carcinoid were % and %, % and % for atypical carcinoid, % and % for lcnec, and % and % for sclc, respectively [ ] . by light microscopy, these tumors display ne architectural features including organoid nesting, a trabecular arrangement, rosette formation, and palisading. these patterns are more prominent in carcinoids than in lcnecs and may or may not be visible in individual sclcs. typical carcinoids contain fewer than mitoses per mm ( hpf) and lack necrosis ( figure . ), while atypical carcinoids show - mitoses per mm ( hpf) or necrosis, which is often punctate [ ] . sclc consists of small, undifferentiated tumor cells with scant cytoplasm and finely granular chromatin and absent or inconspicuous nucleoli ( figure . ). nuclear molding is characteristic, necrosis is common, and the mitotic rate is typically high, with a mean of over mitoses per mm [ ] . combined differences also exist in the characteristics of patients with carcinoids, as compared to patients with sclc and lcnec. patients with carcinoids are typically younger and less likely to smoke than those with sclcs and lcnecs, the vast majority of whom have a current or previous history of tobacco smoking [ , ] . rare patients with carcinoids have the multiple endocrine neoplasia (men ) syndrome, an association that is not seen with sclcs and lcnecs. in addition, an association with diffuse idiopathic pulmonary neuroendocrine cell hyperplasia (dipnech) has been noted for carcinoids but not for sclcs and lcnecs, leading to classification of dipnech as a preinvasive lesion in the most recent version of the who classification scheme [ ] . dipnech is a diffuse proliferation of single cells, small nodules (ne bodies), and linear proliferations of pulmonary ne cells that may reside in the bronchial and/or bronchiolar epithelia ( figure . ) , and may be accompanied by extraluminal proliferations part iv molecular pathology of human disease (tumorlets and carcinoids) [ ] . however, morphologically identifiable precursor lesions for sclc and lcnec have not been established. molecular markers of pulmonary ne tumors include chromogranin a, synaptophysin (figure . ) , and n-cam (cd ). these markers are expressed by all categories of ne tumors, with higher frequencies observed in the carcinoids and atypical carcinoids than in small cell and large cell neuroendocrine carcinomas. gastrin-releasing peptide, calcitonin, other peptide hormones, the insulinoma-associated (insm ) promotor and the human achaete-scute homolog- (hash ) gene have also been reported as overexpressed by these tumors [ , ] . thyroid transcription factor- (ttf- ) is expressed by %- % of sclcs, %- % of lcnecs, and %- % of carcinoids [ ] [ ] [ ] [ ] . sclcs [ ] [ ] [ ] [ ] [ ] [ ] . more than % of sclcs and sqccs demonstrate large, often discontinuous segments of allelic loss on chromosome p, in areas encompassing multiple candidate tumor suppressor genes, including some of those listed previously [ , ] . atypical carcinoids show a higher frequency of loh at p, q, p , and p than typical carcinoids, but not as high as the high-grade ne tumors [ ] . some typical and atypical carcinoids possess mutations of the multiple endocrine neoplasia (men ) gene on chromosome q or loh at this locus [ ] , while these abnormalities occur with lower frequencies in sclcs and lcnecs, supporting separate pathways of tumorigenesis [ ] . men encodes for the nuclear protein menin, which is believed to play several roles in tumorigenesis by linking transcription factor function to histone-modification pathways, in part through interacting with the activator-protein- family transcription factor jund, modifying it from an oncoprotein into a tumor suppressor protein [ ] . oncogenes frequently amplified in sclcs include myc ( q ), mycn ( p ), and mycl ( p ), and additional amplified genes that represent candidate oncogenes include the antiapoptotic genes tnfrsf ( p ), dad ( q ), bcl l ( q ), and bcl l ( q ) [ ] . the myc proteins are transcription factors that are important in cell cycle regulation, proliferation, and dna synthesis, and can induce p arf , leading to apoptosis through p if cellular conditions do not favor proliferation [ ] . tsgs are inactivated in the majority of sclcs. eighty percent to % of sclcs demonstrate tp mutations, as compared to more than % of nsclcs, fewer atypical carcinoids, and virtually no typically carcinoids [ , ] . most of the tp mutations in sclcs are missense point mutations that result in a stabilized p mutant protein which can be easily detected by immunohistochemistry [ ] . p protein overexpression occurs frequently in high-grade ne carcinomas, but is unusual in typical carcinoids and intermediate in atypical carcinoids [ , ] . dysregulation of p produces downstream effects on bcl- and bax. antiapoptotic bcl- predominates over proapoptotic bax in the high-grade ne carcinomas, while the reverse is true for carcinoids [ ] . lcnecs resemble sclcs in their high rates of tp mutation and predominance of bcl- expression over bax expression [ ] . alterations compromising the p ink a /cyclin d /rb pathway of g arrest are consistent in high-grade pulmonary ne carcinomas ( %), primarily through loss of rb protein, but are less frequent in atypical carcinoids ( %) and are uncommon in typical carcinoids [ ] . mutations in the rb gene exist in many sclcs, with associated loss of function of the gene product [ , , ] . in another study, % of the ne carcinomas (excluding carcinoids) versus % of the non-ne carcinomas exhibited loh and loss of rb-protein expression [ ] . the hypophosphorylated form of rb protein functions as a cell cycle regulator for g arrest; cyclin d overexpression and p ink a loss produce persistent hyperphosphorylation of rb with consequent evasion of cell cycle arrest [ ] . recent data also suggest that in sclcs, overexpression of mdm (a transcriptional target of p ) or p arf loss leads to evasion of cell cycle arrest through the p and rb pathway ( figure . ) [ ] . the transcription factor e f- appears to play a role in cellular proliferation by activating genes required for s phase entry. e f- product is overexpressed in % of sclcs and % of lcnecs, and is significantly associated with a high ki index and bcl- :bax ratio > [ ] . a mediator of the proteasomal degradation of e f- , the s phase kinase-associated protein (skp ) f-box protein accumulates in high-grade ne carcinomas ( %), and its overexpression has been associated with advanced stage and nodal metastasis in pulmonary ne tumors [ ] . in the high-grade ne tumors, skp appears to interact with e f- and stimulate its transcriptional activity toward the cyclin e promoter [ , ] . telomeres play an important role in the protection of chromosomes against degradation. telomerases, the enzymes that synthesize telomeric dna strands, serve to counterbalance losses of dna during cell divisions. high telomerase activity has been noted in over % of sclcs and lcnecs [ ] [ ] [ ] versus % or fewer typical carcinoids [ , ] . expression of human telomerase mrna component (hterc) and human telomerase reverse transcriptase (htert) mrna were reported, respectively, in % and % of typical carcinoids; and in % and % of atypical carcinoids, lcnecs and sclcs, and telomere length alterations in lcnecs and sclcs were greater than in typical carcinoids [ ] . aberrant methylation of cytosine-guanine (cpg) islands in promoter regions of malignant cells is an important mechanism for silencing of tsgs (epigenetic inactivation). methylation of dna involves the transfer of a methyl group, by a dna methyltransferase, to the cytosine of a cpg dinucleotide [ ] . rassf a is a potential tsg that undergoes epigenetic inactivation in virtually all sclcs and a majority of nsclcs through hypermethylation of its promoter region [ , ] . ne tumors have lower frequencies of methylation of p , apc, and cdh (h-cadherin) than nsclcs [ ] . sclcs have higher frequencies of methylation of rassf a, cdh (e-cadherin), and rarb than carcinoids [ ] . promoter methylation of casp , which encodes the apoptosis-inducing cysteine protease caspase , was also found in % of sclcs, % of carcinoids, and no nsclcs, suggesting that casp may function as a tsg in ne lung tumors [ ] . although histologically defined precursors for sclc are lacking, a higher incidence of genetic abnormalities is found in the normal or hyperplasic airway epithelium of patients with sclc than nsclc [ ] . by extension, it has been suggested that sclc may arise directly from histologically normal or mildly abnormal epithelium, rather than evolving through a sequence of recognizable histologic intermediary changes [ ] . relatively little is known about molecular abnormalities in precursors of carcinoids. although carcinoids have been viewed as arising from tumorlets, q (int- ) allelic imbalance is significantly more common in carcinoids ( %) than in tumorlets ( %), and may represent an early event in carcinoid tumor formation [ ] . the int- gene lies in close proximity to men , a tumor suppressor gene frequently mutated in ne tumors [ ] . the molecular pathology of dipnech remains to be elucidated. mesenchymal neoplasms included in the who classification scheme (table . ) encompass a spectrum of malignant and benign proliferations that show differentiation along multiple lineages. overall, these tumors are much less common in the lung than are epithelial neoplasms. information about molecular pathogenesis has emerged for some of the mesenchymal neoplasms. pulmonary inflammatory myofibroblastic tumor (imt) is a lesion composed of myofibroblastic cells, collagen, and inflammatory cells that primarily occurs in individuals less than years of age, and is the most common endobronchial mesenchymal lesion in childhood ( figure . ) [ ] . synovial sarcoma is usually a soft tissue malignancy, but uncommonly arises in the pleura or the lung and often takes an aggressive course [ ] . pulmonary hamartomas are benign neoplasms consisting of mixtures of cartilage, fat, connective tissue, and smooth muscle, which present as coin lesions on chest radiographs and are excised in order to rule out a malignancy ( figure . ). many imts demonstrate clonal abnormalities with rearrangements of chromosome p and the anaplastic lymphoma kinase (alk) gene [ ] . the rearrangements involve fusion of tropomyosin (tpm) n-terminal coiled-coil domains to the alk c-terminal kinase domain, producing two alk fusion genes, tpm -alk and tpm -alk, which encode oncoproteins with constitutive kinase activity [ ] . like their soft tissue counterparts, more than % of pulmonary and pleural synovial sarcomas demonstrate a chromosomal translocation t(x; ) (syt-ssx) [ , ] . detection of this translocation can be very helpful for confirming the diagnosis of synovial sarcoma in this unusual location. most pulmonary hamartomas show abnormalities of chromosomal bands p , q - , or other regions [ ] , corresponding to mutations of high-mobility group (hmg) proteins, a family of nonhistone chromatin-associated proteins that serve an important role in regulating chromatin architecture and gene expression [ ] . malignant mesothelioma (mm) is an uncommon, aggressive tumor arising from mesothelial cells on serosal surfaces, primarily the pleura and peritoneum, and less often the pericardium or tunica vaginalis. the most important risk factor for mm is exposure to the subset of asbestos fibers known as amphiboles (crocidolite and amosite) [ ] . the incidence of this tumor in the united states peaked in the early to mid- s, and appears to be declining, likely related to decreases in the use of amphiboles since their peak period of importation in the s [ ] . these tumors are characterized by long latency periods between asbestos exposure and clinical presentation of the tumor, with a mean of - years [ ] . radiation, a nonasbestos fiber known as erionite, and potentially other processes associated with pleural scarring have also been implicated in the causation of smaller numbers of cases of malignant mesothelioma [ ] , and a role for simian virus (sv ) in the genesis of this tumor has been suggested by some, but remains controversial [ , ] . pleural mm most commonly arises in males over the age of . presenting features typically include a hemorrhagic pleural effusion associated with shortness of breath and chest wall pain. weight loss and malaise are common. by the time the tumor is discovered, patients usually have extensive involvement of the pleural surfaces. with progression, the tumor typically invades the lung, chest wall, and diaphragm. lymph node metastasis can cause superior vena caval obstruction, and cardiac tamponade, subcutaneous nodules, and contralateral lung involvement can also occur. from the time of diagnosis, the median survival is months [ ] . treatment may include surgery, chemotherapy, radiotherapy, immunotherapy, or other treatments, often in combination [ ] . the intent of surgery is usually palliative. whether extrapleural pneumonectomy with chemotherapy and radiotherapy can lead to cure is unclear [ ] . new agents are currently under investigation for their potential to improve the life expectancy and quality of life in patients with this aggressive malignancy. gross pathologic features of mm include pleural nodules which grow and coalesce to fill the pleural cavity and form a thick rind around the lung. a firm tan appearance is common, and occasionally the tumor can have a gelatinous consistency (figure . ). extension along the interlobar fissures and invasion into the adjacent lung, diaphragm, and chest wall are characteristic. further spread can occur into the pericardial cavity and around other mediastinal structures, and distant metastases can also develop. histologically, mm manifests a wide variety of histologic patterns. the major histologic categories include epithelioid mesothelioma, sarcomatoid mesothelioma, desmoplastic mesothelioma, and biphasic mesothelioma [ ] . epithelioid mesothelioma consists of round, ovoid, or polygonal cells with eosinophilic cytoplasm and nuclei that are usually round with little cytoatypia (figure . ). these cells most often form sheets, tubulopapillary structures, or gland-like arrangements, and some tumors can have a myxoid appearance due to production of large amounts of hyaluronate. sarcomatoid mesothelioma is composed of malignant-appearing spindle cells occasionally accompanied by mature sarcomatous components (osteosarcoma, chondrosarcoma, others). desmoplastic mesothelioma can be a diagnostic challenge due to its frequently bland appearance and resemblance to organizing pleuritis. it consists of variably atypical spindle cells in a dense collagenous matrix ( figure . ). helpful features for separating figure . malignant mesothelioma. the tan/white tumor involves the entire pleura surrounding and compressing the underlying parenchyma, which appears congested but relatively unremarkable. chapter molecular basis of pulmonary disease this tumor from organizing pleuritis include invasion of chest wall muscle or adipose tissue and necrosis. biphasic mesotheliomas include both epithelioid and sarcomatoid elements, each comprising at least % of the tumor [ ] . pathologic diagnosis of mm has been greatly assisted by the expanded availability of antibodies for use in immunohistochemistry [ ] . mesothelial differentiation can be supported by immunoreactivity with cytokeratin / , calretinin ( figure . ), hbme- , d - , and other antibodies. histologic distinction of epithelioid mesotheliomas from metastatic acs is a common need in practice, and a panel approach using calretinin and cytokeratin / , with other antibodies reactive with acs (cea, moc- , ber-ep , leu m , b . , and others) will usually be successful. electron microscopy can also be helpful in difficult cases by demonstrating long thin microvilli in many mms with an epithelioid component. pan-cytokeratin staining is helpful for supporting a diagnosis of sarcomatoid or desmoplastic mm as opposed to sarcoma, since most (but not all) sarcomas will not stain for pan-cytokeratin. other mesothelial and mesenchymal markers can also be useful for assisting in the differentiation of mm from histologically similar sarcomas. precursor lesions for mm have not been clearly defined from a histologic standpoint, although it is likely that an in situ stage exists [ ] . the term atypical mesothelial hyperplasia has been recommended for surface (noninvasive) proliferations of mesothelial cells of uncertain malignant potential [ ] . exposure to asbestos fibers is believed to trigger the pathobiological changes leading to the majority of mms. currently, it is believed that asbestos may act as an initiator (genetically) and promoter (epigenetically) in the development of mms [ ] . the degree to which tumorigenesis results from direct interactions of the fibers with the mesothelial cells, or through other mechanisms involving oxidative stress (or both), is unresolved [ , ] . multiple chromosomal alterations are often noted in mms, and inactivation of tsgs plays an important part in the pathogenesis of mm [ ] . a variety of genetic abnormalities have been reported including deletions of p - , p , p, q, q, p , q - , q, and proximal q, monosomy , and gains of q, p, p, q - , and q - part iv molecular pathology of human disease [ , ] . the most common genetic abnormality in mm is a deletion in p encompassing the cdkn a locus encoding the tumor suppressors p ink a and p arf , which participate in the p and rb pathways and inhibit cell cycle progression ( figure . ) [ , ] . recent studies have shown that sv large t antigen (present in some mms) inactivates the tsg products rb and p , raising the possibility that asbestos and sv could act as co-carcinogens in mm and suggesting that perturbations of rb-and p -dependent growth-regulatory pathways may be involved in the pathogenesis of mm [ ] . other common findings include inactivating mutations with allelic loss in the tsg neurofibromin (nf ), found at chromosome q [ ] , and inactivation of cdkn a/p arf and gpc (another tsg) by promoter methylation [ ] . loss of cdkn a/ p arf also results in mdm -mediated inactivation of p [ ] . however, in mms, unlike many other epithelial tumors, mutations in the tp , rb, and ras genes are rare [ ] . the wnt signal transduction pathway is also abnormally activated in mms and appears to play a role in pathogenesis [ ] . activation of the pathway leads to accumulation of b-catenin in the cytoplasm and its translocation to the nucleus. interactions with tcf/ lef transcription factors promote expression of multiple genes including c-myc and cyclin d. the mechanism of activation does not appear to involve mutations in the b-catenin gene, but may instead involve more upstream components of the pathway, such as the disheveled proteins [ ] . recent evidence also suggests that the phosphatidylinositol -kinase (pi -k/akt) pathway is frequently activated in mms, and that inhibition of this pathway can increase sensitivity to a chemotherapeutic agent [ ] . the wilms' tumor gene (wt ) is also expressed in most mms, but its role in the pathogenesis of mm is unclear [ ] . finally, egfr signaling in mms has recently become a focus of greater attention, and there are some data showing that the egfr is an early cell membrane target of asbestos fibers and is linked to activation of the mapk cascade [ ] . unfortunately, a phase ii clinical trial of gefitinib treatment in patients with mms did not show effectiveness, despite egfr overexpression in over % of cases [ ] . another study found that common egfr mutations conferring sensitivity to gefitinib are not prevalent in human malignant mesothelioma [ ] . further investigation continues into new, potentially efficacious agents for the treatment of mm. non-neoplastic pulmonary pathology comprises inflammatory and fibrosing diseases of the conducting airways, alveoli, vessels, and lymphoid tissue. this pathology may be localized or diffuse, may either have an obvious etiology or be idiopathic, and may cause injury that is reparable or irreparable. most importantly, an understanding of non-neoplastic lung pathology plays a vital role in the clinical management of these diseases. this section covers the major types of obstructive and interstitial diseases, the vascular lesions, the pneumonias, the occupational diseases, the major histiocytic conditions, and the most common developmental anomalies. this list does not include all of the non-neoplastic diseases that can affect the lung, but it represents those that are responsible for the majority of illness. also, the conditions highlighted within each of these categories are those about which we best understand the molecular biology of the disease mechanisms. obstructive lung diseases are characterized by a reduction in airflow due to airway narrowing. this airflow reduction occurs, in general, by two basic mechanisms: (i) inflammation and injury of the airway, resulting in obstruction by mucous and cellular debris within and around the airway lumen; and (ii) destruction of the elastin fibers of the alveolar walls, causing loss of elastic recoil and subsequent premature collapse of the airway during the expiratory phase of respiration. there are four major obstructive lung diseases: asthma, emphysema, chronic bronchitis, and bronchiectasis. asthma is a chronic inflammatory disease of the airways that affects more than million people worldwide. the prevalence of disabling asthma has increased over % since , ranging from as low as % in rural ethiopia to over % among children in parts of central and south america [ ] . in the united states, asthma affects approximately %- % of the population and is the leading cause of hospitalization among children less than years of age [ ] . clinically, the disease is defined as a generalized obstruction of airflow with a reversibility that can occur spontaneously or with therapy. it is characterized by recurrent wheezing, cough, or shortness of breath resulting from airway hyperactivity and mucus hypersecretion. the hyperresponsiveness is a result of acute bronchospasm and can be elicited for diagnostic purposes using histamine or methacholine challenges. the key feature of these symptoms is that they are variable-worse at night or in the early morning, and in some people worse after exercise. it has previously been assumed that these symptoms are separated by intervals of normal physiology. however, evidence is now accumulating that asthma can cause progressive lung impairment due to chronic morphologic changes in the airways. the treatment strategies for this complex disease are myriad. in atopic individuals, allergen avoidance should be the primary therapy. for example, in children, reducing exposure to house dust mites early in life decreases sensitization and the incidence of disease. for those who do develop the disease, avoidance of allergens later in life improves symptom control. established treatments for asthma flairs include inhaled corticosteroids, and short-acting and long-acting b -adrenoceptor agonists. phosphodiesterase (pde) inhibitors such as theophylline have been used for decades to treat asthmatic bronchoconstriction, but both cardiac and central nervous systems side effects have limited their use. newer pde inhibitors without side effects include non-xanthine drugs such as rofumilast. the pathologic changes to the airways in asthma are very similar to those seen in chronic bronchitis. they consist of a thickened basement membrane with epithelial desquamation, goblet cell hyperplasia, and subepithelial elastin deposition. in the wall of the airway, smooth muscle hypertrophy and submucosal gland hyperplasia are also present ( figure . ). in acute asthma exacerbations, a transmural chronic inflammatory infiltrate with variable amounts of eosinophilia may be present, resulting in epithelial injury and desquamation that can become quite pronounced. one sees clumps of degenerating epithelial cells mixed with mucin in the lumen airway. these aggregates of degenerating cells are referred to as creola bodies and can be seen in expectorated mucin from these patients. also present in these sputum samples are charcot-leyden crystals, rhomboid-shaped structures that represent breakdown products from eosinophil cytoplasmic granules ( figure . ). the changes seen in the walls of these airways represent long-term airway remodeling caused by prolonged inflammation. this remodeling may play a role in the pathophysiology of asthma. the amount of airway remodeling is highly variable from patient to patient, but remodeling has been found even in patients with mild asthma. currently, the effect of the treatment on this chronic pathology is unclear [ ] . the pathogenesis of asthma is complex, and most likely involves both genetic and environmental components. most experts now see it as a disease in which an insult initiates a series of events in a genetically susceptible host. no single gene accounts for the familial component of this disease. genetic analysis of these patients reveals a prevalence of specific hla alleles, polymorphisms of fc erib, il- , and cd [ , ] . asthma can be classified using a number of different schema. most commonly, asthma is divided into two categories: atopic (allergic) and nonatopic (nonallergic). atopic asthma results from an allergic sensitization usually early in life and has its onset in early childhood. nonatopic asthma is late-onset and, though the immunopathology has not been as well studied, probably has similar mechanisms to atopic asthma. although this nosology is convenient for purposes of understanding the mechanisms of the disease, most patients manifest a combination of these two categories with overlapping symptoms. th pathogenetic mechanisms of both types encompass a variety of cells and their products. these include airway epithelium, smooth muscle cells, fibroblasts, mast cells, eosinophils, and t-cells. the asthma response includes two phases: an early response comprising an acute bronchospastic event within - minutes after exposure, and a late response that peaks approximately - hours and that can have prolonged effects. if one wants to understand this complex response, it is best to divide it into three components: (i) a type hypersensitivity response, (ii) acute and chronic inflammation, and (iii) bronchial hyperactivity. type hypersensitivity in general, human asthma is associated with a predominance of type helper cells with a cd þ phenotype. these th -type cells result from the uptake and processing of viral, allergen, and environmental triggers that initiate the episode. the processing includes the presentation of these triggers by the airway dendritic cells to naive t-cells (th ), resulting in their differentiation into populations of th and th . the th differentiation is a result of il- release by the dendritic cells, and the th cells then part iv molecular pathology of human disease further propagate the inflammatory reaction in two ways. first, they release a variety of cytokines such as il- , il- , and il- that mediate a wide variety of responses. il- and il- stimulate b-cells and plasma cells to produce ige, which, in turn, stimulates mast cell maturation and the release of multiple mediators, including histamine and leukotrienes. second, these th cells secrete il- that, together with il- , also stimulates mast cells to secrete histamine, tryptase, chymase, and the cysteinyl leukotrienes causing the bronchoconstrictor response that occurs rapidly after the exposure to the allergen. il- from these lymphocytes also recruits eosinophils to the airways and stimulates the release of the contents of their granules, including eosinophil cationic protein (ecp), major basic protein (mbp), eosinophil peroxidase, and eosinophil-derived neurotoxin. these compounds not only induce the bronchial wall hyperactivity but are also responsible for the increased vascular permeability that produces the transmural edema in the airways. the cells can differentiate into th cells as a result of il- produced by dendritic cells. these th cells produce interferon-gamma (ifn-g), il- , and lymphotoxin, which play a role in macrophage activation in delayedtype hypersensitivity reactions as seen in diseases such as rheumatoid arthritis and tuberculosis [ ] . these th cells are predominantly responsible for defense against intracellular organisms and are more prominent in normal airways and in airways of patients with emphysema than in asthmatics. however, in severe forms of asthma, th cells are recruited and have the capacity to secrete tumor necrosis factor (tnf)-a and ifn-g, which may lead to the tissue-damaging immune response one sees in these airways (figure . ) [ , ] . acute and chronic inflammation the role of acute and chronic inflammatory cells, including eosinophils, mast cells, macrophages, and lymphocytes, in asthma is evident in the abundance of these cells in airways, sputum, and bronchoalveolar samples from patients with this disease. the number of eosinophils in the airways correlates with the severity of asthma and the amount of bronchial hyperresponsiveness. proteins released by these cells including ecp, mcp, and eosinophil-derived neurotoxin cause at least some of the epithelial damage seen in the active form of asthma. neutrophils are prominent in the more acute exacerbations of asthma and are probably recruited to these airways by il- , a potent neutrophil chemoattractant released by airway epithelial cells [ ] . these cells also release proteases, reactive oxygen species (ros), and other proinflammatory mediators that, in addition to the epithelial damage, also contribute to the airway destruction and remodeling that occurs in the more chronic forms of this disease. the susceptibility of the epithelium in asthma to this oxidant injury may be increased due to decreased antioxidants such as superoxide dismutase in these lungs [ ] . finally, mast cells are activated to release an abundance of mediators through the binding of ige to fceri, high-affinity receptors on their surface. allergens bind to ige molecules and induce a cross-linking of these molecules, leading to activation of the mast cell and release of a number of mediators, most notably histamine, tryptase, and various leukotrienes, including leukotriene d (ltd ), and interact with the smooth muscle to induce contraction and the acute bronchospastic response [ ] . allergen bronchial hyperactivity the cornerstone of asthma is the hyperactive response of the airway smooth muscle. the mechanism by which this occurs combines neural pathways and inflammatory pathways. as stated, the inflammatory component of this response comes predominantly from the mast cells. the major neural pathway involved is the nonadrenergic noncholinergic (nanc) system. although cholinergic pathways are responsible for maintaining the airway smooth muscle tone, it is the nanc system that releases bronchoactive tachykinins (substance p and neurokinin a) that bind to nk receptors on the smooth muscle and cause the constriction that characterizes the acute asthmatic response [ ] . in addition to these acute mechanisms, the airway also undergoes structural alterations to its formed elements. in the mucosa, these changes include goblet cell hyperplasia and basement membrane thickening. within the submucosa and airway wall, increased deposition of collagen and elastic fibers results in fibrosis and elastosis, and both the smooth muscle cells and the submucosal glands undergo hypertrophy and hyperplasia. these irreversible changes are a consequence of chronic inflammatory insults on the airways through mechanisms that include release of fibrosing mediators such tgfb and mitogenic mediators such as epidermal and fibroblast growth factors (egf, fgf). the exact mechanisms by which this occurs are not clearly defined, but the similarity of these factors with those involved in branching morphogenesis of the developing lung has led to a focus on the effect of inflammation on the interaction of the epithelium with the underlying mesenchymal cells [ ] . the term chronic obstructive pulmonary disease (copd) applies to emphysema, chronic bronchitis, and bronchiectasis, those diseases in which airflow limitation is usually progressive, but, unlike asthma, not fully reversible [ ] . the prevalence of copd worldwide is estimated at %- % in adults over the age of [ ] . though there are different forms of copd with different etiologies, the clinical manifestations of the most common forms of the disease are the same. these include a progressive decline in lung function, usually measured as decreased forced expiratory flow in second (fev ), a chronic cough, and dyspnea. emphysema and chronic bronchitis are the most common diseases of copd and are the result of cigarette smoking. as such, they usually exist together in most smokers. chronic bronchitis is defined clinically as a persistent cough with sputum production for at least months in at least consecutive years without any other identifiable cause. patients with chronic bronchitis typically have copious sputum with a prominent cough, more commonly get infections, and typically experience hypercapnia and severe hypoxemia, giving rise to the clinical moniker blue bloater. emphysema is the destruction and permanent enlargement of the air spaces distal to the terminal bronchioles without obvious fibrosis [ ] . these patients have only a slight cough, while the overinflation of the lungs is severe, inspiring the term pink puffers. the pathologic features of copd are best understood if one considers the whole of copd as a spectrum of pathology that consists of emphysematous tissue destruction, airway inflammation, remodeling, and obstruction [ ] . the lungs of patients with copd usually contain all of these features, but in varying proportions. the pathologic features of chronic bronchitis include mucosal pathology that consists of epithelial inflammation, injury, and regenerative epithelial changes of squamous and goblet cell metaplasia. in addition, the submucosa shows changes of remodeling with smooth muscle hypertrophy and submucosal gland hyperplasia. these changes are responsible for the copious secretions characteristic of this clinical disease, although studies have reported no consistent relationship between these pathologic features of the large airways and the airflow obstruction [ ] . the pathology definition of emphysema is an abnormal, permanent enlargement of the airspaces distal to the terminal bronchioles accompanied by destruction of the alveolar walls without fibrosis [ ] . the four major pathologic patterns of emphysema are defined by the location of this destruction. these include centriacinar, panacinar, paraseptal, and irregular emphysema. the first two of these are responsible for the overwhelming majority of the clinical disease. centriacinar emphysema (sometimes referred to as centrilobular) represents % of the cases and is a result of destruction of alveoli at the proximal and central areas of the pulmonary acinus, including the respiratory bronchioles ( figure . ). it predominantly affects the upper lobes the remaining two types of emphysema, paraseptal and irregular, are rarely associated with clinical disease. in paraseptal emphysema, the damage is to the distal acinus, the area that abuts the pleura at the margins of the lobules. damage in this area may cause spontaneous pneumothoraces, typically in young, thin men [ ] . irregular emphysema is tissue destruction and alveolar enlargement that occurs adjacent to scarring, secondary to the enhanced inflammation in the area. though this is a common finding in a scarred lung, it is of little if any clinical significance to the patient. though the emphysema in these lungs plays the dominant role in causing the obstruction, small airway pathology is also present. respiratory bronchiolitis refers to the inflammatory changes found in the distal airways of smokers. these consist of pigmented macrophages filling the lumen and the peribronchiolar airspaces and mild chronic inflammation and fibrosis around the bronchioles (figure . ). the pigment in these macrophages represents the inhaled particulate matter of the cigarette smoke that has been phagocytized by these cells. the macrophages in turn release proteases, which destroy the elastic fibers in the surrounding area, resulting in the loss of elastic recoil and the obstructive symptoms. in general, copd is a result of inflammation of the large airways that produces the airway remodeling characteristic of chronic bronchitis as well as inflammation of the smaller airways that results in the destruction of the adjacent tissue and consequent emphysema. the predominant inflammatory cells involved in this process are the alveolar macrophages, neutrophils, and lymphocytes. the main theories of the pathogenesis of copd support the interaction of airway inflammation with two main systems in the lung: the protease-antiprotease system and the oxidant-antioxidant system. these systems help to protect the lung from the many irritants that enter the lung via the large pulmonary surface area that interfaces with the environment. in the protease-antiprotease system, proteases are produced by a number of cells, including epithelial cells and inflammatory cells that degrade the underlying lung matrix. the most important proteases in the lung are the neutrophil elastases, part of the serine protease family, and the metalloproteinases (mmps) produced predominantly by macrophages. these proteases can be secreted in response to invasion by environmental irritants, most notably infectious agents such as bacteria. in this setting, their role is to enzymatically degrade the organism. however, proteases can also be secreted by both inflammatory and epithelial cells in a normal lung to repair and maintain the underlying lung matrix proteins [ ] . to protect the lung from unwanted destruction by these enzymes, the liver secretes antiproteases that circulate in the bloodstream to the lung and inhibit the action of the proteases. in addition, macrophages that secrete mmps also secrete tissue inhibitors of metalloproteinases (timps). a delicate balance of proteases and antiproteases is needed to maintain the integrity of the lung structure. an imbalance that results in a relative excess of proteases (either by overproduction of proteases or underproduction of their inhibitors) leads to tissue destruction and the formation of emphysema. this imbalance occurs in different ways in the two major types of emphysema: centriacinar and panacinar. in centriacinar emphysema, caused primarily by cigarette smoking, there is an overproduction of proteases primarily due to the stimulatory effect of chemicals within the smoke on the neutrophils and macrophages. though the exact mechanism is not completely understood, most studies support that nicotine from the cigarette smoke acts as a chemoattractant, and ros also contained in the smoke, stimulate an increased release of neutrophil elastases and mmps from activated macrophages, leading to the destruction of the elastin in the alveolar spaces [ ] . this inflammatory cell activation may come about through the activation of the transcription factor nfkb that leads to tnfa production [ ] . in addition, the elastin peptides themselves may attract additional inflammatory cells to further increase the protease secretion and exacerbate the matrix destruction [ ] . unlike centriacinar emphysema, panacinar emphysema is most commonly caused by a genetic deficiency of antiproteases, usually due to alpha- anti-trypsin (aat) deficiency, a condition that affects approximately , people in the united states [ ] . aat deficiency is due to a defect in the gene that encodes the protein aat, a glycoprotein produced by hepatocytes and the main inhibitor of neutrophil elastase. the affected gene is the serpina gene (formerly known as p ), located on the long arm of chromosome ( q - . ). the genetic mutations that occur have been categorized into four groups: base substitution, in-frame deletions, frame-shift mutations, and exon deletions. these mutations usually result in misfolding, polymerization, and retention of the aberrant protein within the hepatocytes, leading to decreased circulating levels. aat deficiency is an autosomal codominant disease with over allelic variants, of which the m alleles (m -m ) are the most common; these alleles produce normal serum levels of a lessactive protein [ ] . individuals who manifest the lung disease are usually homozygous for the alleles z or s (zz and ss phenotype) or heterozygous for the m alleles (mz, or sz phenotype) [ ] . an aat concentration in plasma of less than % of normal confers a risk for emphysema [ ] . in individuals with the zz genotype, the activity of aat is approximately one-fifth of normal [ ] . the second system in the lung involved in the pathogenesis of emphysema is the oxidant-antioxidant system. as in the protease system, the lung is protected from oxidative stress in the form of ros by antioxidants produced by cells in the lung. ros in the lung include oxygen ions, free radicals, and peroxides. the major antioxidants in the airways are enzymes including catalase, superoxide dismutase (sod), glutathione peroxidase, glutathione s-transferase, xanthine oxidase, and thioredoxin, as well as nonenzymatic antioxidants including glutathione, ascorbate, urate, and bilirubin [ ] . the balance of oxidants and antioxidants in the lung prevents damage by ros. however, cigarette smoke increases the production of ros by neutrophils, eosinophils, macrophages, and epithelial cells [ ] . evidence that damage to the lung epithelium and matrix is a direct result of ros includes the presence of exhaled h o and -isoprostane, decreased plasma antioxidants, and increased plasma and tissue levels of oxidized proteins, including various lipid peroxidation products. in addition to this direct effect, ros may also induce a proinflammatory response that recruits more inflammatory cells to the lung. in animal models, cigarette smoke induces the expression of proinflammatory cytokines such as il- , il- , tnfa, and il- from macrophages, epithelial cells, and fibroblasts, perhaps through activation of the transcription factor nfkb [ , ] (figure . ) . finally, there is some evidence that cigarette smoke further disturbs the oxidant-antioxidant balance in the lung by depleting antioxidants such as ascorbate and glutathione [ ] . bronchiectasis represents the permanent remodeling and dilatation of the large airways of the lung most commonly due to chronic inflammation and recurrent pneumonia. these infections usually occur because airway secretions and entrapped organisms cannot be effectively cleared. this pathology dictates the clinical features of the disease, which include chronic cough with copious secretions and a history of recurrent pneumonia. the five major causes of bronchiectasis are infection, obstruction, impaired mucociliary defenses, impaired systemic immune defenses, and congenital. these may produce either a localized or diffuse form of the disease. localized bronchiectasis is usually due to obstruction of airways by mass lesions or scars from previous injury or infection. diffuse bronchiectasis can result from defects in systemic immune defenses in which either innate or adaptive immunity may be impaired. diseases due to the former include chronic granulomatous disease (cgd), and diseases due to the latter include agammaglobulinemia/hypogammaglobulinemia and severe combined immune deficiencies. defects in the mucociliary defense mechanism that is responsible for physically clearing organisms from the lung may also cause diffuse bronchiectasis. these include ciliary dyskinesias that result in cilia with aberrant ultrastructure and cystic fibrosis (cf). congenital forms of bronchiectasis are rare but do exist. the most common include mounier-kuhn's syndrome and williams-campbell syndrome, the former causing enlargement of the trachea and major bronchi due to loss of bronchial cartilage, and the latter causing diffuse bronchiectasis of the major airways probably due to a genetic defect in the connective tissue [ , ] . the pathology of bronchiectasis is most dramatically seen at the gross level. one can see dilated airways containing copious amounts of infected secretions and mucous plugs localized either to a segment of the lung or diffusely involving the entire lung as in cystic fibrosis (figure . ) . microscopic features include chronic inflammatory changes similar to those of chronic bronchitis but with ulceration of the mucosa and submucosa leading to destruction of the smooth muscle, and elastic in the airway wall and the characteristic dilatation and fibrosis. these enlarged airways contain mucous plugs comprising mucin and abundant degenerating inflammatory cells, a result of infections that establish themselves in these airways following the loss of the mucociliary defense mechanism. bacteria may be found in these plugs, most notably p. aeruginosa. the pathogenetic mechanism of bronchiectasis is complex and depends on the underlying etiology. in general, the initial damage to the bronchial epithelium is due to aberrant mucin (cystic fibrosis), dysfunctional cilia (ciliary dyskinesias), and ineffective immune surveillance (defects in innate and antibody-mediated immunity), leading to a cycle of tissue injury, repair, and remodeling that ultimately destroys the normal airway. the initial event in this cycle usually involves dysfunction of the mucociliary mechanism that inhibits the expulsion from the lungs of organisms and other foreign substances that invade the airways. this may be due to defects in the cilia or the mucin. ciliary defects are found in primary ciliary dyskinesia, a genetically heterogeneous disorder, usually inherited as an autosomal recessive trait that produces immotile cilia with clinical manifestations in the lungs, sinuses, middle ear, male fertility, and organ lateralization [ ] . over proteins make up the axoneme of the cilia, but mutations in genes, dnai and dnah , which encode for proteins in the outer dynein arms, most frequently cause this disorder [ ] . in cf the main defect affects the mucin. in patients with this autosomal recessive condition, there is a low volume of airway surface liquid (asl) causing sticky mucin that inhibits normal ciliary motion and effective mucociliary clearance of organisms. this is due to a defect in the cystic fibrosis transmembrane conductance regulator (cftr) gene, located on chromosome that encodes a camp-activated channel which regulates the flow of chloride ions in and out of cells and intracellular vacuoles, helping to maintain the osmolality of the mucin. this protein is present predominantly on the apical membrane of the airway epithelial cells, though it is also involved in considerable subapical, intracellular trafficking and recycling during the course of its maturation within these cells. this genetic disease manifests in multiple other organs that depend on chloride ion transport to maintain normal secretions, including the pancreas, intestine, liver, reproductive organs, and sweat glands [ ] . the genetic mutations in cf influence the cftr trafficking in the distal compartments of the protein secretary pathway, and various genetic mutations produce different clinical phenotypes of the disease. over mutations of the cftr gene have been found. however, only four of these mutations occur at a frequency of greater than %. these mutations are grouped into five classes according to their functional deficit: group i, cftr is not synthesized; group ii, cftr is inadequately processed; group iii, cftr is not regulated; group iv, cftr shows abnormal conductance; group v, cftr has partially defective production or processing. approximately % of cf patients are in group ii and have the same mutation, f d cftr, a deletion of phenylalanine at codon [ ] . in these patients, most of the cftr protein is misfolded and undergoes premature degradation within the endoplasmic reticulum, though a small amount of the cftr protein is present on the apical membrane and does function normally. cf patients may have a combination of genetic mutations from any of the five groups. however, those patients with the most severe disease involving both the lungs and pancreas usually carry at least two mutations from group i, ii, or iii [ ] . systemic immune deficiencies cause bronchiectasis through the establishment of persistent infection and inflammation. there are four major categories of immune deficiencies. the first category consists of a number of genetic diseases that cause either agammaglobulinemia or hypogammaglobulinemia. these include xlinked agammaglobulinemia (xla) and common variable immunodeficiency (cvi). xla is caused by a mutation of the bruton's tyrosine kinase (btk) gene that results in the virtual absence of all immunoglobulin isotypes and of circulating b lymphocytes. in cvi there is a marked reduction in igg and iga and/or igm, associated with defective antibody response to protein and polysaccharide antigens. as expected, both of these diseases increase susceptibility to infections from encapsulated bacteria. the second category of immune deficiency is hyper-ige syndrome, a disease with markedly elevated serum ige levels that is characterized by recurrent staphylococcal infections. the third category is chronic granulomatous disease (cgd), a genetically heterogeneous group of disorders that have a defective phagocytic respiratory burst and superoxide production, inhibiting the ability to kill staphylococcus spp. and fungi such as aspergillus spp. finally, severe combined immune deficiency (scid) comprises a group of disorders with abnormal t-cell development and b-cell and/or natural killer cell maturation and function, predisposing these patients to pneumocystis jiroveci and viral infections [ ] . after the initial insult, the subsequent steps in the development of bronchiectasis include destruction of the epithelial cells and bronchial wall connective tissue matrix by the proteases and ros secreted by the neutrophils. this proinflammatory milieu is produced by multiple factors. first, infections can persist in these lungs due to defective host immune systems and mechanisms certain organisms have developed to evade these immune defenses. for example, pseudomonas aeruginosa, changes from a nonmucoid to a mucoid variant and also releases virulence factors to protect against phagocytosis [ ] . second, in the case of cystic fibrosis, neutrophils are directly recruited by proinflammatory cytokines, such as interleukin- (il- ), released from the bronchial epithelial cells as a result of the defective cgft protein [ ] . finally, the necrotic cellular debris and other breakdown products act as chemoattractants that recruit more inflammatory cells to the airway wall, further exacerbating the damage. the final phase of the repair and remodeling begins when macrophages invade and recruit fibroblasts that secrete collagen, leading to the fibrosis seen in the pathology. however, in the absence of effective airway clearance mechanisms, these ectatic airways remain a reservoir of infection that continues the cycle of inflammation and tissue destruction. the idiopathic interstitial pneumonias (iips) comprise a group of diffuse infiltrative pulmonary diseases with a similar clinical presentation characterized by dyspnea, restrictive physiology, and bilateral interstitial infiltrates on chest radiography [ ] . pathologically, these diseases have characteristic patterns of tissue injury with chronic inflammation and varying amounts of fibrosis. by recognizing these patterns, a pathologist can classify each of these entities and predict prognosis. however, the pathologist cannot establish the etiology, since these pathologic patterns can be seen in multiple clinical settings. the pathologic classification of these diseases, originally defined by liebow and carrington in [ ] , has undergone important revisions over the past years with the latest revision by the american thoracic society/european respiratory society in [ ] . the best known and most prevalent entity of the iips is idiopathic pulmonary fibrosis (ipf), which is known pathologically as usual interstitial pneumonia (uip). uip is a histologic pattern characterized by patchy areas of chronic lymphocytic inflammation with organizing and collagenous type fibrosis. these patients usually present with gradually increasing shortness of breath and a nonproductive cough after having had symptoms for many months or even years. imaging studies usually reveal bilateral, basilar disease with a reticular pattern [ ] . therapy begins with corticosteroids, advancing to more cytotoxic drugs such as methotrexate and cytoxan, but most current therapies are not effective in stopping the progression of the disease. the current estimates are that / , males and / , females have the disease, most of whom progress to respiratory failure and death within years [ ] . the pathology is characterized by a leading edge of chronic inflammation with fibroblastic foci that begin in different areas of the lung at different times. these processes produce a variegated pattern of fibrosis, usually referred to as a temporally heterogenous pattern of injury [ ] . because it occurs predominantly in the periphery of the lung involving the subpleura and interlobular septae, the gross picture is one of more advanced peripheral and basilar disease (figure . ) . the progression from inflammation to fibrosis includes interstitial widening, epithelial injury and sloughing, fibroblastic infiltration, and organizing fibrosis within the characteristic fibroblastic foci. deposition of collagen by fibroblasts occurs in the latter stages of repair. the presence of the abundant collagen produces stiff lungs that are unable to clear the airway secretions, leading to recurrent inflammation of the bronchiolar epithelium with eventual fibrosis and breakdown of the airway structure. this remodeling produces mucousfilled ectatic spaces giving rise to the gross picture of honeycomb spaces, which is seen in the advanced pathology ( figure . ) [ ] . theories of the pathogenesis of ipf have evolved over the past decade. early theories favored a primary inflammatory process, while current theories favor the concept that the fibrosis of the lung proceeds independently of inflammatory events and develops from aberrant epithelial and epithelial-mesenchymal responses to injury to the alveolar epithelial cells (aecs) [ ] . the aecs consist of two populations: the type pneumocytes and the type pneumocytes. in normal lungs, type pneumocytes line % of the alveolar wall, and type pneumocytes line the remaining %. however, in lung injury, the type cells, which are exquisitely fragile, undergo cell death, and the type pneumocytes serve as progenitor cells to regenerate the alveolar epithelium [ ] . though some studies have suggested that repopulation of the type cells depends on circulating stem cells, this concept remains to be fully proven. according to current concepts, the injury and/or apoptosis of the aecs initiates a cascade of cellular events that produce the scarring in these lungs. studies of aecs in lungs from patients with ipf have shown ultrastructural evidence of cell injury and apoptosis as well as expression of proapoptotic proteins. further, inhibition of this apoptosis by blocking a variety of proapoptotic mechanisms such the fas-fas ligand pathway, angiotensin, and tnfa production, and caspase activation can stop the progression of this fibrosis [ ] . the result of the aec injury is the migration, proliferation, and activation of the fibroblasts and myofibroblasts that leads to the formation of the characteristic fibroblastic foci of the uip pathology and the deposition and accumulation of collagen and elastic fibers in the alveoli (figure . ). this unique pathology may be a result of the increased production of profibrotic factors such as transforming growth factor-a (tgfa) and tgfb, fibroblastic growth factor- , insulin-like growth factor- , and platelet-derived growth factor. an alternative pathway might involve overproduction of inhibitors of matrix degradation such as timps (tissue inhibitors of matrix production) [ ] . in support of the former mechanism, fibroblasts isolated from the lungs of ipf patients exhibit a profibrotic secretory phenotype [ ] . multiple factors, such as environmental particulates, drug or chemical exposures, and viruses may trigger the initial epithelial injury, but genetic factors also play a role. approximately %- % of patients with ipf have a family history of the disease with an inheritance pattern of autosomal dominance with variable penetrance. two genetic mutations have been implicated in this familial form of ipf. one large kindred has been reported with a mutation in the gene encoding surfactant protein c, and six probands have been a b reported with heterozygous mutations in genes htert or htr, encoding telomerase reverse transcriptase and telomerase rna, respectively, resulting in mutant telomerase and short telomeres [ ] . adult respiratory distress syndrome (ards) represents a constellation of clinical, radiologic, and physiologic features in patients with acute respiratory failure that can occur after a variety of insults. ards is defined by clinical criteria that include a rapid onset of severe hypoxemia that is refractory to oxygen therapy, the presence of abnormal chest radiographs with evidence of bilateral alveolar filling and collapse, increased pulmonary artery occlusion pressure, and a resistance to improved oxygenation regardless of mechanical ventilation therapy [ ] . treatment of ards includes eliminating the underlying cause, protective ventilation strategies that improve oxygenation, and supportive treatment that may include administration of corticosteroids. the pathology of ards is diffuse alveolar damage (dad), whose histologic picture is one of inflammation and fibrosis that diffusely involves all of the structures of the alveolus and is similar throughout the affected areas of the lung [ ] . dad is divided into three major phases that follow each other chronologically after the original insult. these are exudative, proliferative, and fibrotic dad. the initial injury primarily involves the epithelium of the alveolar wall and the endothelium in the capillary, causing the destruction and sloughing of the type pneumocytes into the alveolar space and a breakdown of the tight junctions of the endothelium. in combination, these two events result in the loss of the epithelial-endothelial barrier of the alveolus and leakage of plasma from the capillary into the alveolar space. this flooding of the airspace with fluid markedly decreases oxygen exchange and causes the hypoxia that these patients experience. in addition, acute inflammatory changes of the endothelium also cause thrombi to form in vessels, adding to a decreased amount of blood circulating through the lung and further compromising gas exchange. as air is brought into the alveoli, the positive pressure within the alveolar space forces the plasma against the alveolar wall, producing a membranous morphology referred to as hyalin membranes characteristic of the first phase of dad, referred to as exudative dad (figure . ) . this initial injury is followed by a sequence of events that represent the lung's efforts to repair itself. first, type pneumocytes undergo hyperplasia and re-epithelialize the alveolar wall after the loss of the type cells. this re-establishes the epithelial barrier and, because these cells secrete surfactant, results in increased surfactant production, which lowers the surface tension of the alveolus and inhibits its collapse. because of the increased numbers of type pneumocytes, this is known as the proliferative phase of dad (figure . ) . in the final phase of dad, fibrotic dad, fibroblasts migrate in from the adjacent interstitium to the alveolar space and produce organizing and irreversible fibrosis within both the alveolar space and the interstitium. in addition to this mechanism, fibrosis may also occur in those areas where alveolar walls collapse when surfactant is decreased during the initial insult. the histopathologic picture during this fibrotic phase is one of thickened alveolar septa, intra-alveolar granulation tissue, microcyst formation, and areas of irregular alveolar scarring. in rare cases, these microcysts progress to large cysts, an adult equivalent of bronchopulmonary dysplasia. the cellular events of dad are complex and incompletely understood. in general, the disease can be broken down into two phases. in the first, a large influx of neutrophils and plasma enter the alveolar space. the role the neutrophils play in the initial cellular injury and death is unclear, but it is known that they are necessary for this injury to occur. in addition, clinical studies have shown that within the peripheral blood and bronchoalveolar lavages (bal) of these patients, neutrophils are present along with a myriad of proinflammatory cytokines, such as il- , il- , and tgfa, all of which are capable of recruiting them to the lung. also present in these fluids are mediators that recruit fibroblasts such as tgfb. all of these mediators are probably the result of upregulation of nfkb, a proinflammatory transcription factor, in alveolar macrophages. the adherence of neutrophils to the capillary endothelium in the lung occurs through adhesion molecules such as selectin, integrin, and immunoglobulins. neutrophil adherence and subsequent transmigration through the endothelium of the lung capillaries may cause some endothelial damage. however, most speculate that ros and reactive nitrogen chapter molecular basis of pulmonary disease species (rns) secreted by the neutrophils modulate the majority of this injury [ ] . this is supported by the finding that patients with ards have products of oxidative damage such as hydrogen peroxide (h o ) in the exhaled breath and myeloperoxidase and oxidized aat in the bal. the cell injury and death of the type pneumocytes most likely occurs via two mechanisms: lipopolysaccharide (lps)-induced caspase-dependent apoptosis and hyperoxia-induced cell death through apoptosis and nonapoptotic mechanisms [ ] . in the former, lps, an immunogenic component of the outer membrane of gram-negative bacteria, may trigger innate immune and inflammatory responses via toll-like receptors that bind fas-associated death domain protein and caspase- , leading to epithelial cell death. in hyperoxia-induced cell death, hyperoxia may induce the expression of angiopoietin (ang ) in lung epithelial cells. ang is an angiogenic growth factor that can activate caspase pathways and lead to apoptotic cell death [ ] . cell death in ards is not limited to these mechanisms, and further study of many of pathways by which this can occur is needed. lymphangioleiomyomatosis (lam) is a rare systemic disease of women, usually in their reproductive years (average age of years), that is characterized by a proliferation of abnormal smooth muscle cells giving rise to cysts in the lungs, abnormalities in the lymphatics, and abdominal tumors, most notably in the kidneys. in addition to sporadic cases (denoted as s-lam), lam also affects % of women with tuberous sclerosis (denoted as tsc-lam), a genetic disorder with variable penetrance associated with seizures, brain tumors, and cognitive impairment [ , ] . global estimates indicate that tsc-lam may be as much as -fold to -fold more prevalent than s-lam, though at least some suggest that tsc-lam may have a milder clinical course than s-lam [ ] . clinically, lam patients usually present with increasing shortness of breath on exertion, obstructive symptoms, spontaneous pneumothoraces, and chylous effusions or with abdominal masses consisting of either angiomyolipomas and/or lymphangiomyomas. chest imaging studies characteristically reveal hyperinflation with flattened diaphragms and thin-walled cystic changes. mortality at years from the onset of symptoms is %- % [ ] . lam appears as small, thin-walled cysts ( . - . cm) randomly throughout both lungs [ ] (figure . ) . microscopically, lam lungs contain a diffuse infiltration of smooth muscle cells, predominantly around lymphatics, veins, and venules. most notably, one finds smooth muscle cells in the subpleural with hemosiderin-laden macrophages in the adjacent field, and the macrophages are also seen on bronchoalveolar lavage specimens from these patients. the hemosiderin pigment in these lungs is thought to be secondary to microhemorrhages from the obstruction of the veins ( figure . ) [ ] . the smooth muscle cells in lam react to antibodies to hmb- , a premelanosomal protein. other melanosome-like structures are also found in lam cells, suggesting that these cells have characteristics of both smooth muscle and melanosomes [ ] . the lesional cells in lam are smooth muscle-like with both spindled and epithelioid morphology [ ] . these cells are the same in both s-lam and tsc-lam part iv molecular pathology of human disease and are a clonal population although they lack other features of malignancy [ ] . molecular studies reveal that the abnormal lam cell proliferation is caused by mutations in one of two genes linked to tuberous sclerosis: tuberous sclerosis complex or (tsc or tsc ). these two genes control cell growth and differentiation through the akt/mammalian target of rapamycin (mtor) signaling pathway [ ] . in this pathway, a growth factor receptor (such as insulin or pdgf receptors) becomes phosphorylated when an appropriate ligand binds, resulting in activation of downstream effectors and ultimately akt. the gene products of tsc and tsc are hamartin and tuberin, which act as dimers to maintain rheb (a member of the ras family) in a gdp-loaded state via statins, acting as a break to the akt/mtor pathway, thereby retarding protein synthesis and cell growth. in lam cells, loss-of-function mutations in these two genes remove this inhibition, leading to enhanced rheb activation, mtor activation (with raptor), and subsequent phosphorylation of downstream molecules which result in uncontrolled cell growth, angiogenesis, and damage to the lung tissue ( figure . ) [ ] . the abnormal proliferation of lam cells is thought to damage the lung through overproduction of matrix metalloproteinases (mmps), which degrade the connective tissue of the lung architecture, destroy the alveolar integrity, and result in cyst formation with air trapping [ ] . these destructive capabilities of the lam cells are enhanced by their secretion of the angiogenic factor vegf-c, which is thought to cause the proliferation of lymphatic channels throughout the lung [ ] . sarcoidosis is a multisystemic disease that involves the lung in over % of the cases [ ] . it is most common in the - -year age group and among females. in the united states, african americans are more commonly affected than caucasians [ ] . the clinical picture of sarcoidosis is variable, but most patients present with systemic symptoms including fatigue, weight loss, and fever. the most common finding on chest imaging studies is bilateral hilar lymph node enlargement and reticular, reticulonodular, and focal alveolar opacities within the lung parenchyma [ ] . pulmonary sarcoidosis is characterized by granulomas which consist of activated histiocytes, called epithelioid histiocytes that form nodules ranging in size from - microns (figure . ) [ ] . unlike infectious granulomas that usually contain areas of central necrosis, the granulomas in pulmonary sarcoidosis are predominantly non-necrotizing [ ] . also, the granulomas in sarcoidosis follow a distribution along the lymphatics, which includes the area in the subpleural, along the interlobular septae and around the bronchovascular area containing the bronchiole and branch of the pulmonary artery (figure . ). the granulomas occur much more commonly in the upper lobes, leading to the predominant upper lobe fibrosis and bronchiectasis that can be seen in longstanding sarcoidosis [ ] . despite over years of research on sarcoidosis, the etiology remains unknown. most agree that the disease is probably a result of environmental triggers acting on a genetically susceptible host [ , ] . a genetic basis of sarcoidosis has been suggested by studies that demonstrate familial clustering and racial variation [ , ] . further, complex inheritance patterns for the disease suggest that more than one gene may be involved [ ] . several genes of the major histocompatibility complex (mhc) region of the genome have been implicated. most are clustered on the short arm of chromosome that encompasses the human leukocyte antigen (hla) domain. the hla class i mhc molecules associated with sarcoidosis are the hla-b and hla-b class i alleles [ , ] . hla class ii molecules implicated in susceptibility include the hla-dr alleles [ , ] . genes other than mhc genes thought to regulate the susceptibility to sarcoidosis include those for chemokines such as macrophage inflammatory protein- a and rantes (ccr and ccr ) [ , ] . environmental factors that have been implicated are those that are aerosolized. therefore, these environmental agents have a mode of entry into the lungs and can cause granulomas in the lung, similar to sarcoidosis. these factors can be divided into two major categories, which include infectious and noninfectious agents. the mycobacteria have been the most extensively studied organisms. however, their role in this disease remains controversial due to the difficulty in identifying them by either culture or histochemical stains in sarcoid tissue. recently, molecular techniques have been able to demonstrate mycobacterial nucleic acid in sarcoid tissue [ , ] . however, even studies using this technology have not produced consistent results, and the role of these organisms in the disease requires further study. the immune response in sarcoidosis has two major features: (i) the initial event leading to granuloma formation and (ii) the progression of this granulomatous response to either resolution or fibrosis [ ] . the formation of the granulomas, triggered by activation of tcells and antigen-presenting dendritic histiocytes, results in a release of proinflammatory cytokines and chemokines, and recruitment, activation, and proliferation of mononuclear cells, predominantly t-cells. these activated t-cells are predominantly cd -expressing t-helper (th) cells, which release ifn-g and il- . alveolar macrophages at the site release tnfa, il- , il- , and other growth factors. this results in the granuloma formation and alveolitis, the characteristic morphologic features of the disease [ ] . the second phase of this immunologic response that leads to either resolution of the disease or persistence of the granulomas and fibrosis is less well characterized. ongoing granuloma formation and inflammation may be a result of the persistent presence of antigens, the excessive synthesis of chemotactic factors, or the part iv molecular pathology of human disease persistence of the mononuclear cells within the granulomas. importantly, the role of the t-cells in these granulomas is to secrete cytokines that attract, stimulate, and ultimately deactivate the fibroblasts that are responsible for the fibrosis that is seen in the chronic disease. the balance between the profibrotic mediators such as tgfb, insulin-like growth factor-i, platelet-derived growth factor (pdgf), and the antifibrotic mediators, such as ifn-g, probably dictates the natural history of sarcoidosis in the lung [ ] . genes involved in macrophage-derived cytokines, chemokines, and mediators of fibrosis are all possible candidates for the underlying genetic cause of this complicated disease. pulmonary alveolar proteinosis (pap) is a rare disease of the lungs characterized by accumulation of surfactant in the alveolar spaces. the names alveolar proteinosis, lipoproteinosis, or perhaps most accurately phospholipoproteinosis, apply equally to this entity. pap takes three forms clinically: (i) congenital ( %), (ii) secondary ( %- %), and (iii) idiopathic or primary ( %- %) [ ] [ ] [ ] . pap arises in previously healthy adults with the median age at diagnosis of approximately years and a male-to-female ratio of . : . the clinical presentation is variable and usually includes an insidious onset of slowly progressive dyspnea, a dry cough, and other symptoms of respiratory distress, including fatigue and clubbing. however, almost one-third of patients are asymptomatic and are found clinically by abnormal chest x-rays [ , ] . the secondary form of pap can be found in patients with environmental exposures, including fine silica, aluminum, titanium dioxide, and kaolin dust [ ] . also, secondary pap may be found in patients with malignancies, most commonly hematologic malignancies such as myelogenous leukemia [ , ] . chest imaging studies in both the idiopathic and secondary forms most commonly show fine, diffuse, feathery nodular infiltrates, centered in the hilar areas, sparing the peripheral regions [ ] . on chest computerized tomographs, the infiltrates may have a geometric-type shape, sometimes referred to as crazy paving [ ] . the most prominent microscopic feature of both idiopathic and secondary pap is the filling of the alveoli with finely granular period acid-schiff-positive diastaseresistant (pasd) acellular material (figure chapter molecular basis of pulmonary disease material consists of phospholipids ( %); surfactant proteins a, b, c, and d ( %); and carbohydrate (< %) [ ] . alveolar macrophages (ams) with prominent foamy cytoplasm are commonly seen, while alveolar septa are remarkably normal in appearance. in some alveolar spaces there are denser, more solid clumps of pas-d-positive material. definitive pathologic differences between the idiopathic and secondary forms of pap have not been well documented [ , ] . the etiologies of the two adult forms of pap have been well studied with the most known about the idiopathic variant. theories of the pathogenesis of this form have focused on the abnormal accumulation of the surfactant-like material within the alveolar spaces. since the regulation of surfactant levels in the alveoli depends on appropriate synthesis, recycling, and catabolism, the two opposing hypotheses have included overproduction versus decreased degradation of this material. in normal hosts, surfactant is essential to maintaining the low surface tension needed for proper alveolar inflation and gas exchange. the critical role of maintaining the proper composition and amount of surfactant in the alveoli is performed by two cell types: type pneumocytes and alveolar macrophages [ ] . the type pneumocytes synthesize surfactant in the endoplasmic reticulum and golgi, and store it as lamellar bodies [ ] , which are then delivered to and fuse with the apical plasma membrane, secreting the surfactant into the airways [ ] . catabolism of surfactant is carried out by type pneumocytes and ams. in pap, most evidence suggests that the clearance of surfactant by the am is decreased [ , ] . the first clue as to the underlying mechanism for this defect in am function came in when studies revealed that knockout mice deficient in granulocytemacrophage colony-stimulating factor (gm-csf) develop lung lesions similar to those in patients with pap [ ] . this rather serendipitous finding prompted explorations centered on the am and the effect diminished gm-csf might have on its cellular functions. subsequent studies from humans with pap revealed an autoimmune mechanism by which a circulating neutralizing antibody to gm-csf blocked its binding to the gm-csf receptor, depressing the effect of gm-csf on the ams [ ] [ ] [ ] . neutralizing antibodies to gm-csf have most often been identified in the idiopathic variant of pap. however, recently these antibodies have also been reported in patients with secondary pap [ ] . genes that control many functions in the am are controlled by signaling pathways initiated by gm-csf binding to the am. one pathway is mediated through a transcription factor pu. that controls genes involved in surfactant degradation, among other bactericidal functions [ , ] . another transcription factor, peroxisome-proliferator-activated receptor g (pparg), is also part of a pathway activated by gm-csf. pparg controls the expression of genes involved in intracellular lipid metabolism. ams from patients with pap have a deficiency of this transcription factor, which is correctable by gm-csf therapy [ ] . overall, the lack of gm-csf-initiated signaling in ams from patients with pap leads to inhibition of both pparg and pu. pathways. this results in decreased surfactant catabolism, intracellular lipid metabolism, and the accumulation of surfactant in the alveoli (figure . ). pulmonary hypertension consists of a group of distinct diseases whose pathology is characterized by abnormal destruction, repair, remodeling, and proliferation of all compartments of the pulmonary vascular tree, including arteries, arterioles, capillaries, and veins. the classification of these diseases has undergone a number of revisions. the most recent revision (in ) groups these diseases based on both their pathologic and clinical characteristics [ ] . there are five major disease categories in the current classification system: (i) pulmonary arterial hypertension (pah); (ii) pulmonary hypertension with left heart disease; (iii) pulmonary hypertension associated with lung disease and/or hypoxemia; (iv) pulmonary hypertension due to chronic thrombotic and/or embolic disease; and (v) miscellaneous causes, including sarcoidosis, histiocytosis x, and lymphangioleiomyomatosis. the clinical course of most patients with pulmonary hypertension begins with exertional dyspnea, and progresses through chest pain, syncope, increased mean pulmonary artery pressures and, eventually, right heart failure. the rate of this clinical progression varies among patients, from a few months to many years [ ] . treatment of these diseases focuses on blocking the mediators involved in the pathogenesis of the diseases. however, current therapies rarely prevent progression of the disease, and lung transplantation provides the only hope for long-term survival. the major group of this classification, pah, can be subdivided into familial pah, idiopathic pah, pah associated with other conditions (such as connective tissue diseases, hiv, congenital heart disease), and pah secondary to drugs and toxins (such as anorexigens, cocaine, and amphetamines). in these diseases, the primary pathology is localized predominantly in the small pulmonary arteries and arterioles. however, two other diseases in this group, pulmonary veno-occlusive disease and pulmonary capillary hemangiomatosis, involve predominantly other components of the pulmonary vasculature, the veins, and the capillaries, respectively. the pathologic changes seen in the pulmonary vessels of these patients primarily reflect injury to and repair of the endothelium. early pathologic changes include medial hypertrophy and intimal fibrosis that narrows and obliterates the vessel lumen. these are followed by remodeling and revascularization, producing a proliferation of abnormal endothelial-lined spaces. these structures are known as plexogenic lesions and are the pathognomonic feature of pah (figure . ) . in the most severe pathologic lesions, these abnormal vascular structures become dilated or angiomatoid-like and may develop features of a necrotizing vasculitis with transmural inflammation and fibrinoid necrosis. though the exact pathogenetic mechanism of pah remains unknown, research over the past years has begun to offer some clues. the familial form of pah, with a : female-to-male prevalence, has an autosomal dominance inheritance pattern with low penetrance. the genetic basis for this has been found to be germline mutations in the gene encoding the bone morphogenetic protein receptor type (bmpr ). these mutations account for approximately %- % of familial pah and %- % of patients with sporadic pah [ ] . approximately bmpr mutations have been identified in familial pah, each resulting in a loss of receptor function, either through alteration in transcription of the gene through missense, nonsense, or frameshift alterations in the codon or by rna spicing mistakes [ ] . the mechanism by which a single mutation to the bmpr gene induces vascular smooth muscle proliferation and decreased apoptosis that is not completely understood, but it most likely involves defects in the bmpr signaling pathway. bmpr is a receptor for a family cytokines (bmps) that are members of the tgfb superfamily of proteins that play a role in the growth and regulation of many cells, including those of the pulmonary vasculature. in the vascular smooth muscle cells of the lung, tgfb signaling causes a proliferation of smooth muscle in pulmonary arterioles, while bmpr signaling causes an inhibition of the proliferation of these cells, favoring an apoptotic environment. the bmpr signaling occurs through an activation of a receptor complex (bmpr and bmpr ) that leads to phosphorylation and activation of a number cytoplasmic mediators, most notably the smad proteins (mothers against decapentaplegic). these smad proteins, especially the smad , smad , and smad complex with smad , translocate to the nucleus where they target gene transcription that induces an antiproliferative effect in the cell. in familial pah, the bmrpr gene mutation may lead to insufficient protein product and subsequent decreased protein function, in this case decreased bmpr receptor function, decreased smad protein activation, and decreased antiproliferative effects in the vascular smooth muscle cells. the imbalance between the proproliferative effects of the tgfbs and the antiproliferative effects of the bmps results in the formation of the vascular lesions of pah (figure . ) [ , ] . despite these advances, questions regarding the pathogenesis of pah remain. most notably, why do only %- % of patients with the mutation develop clinical disease? some speculate that genes confer susceptibility but a second hit is required to develop the clinical disease, such as modifier genes or environmental triggers, perhaps drugs or viral infections [ , ] pulmonary vasculitides present as diffuse pulmonary hemorrhage and are usually caused by one of three major pulmonary vasculitis syndromes: wegener's granulomatosis, churg-strauss syndrome, and microscopic polyangiitis. all three diseases have similar clinical presentations and considerable overlap in their pathologic features as small vessel systemic vasculitides that affect the lung as well as other organs, most notably the kidney. wegener's granulomatosis (wg) is an unusual disease that affects the upper and lower respiratory tract and the kidneys. it usually presents between and years of age and is slightly more common in men than women. the clinical presentation depends on the affected organ, but when the lung is involved, hemoptysis is the major presenting symptom. chest imaging studies may show a variety of patterns, most commonly bilateral ground glass opacities with masses, usually in the lower lobes that may cavitate. immunologic testing of peripheral blood or end organ tissue can be helpful in revealing characteristic immunofluorescent staining patterns for antineutrophilic cytoplasmic antibody (anca), an antibody that targets two substances: proteinase (pr ) and myeloperoxidase (mpo). when present in either the blood or the tissue, the pattern of immunofluorescent staining can be cytoplasmic (canca) or perinuclear (panca). the former pattern is more commonly seen in wegener's granulomatosis, and the latter is more commonly seen in microscopic polyangiitis and churg-strauss syndrome (css). css is a systemic disorder defined by the presence of asthma, peripheral blood eosinophilia, and systemic vasculitis. similar to wg, it usually presents between and years of age, and a clinical diagnosis requires a history of asthma, a peripheral blood eosinophilia, neuropathy, an abnormal chest imaging study, and sinusitis. other organs involved include the heart, the central nervous system, kidneys (though less commonly than wg), gastrointestinal tract, and skin. chest imaging usually shows patchy, multifocal infiltrates; masses and cavitation are rare. laboratory tests reveal positive panca tests in % of patients. microscopic polyangiitis (mpa) is similar to both wg and css in that it is a systemic vasculitis that involves the lung and usually presents in the fourth or fifth decade of life. the clinical onset is usually sudden with fever, weight loss, myalgias, and arthralgias. the kidney is the main organ involved, and mpa is the most common cause of pulmonary-renal syndrome. lung involvement occurs in approximately % of the patients, and skin and upper respiratory tract are other common sites. similar to wg and css, anca testing is helpful with positive panca in % of patients. chest imaging usually shows bilateral infiltrates without masses, similar to css. treatment for all three diseases is immunosuppression with glucocorticoids or cyclophosphamide, and all three usually respond well, although wg has a greater relapse rate after treatment than either css or mpa [ ] . the pathology of wg, css, and mpa have overlapping features of an acute and chronic vasculitis that involves medium-and small-sized vessels in the lung. the inflammatory cell infiltrate that destroys the blood vessels is both lymphocytic and neutrophilic, and areas of fibrinoid necrosis are seen. however, in wg, there are characteristic areas of microabscesses that lead to masses of geographic necrosis with basophilia. scattered multinucleated giant cells are present, but no wellformed granulomas are seen. this helps to distinguish it from other vasculitides and infection (figure . ) . similarly, the pathology of css has distinguishing features, with the early pathology characterized by an eosinophilic pneumonia with areas of loosely formed granulomas with central necrosis containing degenerating eosinophils (figure . ). the infiltrate is predominantly eosinophils, but neutrophils, lymphocytes, and plasma cells are also present. capillaritis can be seen in wg, csg, and mpa, and all three have hemosiderin deposition present, both within alveolar macrophages and deposited in the connective tissue of the interstitium and the vessel walls. the pathogenesis of these three pulmonary hemorrhage syndromes is similar to the mechanisms of these diseases in the kidney. in general, these diseases in the lung and the kidney represent immune-mediated these lesions are thought to be the early form of the larger areas of geographic necrosis that produces the mass-like nodules found in these lungs. chapter molecular basis of pulmonary disease necrotizing vasculitides that have few or no immune deposits in the vessels but exhibit the presence of anca autoantibodies to myeloperoxidase (mpo) and proteinase (pr ), the components of primary granules of neutrophils. mpa and css are primarily diseases of mpo antibodies, and wg is primarily a disease of pr antibodies. the mechanism by which the ancas are induced is not known but may be part of an autoimmune response to environmental exposures early in life. these autoantibodies then inflict damage on the vessels through a mechanism that is not yet completely understood. one theory suggests that circulating ancas bind to pr and mpo on the surface of neutrophils and initiate a respiratory burst, degranulation, and apoptosis. ros and proteases are released and inflict endothelial and tissue damage on the adjacent vessel. the anca binding may also induce the release of proinflammatory cytokines and chemokines such as il- and tnfa that further contribute to the vascular inflammation. the second theory postulates that circulating immune complexes of excess anca antigen (mpo or pr ) and anca autoantibodies attach to the vascular endothelium and activate complement that results in the chemotaxis and adhesion of inflammatory cells, causing these cells to undergo a respiratory burst and, as in the first theory, release of ros and proteases that cause the vascular endothelial damage. in both theories, it is important to remember that mpo and pr are also present in monocytes and that anca autoantibodies may be involved with monocytes in similar ways to release inflammatory mediators [ ] . infectious diseases of the lung are a common cause of pulmonary disease given the constant exposure of the lungs to the environment. various organisms are capable of causing these infections, including common viruses and bacteria, as well as more uncommon fungi, parasites, and protozoa. the diagnosis of the specific etiologic agent can be challenging given that most have similar clinical features and many are difficult to identify in the lung tissue. this brief overview of the defense mechanisms the lung uses to protect itself will serve to introduce the pathology of these lung infections. the lung has multiple anatomic mechanisms by which it defends itself against invasion by various pathogens. first, the upper nasal cavities and respiratory tract serve as anatomic barriers to inhaled organisms. the ciliated epithelium and torturous cavities of the sinuses screen large organisms (typically larger than microns). for those particles that venture further down the respiratory tract, the cough reflex that the upper trachea elicits serves to expel them up and out. second, the mucociliary tree of the upper respiratory tract captures organisms that evade these two mechanisms. the bronchial epithelium contains cilia of up to microns in length that extend into the air surface liquid (asl). the asl is a bilayer of - microns in thickness consisting of a low-viscosity or watery lower layer that is covered by a high-viscosity or gel upper layer secreted by adjacent goblet cells. this sticky upper layer serves to trap organisms, and the coordinated beating of the cilia moves these entrapped invaders up this mucociliary escalator to the larynx, where they can be expectorated. present in the secretions of the large airways and within the surfactant lining the alveolar walls are soluble mediators secreted by various cells. these mediators include lysozyme and lactoferrin, which lyse bacteria and inhibit their growth; the defensins and cathelicidins, small peptides both with microbicidal properties; and surfactant proteins a and d at the alveolar level, which bind to microorganism and enhance phagocytosis and also have direct bactericidal activity [ ] . the major cells of the innate immune response of the lung are the alveolar macrophages (am) and the polymorphonuclear leukocytes (pmn). neutrophils phagocytize and destroy bacteria such as s. aureus, s. pneumoniae, and h. influenzae through a respiratory burst that generates nadph oxidase-dependent ros. in some instances, ams may ingest but not kill an organism. this occurs with such organisms as mycobacterium spp., nocardia spp., and legionella spp. because of the ability of these organisms to continue to replicate within part iv molecular pathology of human disease the am, cell-mediated immunity is required for their complete elimination. patients with defects in nadph oxidase are especially prone to respiratory infections by such organisms as s. aureus, nocardia spp. and aspergillus spp. bronchial epithelial cells are important in innate immunity through secretion of cytokines and molecules including il- , il- , il- , il- , and granulocytemacrophage colony-stimulating factor (gm-csf). these molecules attract macrophages as well as neutrophils and other inflammatory cells to the area to enhance the inflammatory response to the organism [ ] . bronchial epithelial cells also serve an important role in recognizing pathogens through patternrecognition receptors (prrs). natural killer (nk) cells are involved in the innate immune response with surface receptors that recognize cells infected with viruses such as rsv, influenza, parainfluenza, and rhinovirus. the nk cells release ifn-g, which recruit other immune cells to add to the antiviral response. dendritic cells are tissue histiocytes positioned around the airways and lymphatics in the lung that recognize pathogens and their antigens and trigger the proliferation and amplification of antigen-specific tcells. this immune response bridges the innate immune response to the adaptive immune responses and is especially important in fungal infections. this mechanism is mediated through toll-like receptors (tlrs) that are able to distinguish pathogens from self-components by triggering cytokine production through nfkb and ap- and expressing co-stimulatory molecules necessary for this t-cell activation [ ] . for those organisms that evade the basic, innate immunity of the lung, there are adaptive immune mechanisms that encompass both humoral and cellular immune mechanisms. humoral immunity is an important defense against encapsulated bacteria, most notably s. pneumoniae, and for other pyogenic bacteria such as h. influenzae, and staphylococci spp., and resolution of these infections requires the production of igg antibodies to the organisms. cellular immunity is especially important against such respiratory viral infections as influenza, rsv, cmv, varicella, and also against opportunistic infections. these viruses induce a cd þ and cd þ t-cell response that clears the lung of these viruses within - days post infection. granulomas are a common inflammatory response to both pathogens and foreign material. the most notable granulomatous infections in the lung are due to mycobacteria and fungal organisms. activation of cd þ t-cells by these organisms leads to proliferation and differentiation of these cd þ t-cells into t-helper- cells. the release of ifn-g by the th- cells activates lung macrophages to form epithelioid macrophages that have an increased ability to kill the microorganisms and express surface molecules that promote cell-to-cell fusion into giant cells. in addition, activation of these macrophages results in the release of numerous cytokines including ifn-g and tnfa. in patients who are deficient in cd þ t-cells or ifn-g, granuloma formation is very poor, altering the pathologic picture of these infections. this effect is most obvious in the nontuberculous mycobacterial infections, which have numerous patterns of injury depending on the immune status of their host. pneumonias can be broadly categorized into one of five major clinicopathologic categories, including (i) community-acquired pneumonias (acute and atypical), (ii) nosocomial pneumonias, (iii) aspiration pneumonias and lung abscess, (iv) chronic pneumonias, and (v) pneumonias in immunocompromised hosts. each type presents with a characteristic clinical pattern and may be caused by any of several pathogens so that treatment is many times empiric. the first category comprises community-acquired pneumonias (cap). these represent the majority of the lung infections that receive medical treatment, usually on an outpatient basis, with low (< %) mortality. patients hospitalized for these infections typically have other comorbidities. the responsible organisms include respiratory syncytial virus (rsv); rhinovirus, parainfluenza, and influenza virus; bacteria, including mycoplasma pneumoniae and rickettsia; and most notably chlamydia pneumonia. chlamydia causes what is termed atypical pneumonia with a clinical course characterized by a progressive onset of fever without chills, a dry cough, and chest imaging that reveals focal infiltrates. acute or typical cap presents abruptly with high fever, chills, productive cough, and radiographs with lobar or segmental consolidation. the most common pathogens are streptococcus pneumoniae, haemophilus influenza, staphylococcus aureus, and moraxella catarrhalis. the second category, nosocomial pneumonias, consists of infections acquired within the hospital or from healthcare associated facilities. these infections are usually found in patients with predisposing risk factors and are a major source of morbidity and mortality, with some studies reporting a mortality range of %- %. the most common risk factors include respiratory ventilation, artificial airways, nasogastric tubes, supine positioning, and medications that alter gastric emptying. the responsible organisms include klebsiella spp., legionella spp., staphylococcus aureus, and pseudomonas aeruginosa. the third category includes aspiration pneumonias and lung abscesses. these infections occur in the setting of patients with aberrant swallow or gag reflexes that allow gastric or oral contents into the airways. the organisms where necrosis and cavity formation occurs include s. aureus, k. pneumoniae, the anaerobic oral flora, and mycobacteria. clinically, these infections may have an acute course with fever and dyspnea or a more insidious course, many times with patients first presenting with lung cavities, empyemas, or necrotizing pneumonias. the fourth category, chronic pneumonias, includes indolent infections that cause a localized mass-like lesion in an otherwise healthy host. nocardia and actinomyces spp. are the most common pathogens, but mycobacteria and fungi may also cause these pneumonias. the fifth category includes pneumonias that occur in the setting of an immunocompromised patient. these include a number of organisms that otherwise would not act as pathogens such as the viruses cmv and hsv, the fungi aspergillosis and pneumocystis pneumonia, and the bacterium mycobacterium avium complex. streptococcus pneumoniae streptococcus pneumoniae, a gram-positive diplococcus also known as pneumococcus or diplococcus pneumonia, is a common cause of bacterial pneumonia in infants and elderly patients, alcoholics, diabetics, and patients with immunosuppression. this pneumonia usually presents abruptly with chills, a cough with rust-colored sputum and pleuritis, with high fevers, tachycardia, and tachypnea. the characteristic gross pathology is a lobar pneumonia that progresses from a red acute phase to a gray organizing phase. a fibrinous pleuritis is common, which eventually organizes to entrap the lung parenchyma in a fibrous capsule [ ] . the microscopic examination reveals abundant fibrin, neutrophils, and extravasated red blood cells within the alveolar space and congested capillaries. hemophilus influenzae hemophilus influenzae is a gramnegative bacillus that inhabits the upper respiratory tract and can cause otitis media, epiglottitis, and meningitis, and usually enters the lung through aspiration or hematogenous spread. six serotypes are defined based on their capsular antigens, with type b the most common cause of pneumonias. this type of pneumonia is most commonly found in children or in the elderly with underlying chronic lung disease such as emphysema, cystic fibrosis, bronchiectasis, in patients with hiv infection, or in alcoholics. this bacterial pneumonia is usually preceded by a viral or mycoplasma infection that damages the mucociliary elements in the airways and allows for colonization by h. influenzae. the symptoms include fever; a productive, purulent cough; and myalgias. the incidence of this pneumonia as a common community-acquired pneumonia in children is quite low due to the advent of effective vaccines. however, it is increasing in incidence as a nosocomial infection [ ] . like pneumococcal pneumonia, the pathology of h. influenzae pneumonia is in a lobar distribution with a neutrophilic-rich infiltrate and a pleural effusion. necrosis and empyema may occur but are uncommon. staphylococcus aureus staphylococcal pneumonia is caused by staphylococcus aureus, gram-positive cocci that usually spread to the lung through the blood from other infected sites, most often the skin. though a common community pathogen, it is found twice as frequently in pneumonias in hospitalized patients. it often attacks the elderly and patients with cf and arises as a co-infection with influenza viral pneumonia. the clinical course is characterized by high fevers, chills, a cough with purulent bloody sputum, and rapidly progressing dyspnea. the gross pathology commonly reveals an acute bronchopneumonia pattern (figure . ) that may evolve into a necrotizing cavity with congested red/purple lungs and airways that contain a bloody fluid and thick mucoid secretions. the histologic pattern is characterized by a bronchopneumonia that spreads distally from the small airways into to the alveolar spaces (figure . ) to form abscesses that connect with the pleural surface and may result in empyemas. the treatment of this organism has become increasingly problematic due to antibioticresistant strains, most notably methicillin-resistant s. aureus. legionella pneumophila legionella are gram-negative bacilli found predominantly in aquatic habitats such as lakes, rivers, and ponds. standing pools of water from humidifiers and other water outlets may be other sources. approximately % of air conditioners contain these bacilli. though serogroups of legionella have been identified, cause the overwhelming majority of human pneumonia. the clinical disease takes two forms: (i) legionnaires' disease, named after the outbreak of pneumonia at the american legion convention in philadelphia; and (ii) pontiac fever, a self-limiting flu-like disease with nonspecific symptoms. legionella pneumonia presents as a severe infection of the lung with chills and rigors with a nonproductive cough. it can progress rapidly to systemic symptoms of nausea, vomiting, and diarrhea and can lead to renal failure and death without immediate antibiotic therapy. the infected lungs are remarkably red and congested and appear to be distended with fluid. the microscopic picture reveals fibrinopurulent exudates that fill the alveolar space mixed with a necrotic, cellular infiltrate of degenerating neutrophils and monocytes (figure . ). hyaline membranes may form in the periphery of the lesions, and pleural effusions consisting of fibrinoserous exudates are common. pseudomonas aeruginosa pseudomonas aeruginosa is a gram-negative bacillus that is found throughout the environment and in % of the airways of hospitalized patients. it usually enters the body through a disruption of the epithelial surface by cuts, burns, or therapeutic devices such as mechanical ventilators or intravascular catheters. pneumonias caused by this organism are usually found in intensive care units of hospitals and burn units, in patients with underlying chronic lung diseases including cystic fibrosis, emphysema, and in patients with prolonged hospitalization. the pathology is necrosis with a bronchopneumonia pattern that usually consists of an area of congestion and hemorrhage that is surrounded by a halo of tan/white consolidation (figure . ) . a necrotizing vasculitis with abundant organisms in vessel walls can be seen, and cavitation is common ( figure . ). in treated lungs, healed cavities or pneumatoceles may appear as smooth-walled fibrous cysts. other gram-negative bacilli gram-negative bacilli such as klebsiella pneumoniae, acinetobacter, and various enterobacteriaceae spp. are common nosocomial pathogens. similar to p. aeruginosa, these pathogens colonize the oropharynx and are usually introduced into the lung by inhalation or aspiration of oral contents. the most notable of these is friedlander's pneumonia caused by k. pneumoniae, the most common cause of gramnegative bacterial pneumonia. this typically occurs in men over years of age, usually in the setting of alcoholism, diabetes mellitus, or chronic lung disease. these patients produce large amounts of thick, bloody sputum, a product of the viscous mucopolysaccharide capsule of the organism, and present with severe systemic symptoms of hypotension and generalized weakness. the pathology of these pneumonias is similar to pseudomonas pneumonia with marked cavitation and abundant organisms on microscopic examination. nocardia spp. nocardiosis of the lung is caused by nocardia asteroides, a gram-positive rod found in the soil or organic matter. this infection is most common in immunocompromised adult patients and can be seen in the setting of pulmonary alveolar proteinosis, chronic lung diseases, and mycobacterial and other granulomatous diseases that affect the lung. its clinical course is indolent and usually begins - weeks before the patient presents for medical therapy. cough is common, often with thick, purulent sputum. in the immunocompromised setting, fever, chills, dyspnea, and hemoptysis are common, and weight loss may occur as the disease progresses. the pathology is remarkable for suppurative abscess formation with multiple cavities filled with green, thick pus. the inflammatory infiltrate consists of neutrophils, macrophages, and abundant necrotic debris with epithelioid histiocytes and giant cells within the wall of the cavity (figure . ). empyema and pleura involvement occur in the majority of cases. mycoplasma and rickettsia pneumonias mycoplasma pneumoniae pneumonia is among the most common infections of the lower respiratory tract and usually occurs in small epidemics in closed populations. it often presents with atypical features of a progressive onset, fever without chills, a dry cough, diffuse crackles on physical examination, and chest imaging studies that reveal patchy interstitial infiltrates. the pathologic features are a result of the attachment of the organisms to the bronchiolar epithelium where they cause epithelial injury and ulcerations through secretion of peroxide [ ] . in cases of severe infection, diffuse alveolar damage may be present. chlamydial pneumonia chlamydia spp. causes pneumonia in a variety of clinical settings. chlamydia trachomatis is an infection found predominantly in the postnatal period, chlamydia psittaci is the result of direct transmission from infected birds, including parakeets, parrots, and pigeons. chlamydia pneumoniae is the most common of the three and is a frequent cause of community-acquired pneumonia. it typically causes a very mild or asymptomatic infection with fever, sore throat, and nonproductive cough. the course of this infection may be severe in the elderly. chest imaging studies show alveolar infiltrates, and pleural effusions are present in the majority of cases. the pathology has not been well defined since the infection is usually self-limited. however, in experimental animal models there is a neutrophilic response in the early stages, and an interstitial, peribronchiolar, and perivascular infiltrate of lymphocytes, macrophages, and plasma cells in the latter stages of the infection. mycobacteria, a major cause of lung infections, are nonmotile, aerobic, catalase-producing, acid-fast bacilli. clinically significant lung infections can be caused by m. tuberculosis and by a group of nontuberculous mycobacteria (ntm). the latter group consists of over species, of which three cause the overwhelming majority of pulmonary disease. these are m. avium-intracellulare (m. avium complex), m. kansasii, and m. fortuitum-chelonae. throughout history, tuberculosis (infection with m. tuberculosis) was the major disease caused by these organisms and was responsible for worldwide morbidity and mortality. however, over the past two decades lung diseases caused by ntm have become much more common and now represent the majority of the pulmonary mycobacterial disease. mycobacterium tuberculosis pulmonary tuberculosis is spread by interpersonal contact through aerosolized droplets. once in the alveoli, the bacteria cause a cell-mediated inflammatory response that is capable of inducing granuloma formation and necrosis. as in all infections, the extent of the disease is a function of the host's immune response. the most susceptible part iv molecular pathology of human disease patients are those with certain conditions that include immunosuppression, diabetes, malignancy, renal failure, among others. clinically, an infected patient has a productive cough, fever, and weight loss, and may develop hemoptysis as the cavitation progresses and erodes into the pulmonary vessels. extensive involvement of the lung can produce significant dyspnea and pleuritic chest pain. the pathology of tuberculosis is primarily that of granuloma formation and acute pneumonia. the granulomas are predominantly necrotizing, and the pneumonia usually contains abundant fibrin and neutrophils that fill the alveolar spaces. the gross lesions are referred to as caseous or cheese-like, because of the amount of necrosis present. this caseous material can extend into airways and is commonly coughed up during the active disease. in chronic forms of the disease, the area can undergo fibrosis and involute into a firm, hard scar. there are three major clinicopathologic variants of the disease: (i) primary tuberculosis, (ii) postprimary or reactivation tuberculosis, and (iii) progressive fibrocavitary disease. primary tuberculosis. in this form of the disease, the initial site of infection can be anywhere in the lung, but is usually in the lower lobe or anterior segment of the upper lobe, the areas that receive the most ventilation. the lesion usually consists of a dense consolidation with acute pneumonia and necrotizing granulomas. cavitation may occur, especially in the setting of immunocompromised hosts. from these foci, the organisms may spread through the lymphatics to elsewhere in the lung, the hilar lymph nodes, and the bloodstream, and lay dormant for long periods of time. the combination of the primary site of infection and the involved hilar lymph nodes is known as a ghon complex [ ] . postprimary tuberculosis. this form of tuberculosis represents reactivation of old, scarred primary lesions long after the initial insult. the lesion can occur anywhere in the lung where the bacteria from the primary lesion have spread, but is usually apical. it consists of a focus or organizing pneumonia and fibrosis with central caseation. in an active lesion, the typical parenchymal pattern is an acute pneumonia with cavitation that expands to include the surrounding lung with aggregates of granulomas. the controversy surrounding this lesion arises as some evidence suggests that these lesions represent exogenous reinfection. the pathology of reactivation or reinfection may be indistinguishable, although reactivation tuberculosis may appear to arise out of a fibrotic, calcified chronic lesion [ ] . progressive fibrocavitary disease. this form of the disease may arise out of either primary or postprimary tuberculosis. however, the latter is the more common scenario. the cavities that develop in this form of the disease begin as a slowly progressive, necrotizing pneumonia with abundant granulomas (figure . ). the active disease may spread through the airways, causing ulceration, necrosis, and fibrosis of the surrounding bronchi and bronchioles. the extension of the disease in this way depends on the host, and patients with depressed immune systems can have large areas of the lung involved with massive pulmonary necrosis. usually, a fibrous capsule develops in the area of the cavitation, although inspissated necrotic material into the adjacent airways remains a continuous source of inflammation that can lead to reinfection and ongoing scarring [ ] . nontuberculous mycobacteria the nontuberculous mycobacteria (ntm) are ubiquitous inhabitants of our environment, isolated from soil, fresh and brackish water, house dust, birds, animals, and food, and are increasingly important in causing pulmonary disease. there are currently more than ntm species known. those organisms thought to be pathogenic to the lung include the clinical presentation of these lung infections can vary from minimally symptomatic small lesions discovered by routine radiography to sudden hemoptysis from advanced disease with severe cavitation (table . ). the two most characteristic lesions are those of diffuse infiltrates in an immunocompromised patient, seen most commonly in the hiv-positive population and an viruses most pulmonary infections are due to viruses from four major groups: influenza, parainfluenza, respiratory syncytial virus (rsv), and adenovirus (table . ) [ ] . the clinical presentations of these infections have some common features, including insidious onset, nonproductive cough, fever, and chest pain. chest imaging studies usually reveal bilateral, multifocal infiltrates, most without evidence of cavitation or pleural involvement. these infections are mild, self-limiting, and require no more than supportive therapy except in immunocompromised hosts, where the clinical course can be much more serious. also, immunocompromised patients are susceptible to other viruses such as herpesvirus and cytomegalovirus pneumonias, which are not common pathogens in normal hosts [ ] . since the s, a subset of pulmonary viral infections has emerged with a much more aggressive clinical course, most notably sars, coronavirus, and hantavirus. these viruses present with systemic symptoms of headache, myalgias, and weakness followed by a deteriorating clinical course with respiratory distress, shock and, in over % of the cases, death [ , ] . therapy for most respiratory viral infections is supportive, although antivirals are available for some viruses, mostly used in the setting of immunocompromised patients. ribavirin, a guanosine analogue, is the main antiviral used for rsv; m inhibitors or adamantanes (amantadine and rimantadine) are used against influenza a and neuraminidase inhibitors (oseltamivir and zanamivir) are used against both influenza a and b [ ] . cytomegalovirus is treated with ganciclovir, foscarnet, or cidofovir, while herpesvirus is treated with acyclovir [ ] . the pathologic patterns of injury for most viruses are similar, making morphologic distinctions among them difficult. however, some characteristic patterns emerge, most notably in those viruses that cause cytopathic changes. influenza, adenovirus, sars, coronavirus, and hantavirus all cause an acute lung injury pattern with diffuse alveolar damage, and in the case of the latter two viruses, evidence of hemorrhage and edema. influenza and adenovirus will also cause a necrotizing bronchiolitis due to their preferential infection of bronchial epithelial cells. finally, some viral infections can be distinguished by their characteristic cytopathic inclusions. adenovirus can be identified by characteristic smudge cells that present in advanced stages of the disease and represent adenovirus particles in the nucleus of an infected cell (figure . ). cytomegalovirus has both nuclear owl's eye inclusions, as well as cytoplasmic inclusions (figure . ). herpesvirus has glassy intranuclear inclusions and can also have multinucleation (figure . ). fungi are larger and more complex than bacteria, and their patterns of injury in the lung are different and in general more destructive. these pathogens are common in our environment and enter the lungs through inhalation. though many fungi are capable of causing pulmonary disease, most only inhabit the lung as colonizers. those of most concern for causing clinical disease include the endemic fungi of north america-histoplasma capsulatum, blastomyces dermatitidis, and coccidioides immitis-and two fungi that are commonly seen in immunocompromised hosts-aspergillus fumigatus and pneumocystis jiroveci. histoplasma capsulatum histoplasma capsulatum is a dimorphic fungus most prevalent in the middle portion of the united states from the great lakes to tennessee. the fungus is present in soil that has been contaminated with guano and other debris by nesting birds, most commonly blackbirds and chickens, and by bats. the organism lives in the environment as spores or conidia and germinates to form hyphae. these structures divide to create the yeast forms, which, when inhaled, induce granuloma formation in the lung. approximately % of people have skin tests that are positive for exposure to h. capsulatum, but most exposures do not cause clinical disease. disease typically occurs in people exposed to large amounts of organisms, such as construction workers who move large volumes of dirt or spelunkers who venture into bat-ridden caves. the acute disease has flu-like symptoms which are self-limiting. healed disease may leave behind calcified granulomas in the lung that appear as buckshot on chest imaging studies. the most chronic forms of this disease may slowly progress, giving rise to cavitating and fibrous lesions. in the immunocompromised host, disseminating histoplasmosis can be seen, although reactivation is uncommon [ ] . the pathology reveals characteristic necrotizing granulomas distributed around the airways (figure . ), which contain silver-positive yeast forms of - microns. these granulomas may resolve into scarred nodules, which can calcify and produce the characteristic chest images. cavities may form in the apices with progression of the disease, and the disseminated form of the disease has an abundance of organisms both within macrophages in the lung and throughout many organs in the body. blastomyces dermatitidis blastomyces dermatitidis is also endemic to the middle united states, including the ohio and mississippi river valleys. it is found in wooded terrain, usually during the wet seasons, putting campers and outdoorsmen at risk. the clinical disease takes two forms, cutaneous and systemic, the latter beginning in the lungs through inhalation. the acute pulmonary infection takes a nonspecific form with fever, malaise, and chest pain. imaging studies may show either infiltrates or a mass-like infiltrate. thus, blastomyces infection may mimic other diseases, and the diagnosis may be delayed. some patients go on to chronic disease with cavitation or progressive pulmonary blastomycosis, which manifests as acute respiratory distress syndrome, cavitary lesions, and a poor prognosis [ ] . the pathology of blastomyces infection is similar to histoplasmosis with necrotizing granulomas. however, the lesions are larger, showing more neutrophilic necrosis. the organisms are also larger ( - microns), with prominent broad-based budding, and are apparent on routine hematoxylin and eosin staining (figure . ). coccidioides immitis coccidioides immitis is found in the semi-arid desert climate of the southwestern united states. the organisms are inhaled as spores, causing an acute disease characterized by fever, chills, chest pain, dyspnea, and hemoptysis. chest imaging studies typically show consolidation and cavitation, and hilar lymphadenopathy is common. reactivation and dissemination are possible in patients with previous infection, whether or not they are immunocompromised patients [ ] . the pathology of pulmonary coccidioidomycosis is neutrophilic, suppurative, and granulomatous. the organisms appear as large spherules containing endospores, visible on silver stains. the spherules are - microns in diameter and the endospores that are released into the surrounding tissue proceed to mature into new spherules (figure . ) . as in histoplasmosis, cavitating lesions may have hyphal forms that begin to germinate. aspergillus fumigatus aspergilli are asexual mycelial fungi that are ubiquitous in the environment as airborne aspergillus spores. they are weak pathogens that produce invasive infections predominantly in immunocompromised hosts or in those with significant chronic lung diseases. in tissue, aspergilli form septate hyphae, - microns in diameter, with characteristic acute-angle, dichotomous branching (figure . ) . these organisms affect the lung in three major ways: (i) saprophytic growth in bronchi or pre-existent cavities; (ii) as an allergic or hypersensitivity reaction, predominantly in asthmatics; and (iii) invasive aspergillosis in immunocompromised hosts [ , ] . as a saprophyte, aspergillus produces surface growths or minute masses of hyphae, usually in bronchiectatic cavities, emphysematous bullae, or scars from previous lung diseases such as tuberculosis or sarcoidosis. the pathology is usually that of a fibrous-walled cavity containing degenerating hyphae (figure . ). in this setting, hyphae do not invade into the lung tissue, but surface erosion of a vascularized cavity may cause hemoptysis. aspergillus causes an immunologic response resulting in mucoid impaction or eosinophilic pneumonia in asthmatics, an entity known as allergic bronchopulmonary aspergillosis (abpa). pathologically, one sees mucoid plugs and superficial erosions of the airways with histiocytic inflammation, with only rare hyphal fragments present. the final form of the disease, invasive pulmonary aspergillosis, is found in severely immunocompromised, neutropenic patients. the hyphae, which disseminate through the blood, invade the blood vessels causing thrombosis, hemorrhage, and infarction to form typical targetoid lesions. this form of the disease has a poor prognosis despite aggressive antifungal therapy. pneumocystis jiroveci the taxonomy of pneumocystis jiroveci (formerly pneumocystis carinii) has changed over the past decade. previously thought to be a protozoan based on the histological characteristics of its trophozoite and cyst life forms, it has recently been placed in the fungal kingdom after ribosomal rna was found to have sequences compatible with the ascomycetous fungi [ ] . the inability to culture pneumocystis jiroveci has slowed the understanding of this organism. animal models have helped in defining the antigenic and genotypic differences among the various pneumocystis organisms, which has led to the proposal for species-specific strains, with p. jiroveci found in human infections [ ] . the molecular methods used for the typing these species examine a number of gene loci. most importantly, sequence analysis of the thymidylate synthase (ts) and superoxide dismutase (soda) gene loci, the epsp synthase domain of the multifunctional arom gene, and the mitochondrial small subunit ribosomal rna (mtssu rrna) locus have been used to distinguish the various pneumocystis species that infect different mammalian hosts [ ] . clinically, p. jiroveci causes disease predominantly in the immunocompromised setting. pneumocystis pneumonia (pcp) has been found during recent times most commonly in the aids population, but prior to this epidemic, it was found in malnourished infants and other severely immunocompromised hosts. because this organism has not been cultured, the diagnosis of pcp continues to be challenging. the clinical characteristics are nonspecific and vary with the patient's immune status. in the hiv population, patients typically develop a subacute onset of progressive dyspnea, a nonproductive cough, malaise, and a low-grade fever. in the non-hiv population, the presentation is more acute, with fulminant respiratory failure associated with cough and fever, and usually requiring mechanical ventilation [ ] . chest imaging studies typically show bilateral, symmetric, fine reticular interstitial infiltrates involving the perihilar area, which spread to involve the entire lung. figure . aspergillosis. aspergillus fumigatus grows within necrotizing cavities of the lung as branching septated fungal hyphae, as seen on this grocott methenamine silver stain. figure . aspergilloma. fungal hyphae from aspergillus fumigatus can colonize chronically inflamed lungs with cavities and may grow to form fungal balls with a dark, green color that are treated by surgical resection, as seen in this case of a lobectomy specimen. treatment is usually with trimethoprim/sulfamethoxazole and intravenous pentamidine. survival is %- % even in severely immunocompromised patients. the life cycle of p. jiroveci consists of three stages: trophozoite, cyst, and sporozoite. the trophozoite form, which adheres to the type epithelium, replicates and enlarges through three precyst stages before maturing into a cyst form that is found in the alveolar space. sporozoites develop within immature cysts through meiosis and mitosis. the mature cyst contains eight haploid sporozoites. the rupture of the cyst wall releases sporozoites into the surrounding environment where they mature into trophozoites. the pathology of the infection is predominantly due to the interaction of the organism with the epithelium. the attachment of the organism to the lung epithelium is via glycoprotein a present on the surface of the organism. the binding of the organism to the type cell occurs via surface receptors on the type cell that include macrophage mannose receptors. these interact with glycoprotein a and activate pathways in the organism that induce genes encoding for pathways that induce mating and proliferation responses, and for the formation of pheromone receptors, transcription factors, and heterotrimeric g-protein subunits [ ] . in addition to these genetic effects, the cyst wall contains chitins, polymers, and other substances, in particular, , -glucan, that maintain its integrity and induce the inflammatory response of the host. the , -glucan in the wall of the organism stimulates the release by the macrophages of reactive oxidant species and the generation of potent proinflammatory cytokines, such as tnfa, which bind to the organism and exert a toxic effect. once inside the macrophage, the organism is incorporated into the phagolysosome and degraded. tnfa also directly recruits other inflammatory cells including neutrophils, lymphocytes, and circulating monocytes, and induces the release of il- and ifn-g that recruit and activate inflammatory cells [ ] . in aggregate, the recruitment of these inflammatory cells and the mediators they release is responsible for the damage to the lung epithelium and endothelium that is seen in this disease [ ] . the pathology of pcp has typical and atypical variants. typically, the lung contains a dense interstitial plasma cell pneumonia that expands alveolar walls. the epithelium consists predominantly of type pneumocytes, and the alveolar spaces contain an eosinophilic, frothy exudate, which contains fine, hemoxylin-stained dots that represent a thickening in the cyst wall (figure . ) . in this form of the disease, the organisms are abundant and the diagnosis can usually be made by bronchoalveolar lavage. atypical pathologic variants include a necrotizing variant that has a pattern similar to the typical form with exudative alveolar infiltrates, but which undergoes necrosis and cavity formation. these cavities heal into fibrous-walled cysts, similar in gross appearance to those found in pseudomonas pneumonia. a third variant has wellformed granulomas involving the airways, a pattern common to histoplasmosis and tuberculosis. in this form, the organisms are rare and very difficult to find, even with tissue organismal stains. in general, the pathologic pattern of injury depends on the host's immune status, with the typical pathology found in severely immunocompromised hosts as the aids population and the atypical forms found in hosts with immune systems that are less compromised. pulmonary langerhans cell histiocytosis (plch) and erdheim-chester disease are histiocytic diseases that primarily affect the lung. other histiocytic diseases may affect the lung, such as niemann-pick disease, gaucher disease, hermansky-pudlak and rosai-dorfman disease, but these are not considered primarily lung histiocytic diseases. pulmonary langerhans' cell histiocytosis (plch) is a disease of the dendritic histiocytes of the lung referred to as langerhans' cells (lcs). this disease is part of a group of diseases that are characterized by a proliferation of langerhans cells in organs throughout the body that range from a malignant systemic disease as is seen in children [ ] to the pulmonary variant that is seen in adolescents and adults. plch is usually the result of inflammatory or neoplastic stimuli in lungs of smokers or in lungs involved by certain neoplasms [ ] . chest radiographs from patients with plch usually reveal bilateral nodules, predominantly in the upper lobes, which are worrisome for metastatic disease. treatment involves smoking cessation and steroid therapy. typically, the disease undergoes spontaneous regression. approximately %- % of patients will progress to irreversible end-stage fibrosis [ ] . the pathology of plch consists of airway-based lesions with a proliferation of lcs. the early cellular lesions contain a mixture of cells including langerhans' cells, lymphocytes, plasma cells, and eosinophils ( figure . ). though it was previously referred to as eosinophilic granuloma, eosinophils are not the major cell type present, and the lesion is, at best, a loosely formed granuloma. immunohistochemistry reveals the lcs to be diffusely, strongly immunoreactive to s- protein and cd a. ultrastructural analysis reveals intracytoplasmic organelles called birbeck granules, a normal constituent of langerhans' cells, in greater numbers in plch [ ] . the pathogenetic mechanisms of plch focus on defects in the homeostasis of dendritic cells (dcs) in the lungs of smokers and the role tobacco smoke may play in stimulating the proliferation of these cells [ ] . some studies suggest that stimulation of alveolar macrophages by chemicals in smoke results in secretion of such cytokines as gm-csf, tgfb, and tnfa [ ] . in transgenic mice, accumulation of dcs around airways may be a result of excess gm-csf [ ] . other theories suggest that cigarette smoke stimulates the secretion of bombesin-like peptide by the neuroendocrine cells in the bronchiolar epithelium and leads to a similar stimulation of alveolar macrophages and a cytokine milieu that promotes the proinflammatory proliferative changes [ ] . not all smokers get plch, leading to the suggestion that only smokers with an underlying genetic susceptibility will develop the disease. studies have established that in some cases the lcs in plch are clonal, suggesting that cellular abnormalities must play some part in the pathogenesis of the diseases [ ] . to support this, studies have shown genetic mutations and allelic loss of tumor suppressor genes in smokers with plch [ ] . the mechanisms by which this proliferation of lcs leads to the destruction of the bronchiolar epithelium and the other observed pathology are unclear. lcs in normal lungs have little ability to interact with t-cells or act as effective antigen-presenting cells, but the lcs of plch have a mature immunophenotype, expressing b - and b - , the co-stimulatory molecules needed for lymphostimulatory activity [ ] . whether this more mature immune phenotype leads to an unregulated immune response and destruction of the bronchial epithelial cells is not known. however, some studies have shown that bronchiolar epithelial cells may induce the expression of this mature phenotype by secreting cytokines in response to environmental stimulants such as cigarette smoke or viral infections, or by the development of hyperplastic or dysplastic lesions that express new foreign antigens [ ] . erdheim-chester disease (ecd) is a systemic non-langerhans' cell histiocytosis of adults that most commonly involves the long bones. involvement of other organs, including the lung, has been reported. lung involvement occurs in approximately %- % of the cases, and the patients usually present with cough, dyspnea, rhonchi, and pleuritic pain. radiographically, the lungs reveal infiltrates in a lymphatic distribution, predominantly upper lobe, with prominent interstitial septal markings that can mimic sarcoidosis [ ] [ ] [ ] [ ] [ ] [ ] [ ] . pulmonary involvement by ecd may have an unfavorable prognosis, and the fibrosis that ensues is one of the most frequently reported causes of death [ , ] . the treatment of ecd is variable with corticosteroids, chemotherapy, surgical resection, and radiation therapy reported [ ] . non-langerhans' cell histiocytes of dendritic cell phenotype are the main cells present in this disease. this infiltrate contains foamy histiocytes with scattered giant cells, a scant number of lymphocytes or plasma cells, and some fibroblasts. the histiocytes express cd (macrophage antigen) and factor xiiia (dendritic cell antigen), but express s- protein weakly or not at all, and do not express cd a. ultrastructural analysis reveals phagolysosomes, but no birbeck granules are present [ ] . this infiltrate that involves the lung is usually present in the pleura and subpleura, within the interlobular septa and around the bronchovascular structures. the remainder of the lung parenchyma is unremarkable, though fibrosis and paracicatricial emphysema can appear in the late stages of the disease [ ] . the etiology of ecd is not known, but this rare disease has been established as primarily a macrophage disorder [ ] . these histiocytes have abundant phagolysosomes and express the antigen cd a and are consistent with a phagocytic cell, most likely closely related to alveolar macrophages. the peripheral monocytosis and the proinflammatory cytokine profile that is found in these patients might suggest that the histiocytic infiltrate is a result of systemic monocytic activation and invasion of circulating monocytes into the tissues throughout the body [ ] . recently, an ecd patient was successfully treated by an agent toxic to monocytes, supporting the theory that these cells play a part in the disease [ ] . alternatively, end organ cytokine production by local inflammatory cells resulting in proliferation and differentiation of resident immature histiocyte populations may produce a similar picture. another interesting observation is that erdheim-chester has been reported to occur in patients with langerhans' cell histiocytosis [ ] , which may suggest that this is a disease where macrophages transition between two different phenotypes along the differentiation spectrum of tissue dendritic cells [ ] . whether this is a benign or malignant proliferation has not been established. of patients studied, clonality has been demonstrated in by polymerasechain reaction [ ] . environmental exposures are a major cause of lung disease and can cause a wide spectrum of both acute and chronic pathology. many organic and inorganic materials can cause lung damage, and because of their similar patterns of injury and long latent periods, it can be difficult to isolate the exact offending agent without a thorough clinical history. the two occupational lung diseases presented here-asbestosis and silicosis-represent pneumoconiosis, which are defined as diseases which result in diffuse parenchymal lung injury due to inhaled inorganic material. both have many pathologic patterns of injury that depend on the amount and length of time of exposure, and both can also cause neoplastic diseases of the lung. asbestos fibers are naturally occurring silicates that are commonly used in construction materials such as cement and insulation and in many textiles. they can be separated into two groups based on their mineralogic characteristics. serpentine fibers, named as such because they are long and curly, include chrysotile asbestos. amphibole fibers, more straight and rodlike, include predominantly amosite and crocidolite asbestos. in the united states most of the asbestos is chrysotile. the amphiboles are more pathogenic and are responsible for most of the neoplastic and non-neoplastic pulmonary diseases associated with asbestos exposure. by definition, asbestosis is bilateral diffuse interstitial fibrosis of the lungs that can be attributed to asbestos exposure. the disease, which mostly affects textile and construction workers, is usually the result of direct exposure over - years. the latency to clinical disease is inversely proportional to the level of exposure. the symptoms are a gradual onset of shortness of breath, a cough with dry rales at the bases on inspiration, and digital clubbing. in the early disease, the chest x-ray shows basilar disease that begins predominantly as thickening of the subpleural, but progresses as infiltrates and fibrosis that involve the middle zone, eventually leading to thickening of the airways and traction bronchiectasis. the apex of the lung is usually spared. the clinical findings are nonspecific and have considerable overlap with uip, so the diagnosis is usually made only when a history of significant exposure is discovered. the gross picture includes a bilateral lower lobe gray/tan fibrosis with honeycomb changes in late disease. microscopically, asbestosis can cause many patterns of injury in the lung, but the most common is collagenous deposition in the areas of the lymphatics where the fibers are in the highest concentration. these areas include the subpleural, interlobular septae, and around the bronchovascular areas that contain a bronchiole and a branch of the pulmonary artery. hyalinized pleural plaques are a common manifestation of asbestos exposure but are not specific for asbestos and can be found in the absence of pulmonary parenchymal disease. eventually, the fibrosis involves the alveoli beyond the bronchioles and causes distortion of the lung architecture to form remodeled, dilated airspaces similar to those seen in uip. distinguishing this fibrosis from other forms of fibrosing lung disease can be difficult, but the presence of ferruginous bodies, asbestos fibers coated by iron, proteins, and a mucopolysaccharide coat are indicative of significant asbestos exposures and support this diagnosis (figure . ) [ ] . figure . asbestosis. this cytopathologic preparation from a bronchoalveolar lavage specimen illustrates an asbestos fiber coated by an iron-protein-mucopolysaccharide substance and appears as a golden brown, beaded structure known as a ferruginous body. silicosis results from chronic, high-dose exposure to crystalline silica, which consists of silicon and oxygen with trace amounts of other elements, usually iron. the most common silica is quartz, which is present in large amounts in such rocks as granite, shale, and sandstone and is among the more fibrogenic of all silica types. thus, occupations most at risk for silicosis include sandblasting, quarrying, stone dressing, and foundry work where exposure to quartz is high. the disease takes three major clinical and pathologic forms that have different clinical characteristics. simple or nodule silicosis is marked by the presence of fine nodules cm, on chest imaging studies, usually in the upper lobes. patients with this condition are typically asymptomatic, with normal respiratory physiology. the pathology in these lungs reveals discrete, hard nodules that have a green/gray color, centered either on the small airways or in the subpleura. microscopically, these nodules have an early stellate shape that eventually transforms to a more whorled appearance with dustladen macrophages scattered throughout it. polarized light examination reveals weakly birefringent material. complicated pneumoconiosis represents similar pathologic findings only with larger and more circumscribed nodules, which coalesce into a large upper lobe mass, a condition known as progressive, massive fibrosis ( figure . ) . these patients are symptomatic with a productive cough and mixed pulmonary function tests with a reduced diffusing capacity as the fibrosis increases. diffuse interstitial fibrosis may occur; however, unlike asbestosis, this pattern is found in pneumoconiosis. when complicated pneumoconiosis is found with rheumatoid nodules in the setting of a patient with rheumatoid arthritis, this is known as caplan's syndrome. the pathogenesis of both asbestosis and silicosis depends upon inflammation and fibrosis caused by the inhaled fibers. in humans, the amount of fiber needed to cause fibrosis varies from person to person. this may be related to a difference in fiber deposition based on the size of the lungs or to the efficacy with which the lung clears these fibers [ ] . some studies have also suggested that fiber length determines the amount of pathology. however, this association has not been confirmed in humans for either asbestosis or silicosis. in both diseases, it is known that other factors increase the risk of developing disease. for example, smokers exhibit worse disease than nonsmokers with similar exposures to asbestosis. the mechanism for this effect is unclear, although speculation centers on the inhibition of fiber clearance in smokers. also, it is known that smoking enhances the uptake of fibers by pulmonary epithelial cells and in this way may increase the fibrogenic and inflammatory cytokine production by these cells. the cellular mechanisms by which both asbestos and silica fibers induce the inflammation and fibrosis are mediated predominantly through alveolar macrophages. in the case of silica, it is known that the uptake of these fibers into the alveolar macrophages is by way of a scavenger receptor expressed on the surface of the cell known as marco (macrophage receptor with a collagenous structure). once inside the cells, the fibers activate the release of ros that can lead to cellular and molecular damage through a number of pathways. first, ros can directly cause lipid peroxidation, membrane damage, and dna damage. second, silicainduced free radicals can trigger phosphorylation of cellular proliferation pathways through mitogen-activated protein kinases (mapks), extracellular signal regulated kinases (erks), and p . these pathways are also involved in the proliferation of fibroblasts in asbestosis and of mesothelial and epithelial cells in the neoplastic diseases associated with the inhalation of these fibers [ ] . in addition, these fibers can activate proinflammatory pathways controlled by such transcription factors as nuclear nfkb and activator protein (ap- ). these pathways result in the activation of the early response genes c-fos and c-jun and the release of proinflammatory cytokines such as il- as well as fibrogenic factors such tnfa [ ] . tnfa plays a prominent role in both diseases, and its regulation has been studied in animal models exposed to silica. it is now known that a transcription factor labeled nuclear factor of activated t-cells (nfat) plays a key role in the regulation of tnfa. figure . complicated pneumoconiosis/ progressive massive fibrosis. this sagittal cut section of lung reveals a large gray/black mass that extends from the apex to include the majority of the lung. the patient had a long history as a coal mine worker, and the microscopic sections revealed abundant anthracotic pigment and scarring in this area. binding sites for nfat have been found in the promoter region of the tnfa gene. the mediation of silica-induced tnfa transcription is probably via o -but not h o [ , ] . atresia of the lung represents a premature closure of the airway at any level of the bronchial tree including the lobar, segmental, or subsegmental airways. clinically, these children usually present between and years of age for symptoms of dyspnea, wheezing, recurrent pneumonias, or for incidental findings on a chest imaging study. these lesions are more common in the proximal segmental bronchi, right more often than left. when atresia is associated with anomalies of the vascular supply to the affected airway, the lesion represents a separate, aberrant segment of lung known as a sequestration, either intralobar or extralobar type. the pathology of bronchial atresias and sequestrations represents sequelae of chronic inflammation due to the accumulation of secretions in these blind-end airways. these features consist of cystically dilated airways with mucus and parenchymal fibrosis with honeycomb changes. in intralobar sequestrations (ils), the anomalous vessel is a muscular artery that enters through the pleura from an aortic source, usually from the thoracic area. ils are separate, isolated areas of lung invested with the normal visceral pleura without bronchial or arterial connections (figure . ). extralobar sequestrations (els) are pyramid-shaped accessory pieces of lung that have their own pleura with an artery from the lung but without airway connections. the category of congenital pulmonary cystic diseases represents the majority of congenital pulmonary disease and includes foregut cysts and cystic adenomatoid malformations. foregut cysts include bronchogenic, esophageal, and thymic cysts that form from defects in the foregut branching. clinically, these cysts are usually incidental findings on chest imaging studies, but they can present with complications due to infection or hemorrhage. pathologic features of these cysts include subtle differences that are usually only apparent after microscopic examination. grossly, these cysts usually arise proximally either within the mediastinum (over %) or in the proximal regions of the lungs, right more commonly than left, along the esophagus, and rarely within the lung parenchyma or below the diaphragm [ ] . microscopically, each cyst contains a simple cuboidal or columnar epithelium, ciliated or nonciliated, that may undergo squamous metaplasia. distinguishing among the three types of cysts requires the presence of other elements. bronchogenic cysts have submucosal glands and/or hyaline cartilage within their walls, and thymic cysts may contain residual thymus. congenital cystic adenomatoid malformations (ccam), now more commonly referred to as congenital pulmonary airway malformations (cpam), are segments of lung with immature airways and alveolar parenchyma. these are usually classified by their predominant cyst size into types - . type cysts, which contain a main large cyst of up to cm, are the most common. these cysts are distinguished from foregut cysts upon the recognition in the cpam of immature alveolar duct-like structures connecting to the surrounding lung parenchyma. this type of cpam is also notable, as it is known to undergo malignant transformation, usually to mucinous bronchioloalveolar cell adenocarcinomas. these anomalies arise due to defects during the various stages of development and are best considered within these developmental stages. the embryonic stage occurs within the first - weeks of life when the ventral wall of the foregut separates into the trachea and esophagus and branches to form the left and right lungs. the splanchnic mesenchyme that surrounds this foregut forms the vascular and connective tissues of the lungs. defects in this phase result in complete lack of lung development known as pulmonary agenesis and incomplete separation of the trachea and esophagus, causing tracheal-esophageal atresias and fistulas. the pseudoglandular stage, between weeks - of development, is a time of rapid development of the conducting airways including the bronchi and bronchioles and the expansion of the peripheral lung into the acinar buds. the mesenchymal tissue figure . intralobar sequestration. the tan and white mass involving this left lower lobectomy specimen represents chronic pneumonia and fibrosis in the sequestered area of the lung. the dilated airways are features of an endstage fibrosis that is commonly found in this entity. part iv molecular pathology of human disease that surrounds these buds begins to thin, becomes vascularized, and forms the cartilage that surrounds the more proximal branching airways. during the canalicular (week [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , saccular (weeks [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , and alveolar (weeks to maturity) stages of development, the acinar buds continue to expand, and the mesenchyme surrounding this continues to thin. during the canalicular stage, the pulmonary vascular bed begins to organize, the distance between the blood in the vascular spaces and the air in the alveoli narrows, and the respiratory epithelium begins to form. the gas exchange unit of the alveolus becomes functional during the saccular stage with further differentiation of the respiratory epithelium to include clara cells, ciliated and nonciliated cells, and type cells with the first production of surfactant occurring during this period. this gas exchange unit continues to mature during the alveolar stage with the growth and septation of the alveoli. this process continues postnatally through - years of age. the different types of cpams arise at different stages of development. cpams , , and are a result of defects during the early embryonic and pseudoglandular stages of development, producing pathology with features of primitive alveolar buds and immature and abnormal airway cartilage structures. cpams and result from abnormal formation of the more distal airways and pulmonary parenchyma during the canalicular, saccular, and alveolar phases, causing pathology with immature alveolar, or alveolar simplification with enlarged alveoli [ ] . various genetic defects in the pathways that control lung morphogenesis have been associated with these congenital lung diseases. two major transcription factors are responsible for the normal branching morphogenesis. the first, thyroid transcription factor- (ttf- ), is a member of the nkx . family of hemeodomain-containing transcription factors. this factor plays a role in the lung epithelial-specific gene expression and proper lung bud development in the embryonic stage, as well as in the maturation of the respiratory epithelium. the second major factor is somatic hedgehog (shh)/gli, expressed by endodermally derived cells and required for branching morphogenesis. the development of the lung bud from the foregut endoderm depends on the appropriate expression of these lung-specific genes at the correct time in development. in the presence of genetic defects, aberrant lung development may occur. for example, mutations of various types in the shh/gli gene have been found to cause tracheoesophageal fistulas, anomalous pulmonary vasculature, and aberrant airway branching. also, deletions in the ttf- gene are associated with tracheoesophageal fistulas and a variety of forms of lung dysgenesis [ ] . finally, factors present in the surrounding mesenchyme play a role in inducing the proper development of the pulmonary endoderm. a prominent mesenchymal factor in this process is fibroblast growth factor (fgf), which modulates both the proximal and distal lung branching morphogenesis. deletions in this gene may cause lung agenesis and tracheal malformations [ ] . surfactant dysfunction disorders represent a heterogenous group of inherited disorders of surfactant metabolism, found predominantly in infants and children. pulmonary surfactant includes both phospholipids and surfactant proteins, designated surfactant proteins a, b, c, and d (sp-a, sp-b, sp-c, sp-d), synthesized and secreted by type cells beginning in the canalicular stage of lung development. damage to type cells during this time period can lead to acquired surfactant deficiencies. however, more commonly these deficiencies are the result of genetic defects of the surfactant proteins themselves. the major diseases are caused by genetic defects in the surfactant protein b (sftpb, chromosome p -p . ); surfactant protein c (sftpc, chromosome p ); and adenosine triphosphate (apt)-binding cassette transporter subfamily a member (abca , chromosome p . ). defects in sftpb and abca have an autosomal recessive inheritance pattern, and defects in sftpc have an autosomal dominant pattern. sp-b deficiency is the most common. it presents at birth with a rapidly progressive respiratory failure and chest imaging studies showing diffuse ground glass infiltrates. the gross pathology in these lungs consists of heavy, red, and congested parenchyma with microscopic features that range from a pap-like pattern to a chronic pneumonitis of infancy (cpi) pattern. in sp-b deficiency, the pap pattern predominates with a histologic picture of cuboidal alveolar epithelium and eosinophilic pas-positive material within the alveolar spaces that appears with disease progression. in the late stages of the disease, the alveolar wall thickens with a chronic inflammatory infiltrate and fibroblasts. this alveolar proteinosis-type pattern of injury can be confirmed with immunohistochemical studies that establish the absence of sp-b within this surfactant-like material. diseases due to abca or sftpc deficiency may present within a week of birth or years later; the former has a poor prognosis, but the latter has a more variable prognosis with some patients surviving into adulthood. indeed, sp-c mutations have also been recognized in some families as a cause of interstitial pneumonia and pulmonary fibrosis in adults [ ] . the pathology of sp-c deficiency has more cpi features and less proteinosis. in contrast, abca deficiency can have either pap or cpi features, with the former present early in the disease and the latter present in more chronically affected lungs [ ] . the sp-b gene (sftpb) is approximately kb in length and is located on chromosome . there are over recessive loss-of-function mutations associated with the sftpb gene. however, the most common mutation is a gaa substitution for c in codon , found in about % of the cases. the lack of sp-b leads to an abnormal proportion of phosphatidylglycerol and an accumulation of a pro-sp-c peptide, leading to the alveolar proteinosis-like pathology. sp-c protein deficiency is due to a defect in the sftpc gene localized to human chromosome . there are approximately dominantly expressed mutations in sftpc that result in acute and chronic lung disease. approximately % of them arise spontaneously, and the remainder are inherited. the most common mutation is a threonine substitution for isoleucine in codon (i t), found in % of the cases, including both sporadic and inherited disease [ ] . this mutation leads to a misfolding of the sp-c protein, which inhibits its progression through the intracellular secretory pathway, usually within the golgi apparatus or the endoplasmic reticulum [ ] . the absence of sp-c within the alveolar space causes severe lung disease in mouse models. infants with documented mutated prosp-c protein, the larger primary translation product from which sp-c is proteolytically cleaved, can have respiratory distress syndrome (rds) 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world health assembly endorsed a plan to completely eradicate polio in , the large-scale use of the attenuated oral poliovirus vaccine (opv) has drastically decreased the number of polio cases. however, the opv vaccine brings rare but serious adverse consequences, especially in the type vaccine strains. most vaccine-associated paralytic poliomyelitis (vapp) outbreaks are associated with type polio vaccine strains, and approximately - % of genetically divergent vaccine-derived polioviruses (vdpvs) cases are associated with the type component of opv. [ , ] other than vapp cases and vdpvs, type polio vaccine strains can also cause a variety of illnesses. [ ] to the best of our knowledge, no cases of pneumonia resulting from type polio vaccine strains have been reported. however, here we report an infant case associated with the type polio vaccine strain. a -month-old male infant with no underlying diseases was admitted to beijing haidian hospital on july , , where he was diagnosed with lobular pneumonia exactly days after he had received his second dose of trivalent opv (topv). the infant was born from a second regular pregnancy by normal delivery ( / weeks gestation, birth weight: g). the infant also received two birth dose vaccinations (the bacillus calmette-guerin vaccine and hepatitis b vaccine), and no adverse reactions to the vaccinations were reported. the infant had no signs of immunodeficiency. his family had no history of travel in the months before he became ill. on july , , days after his second dose of topv, he developed a fever, concomitant cough, some phlegm, and his body temperature reached °c. an antibiotic was given by intravenous drip for days in a local hospital in anhui province, but a low-grade fever and cough persisted until his hospitalization in beijing haidian hospital. blood tests in the haidian hospital revealed the following results: the total white blood cell count was . × /l (normal range: . × /l- . × /l); n: . %; the total platelet count was × /l; and hemoglobin was g/l. the laboratory tests showed that the c-reactive protein was . mg/l (normal range ≤ . mg/l). chest radiographs showed thickness or turbulence in the texture in both lungs and blotches of shadows in the right lung. further clinical features were respiratory sounds and pulmonary rales. in the hospital, treatments included antibiotic therapy and respiratory management such as aerosols, suctioning, back therapy, and body positioning. he was given intravenous tazobactam sodium g/d and ambroxol hydrochloride and ge injections mg/d. after days of treatment, his body temperature returned to normal and his cough was mild. he was released on august after his cough and pulmonary rales disappeared and after the chest films revealed significant absorption of the infected lesions. except for the presence of the pneumonia symptoms, he had no problems with his growth and development. a nasopharyngeal swab sample was collected at the time of admission. total nucleic acid (rna and dna) was extracted from the clinical specimens using a thermo the results were positive for the enterovirus rna, but no other respiratory pathogens were detected. subsequently, a type polio vaccine strain was identified with cycle threshold values of using the enterovirus molecular serotyping method and the poliovirus intratypic differentiation method. [ ] to further characterize this virus strain, the entire vp region was determined directly from the nasopharyngeal swab with the method provided by national polio laboratory of china. the vp sequences were compared with the sequences available in the genbank database using the basic local alignment search tool. the results showed that the sequence had % nucleotide similarity compared with that of the parental sabin type strains. meanwhile, the poliovirus nucleic acid was identified in the patient's nasopharyngeal swab sample using an ion torrent pgm deep sequencing instrument for a viral metagenome analysis. several other viruses were identified in this sample, including the fowl adenovirus c, the streptococcus phage, the human endogenous retrovirus, and the choristoneura occidentalis granulovirus, but none were associated with this pneumonia. the virus was also isolated from the patient's nasopharyngeal swab sample using rhabdomyosarcoma (rd) (human rd cell) and l b (murine cell lines expressing the human poliovirus receptor) cell lines provided by national laboratory for poliomyelitis in china cdc. this case investigation revealed that the patient received his first dose of topv on june and the second dose on july , in accord with china's immunization schedule. he was hospitalized in the capital institute of pediatrics with pneumonia on june , which was days after taking the first topv, and he had similar respiratory clinical symptoms within days after his second dose of topv. unfortunately, the clinical specimens could not be obtained from his first hospitalization. the results of the clinical and laboratory tests indicated that this pneumonia was caused by the polio virus. to the best of our knowledge, the relationship between the polio vaccine strain and pneumonia has not been previously reported. the polio vaccine strain can propagate and excrete in the upper respiratory system within several days after the opv administration. in this case, it was unclear whether the polio vaccine strain was a provoking factor or only a contributing factor to the pneumonia onset and development. the clinical and virologic investigation revealed that respiratory illnesses were diagnosed in many patients but only the sabin poliovirus could be detected in their respiratory tracts, suggesting that ingested opvs spread and cause diseases beyond the gastrointestinal tract. [ ] the poliovirus caused outbreaks of human acute respiratory diseases or "minor illnesses" without clinical symptoms of the involvement of the central nervous system. a search of the database revealed that opvs spread through the nasopharynx which is detected by serum neutralization from patients with acute respiratory infections. [ ] notably, an epidemiological investigation found that an infant had similar respiratory clinical symptoms after each dose of topv. thus, these reports, along with the case in this study, support the causal relationship between the type polio vaccine and pneumonia. additional studies are necessary to better understand the role of opv in the pathogenesis of respiratory tract infections. however, this report provides an initial case of pneumonia, the outcomes associated with type polio vaccine strains, and the implications for the safety of attenuated opv in the absence of wild virus diseases. this report also offers clinical support for the world health organization's plan to eliminate the type component of opv in by removing the topv and using the bivalent opv, which contains only type and components of opv. there are no conflicts of interest. vaccine-derived polioviruses polio endgame: the global introduction of inactivated polio vaccine investigations of clinical isolations of oral poliovirus vaccine strains between and in southern taiwan genotyping of enteroviruses isolated in kenya from pediatric patients using partial vp region non-rhinovirus enteroviruses associated with respiratory infections in peru ( - ) key: cord- - f wq authors: ory, marcia g.; lee, shinduk; towne, samuel d.; flores, starr; gabriel, olga; smith, matthew lee title: implementing a diabetes education program to reduce health disparities in south texas: application of the re-aim framework for planning and evaluation date: - - journal: int j environ res public health doi: . /ijerph sha: doc_id: cord_uid: f wq health disparities in diabetes management and control are well-documented. the objective of this study is to describe one diabetes education program delivered in the united states in terms of the re-aim (reach, effectiveness, adoption, implementation, and maintenance) planning and evaluation framework. questionnaires, clinical data, and administrative records were analyzed from adults with diabetes living in south texas, an area characterized by high health disparities. the diabetes education program delivered was a professionally led -month program involving h of in-person workshop education followed by quarterly follow-up sessions. changes in average blood glucose levels over the past months (e.g., a c levels) were the primary clinical outcome. descriptive and multiple generalized linear mixed models were performed. this community-based initiative reached a large and diverse population, and statistically significant reductions in a c levels (p < . ) were observed among participants with type diabetes at months. these reductions in a c levels were sustained at -, -, and -month follow-up assessments (p < . ). however, considerable attrition over time at follow-up sessions indicate the need for more robust strategies to keep participants engaged. for this diabetes education program, the re-aim model was a useful framework to present study processes and outcomes. health disparities are often geographically bound and occur more frequently in impoverished populations characterized by low socio-economic status and a dearth of available healthcare resources [ ] [ ] [ ] [ ] . the u.s.-mexico border is impacted by extremely high disparities in income, education, and healthcare access, and these social determinants of health make this region among the nation's figure illustrated the counties formally included in the healthy south texas initiative [ ] , and the counties in which the diabetes education program was offered were marked with a red dot. counties along the us-mexico border were included, as well as areas adjacent to border counties, which were all referred to as south texas. including urban, small town, and rural areas, the overall estimated population in these counties in was approximately . million, and these areas were among the most impoverished in the nation in terms of socioeconomic status and lack of healthcare services [ , ] . as a community-driven initiative, inclusionary criteria were broad with the intent of serving those both directly and indirectly involved in a person's diabetes prevention and management. while the focus was on adults with type diabetes, persons with pre-diabetes and type diabetes were invited, as well as family members or friends providing care for persons with diabetes. as indicated in figure , the formal diabetes education program was offered in of the south texas counties in two primary hubs clustered around nueces and hidalgo counties. given the community-based nature of this initiative and the desire to reach as many participants as possible to show widespread program penetration, participants were recruited from a variety of sources including screenings at health fairs, referrals from healthcare facilities, outreach to community partnerships with flyers and other social media, and self-referrals. although there was no attempt to standardize referral sources, which differed by organizational sponsorship and location, promotional materials (e.g., flyers) were standardized with a uniform healthy south texas brand. the diabetes education program was a recognized american diabetes association (ada) program that was professionally led but also included community health workers for outreach and programming assistance [ ] . all ada-recognized programs provided quality education for people with diabetes and followed the national standards for diabetes self-management education and support (dsmes) guidelines [ ] . ada-recognized diabetes education programs were eligible for reimbursement through many federal and private u.s. insurers [ ] . offered in both spanish and english, the program consisted of hours of face-to-face educational workshop sessions led by at least one trained health professional (e.g., registered nurse (rn), registered dietician (rd), pharmacist, or certified diabetes educator). workshop sessions were followed with brief (e.g., - min) in-person individualized follow-up educational and support sessions offered on a quarterly basis for a year. focal workshop topics included a discussion of, as well as hands-on experiential learning about what diabetes is, blood glucose monitoring, given the community-based nature of this initiative and the desire to reach as many participants as possible to show widespread program penetration, participants were recruited from a variety of sources including screenings at health fairs, referrals from healthcare facilities, outreach to community partnerships with flyers and other social media, and self-referrals. although there was no attempt to standardize referral sources, which differed by organizational sponsorship and location, promotional materials (e.g., flyers) were standardized with a uniform healthy south texas brand. the diabetes education program was a recognized american diabetes association (ada) program that was professionally led but also included community health workers for outreach and programming assistance [ ] . all ada-recognized programs provided quality education for people with diabetes and followed the national standards for diabetes self-management education and support (dsmes) guidelines [ ] . ada-recognized diabetes education programs were eligible for reimbursement through many federal and private u.s. insurers [ ] . offered in both spanish and english, the program consisted of h of face-to-face educational workshop sessions led by at least one trained health professional (e.g., registered nurse (rn), registered dietician (rd), pharmacist, or certified diabetes educator). workshop sessions were followed with brief (e.g., - min) in-person individualized follow-up educational and support sessions offered on a quarterly basis for a year. focal workshop topics included a discussion of, as well as hands-on descriptive statistics (mean and standard deviation or frequency and percentage) were used to describe the characteristics of participants. not all implementation sites collected the exact same set of variables; thereby, the descriptive statistics reflected available data (i.e., not all variables had the same number of missing cases). bivariate analyses (e.g., independent t-tests or chi-square tests) were performed to compare the characteristics of participants recruited during the first biennium and second biennium. next, retention rates were estimated for each follow-up session. as a part of retention analysis, characteristics of participants who attended and did not attend each follow-up session were described and were then compared using bivariate analyses. this study used data collected between september and december . although having four additional data collection months after the second biennium ended in august allowed for more follow-ups, not all participants had an opportunity to complete all their follow-up sessions. intervention delivery dates were used to identify and exclude participants from the retention analyses based on their eligibility to participate in the follow-up. for example, participants who participated in the workshop after june were excluded from the retention analyses for the -month follow-up. similarly, participants who participated in the workshop after march were excluded from the retention analyses for the -month follow-up; and those who participated in the workshop after december were excluded from the retention analyses for the -month follow-up. multiple generalized linear mixed models with participant-level random intercepts were fitted to examine changes in a c level over time among participants with pre-diabetes or type diabetes. persons with type diabetes (n = ) or gestational diabetes (n = ) were not included in the regression models due to small sample sizes. separate models were performed for the first and second biennia. the first set of models examined changes in a c levels over time in participants with type diabetes (n = in the first biennium and n = in the second biennium). the second set of models examined changes in a c level over time in participants with pre-diabetes (n = in the first biennium and n = in the second biennium). the third set of models examined any differences in the changes in a c levels over time based on diabetes type (pre-diabetes or type diabetes) (n = in the first biennium and n = in the second biennium). the next set of models examined changes in a c level among participants with type diabetes by their baseline a c level (i.e., . - . %, . - . %, . - . %, . - . %, . - . %, . - . %, and . % or higher) (n = in the first biennium and n = in the second biennium). in addition, a separate regression model was used to examine any racial/ethnic differences in changes in a c level (n = in the first biennium and n = in the second biennium). all regression models controlled for covariates including age, sex, race/ethnicity, education, language, and baseline bmi category. given that there were only participants reported speaking "other" as their primary language, they were excluded from the regression analyses. a significance level of . was used. this study involved retrospective reviews and analyses of limited data, and this study was reviewed and approved by the institutional review board at texas a&m university (irb - d). results were presented based on the five re-aim elements to provide a case study of this applied research about diabetes self-management education [ ] . reach was defined as "the absolute number, proportion, and representativeness of individuals who are willing to participate in a given initiative, intervention, or program, and reasons why or why not" [ ] . the number of persons who participated in the program and their general characteristics were tracked (table ). the majority of program participants were aged between and years ( . %), female ( . %), hispanic ( . %), and had high school or less education ( . %). most participants reported english as their primary language ( . %) ( table ). the intervention could be attended by individuals with pre-diabetes or diabetes as well as their family and friends. among the program participants with a recorded diabetes type, nearly % had pre-diabetes and more than % had type diabetes. the mean a c level was . % among those with pre-diabetes, . % among those with type diabetes, and . % among those with type diabetes. * p < . ; ** p < . ; a . p-values from bivariate analyses (e.g., independent t-tests or chi-square tests) comparing the characteristics of participants recruited during the first biennium and second biennium; b . chi-square comparison was performed after excluding "other" language (n = ); c . in total, . % of diabetes type records were missing, and frequency and percentage were calculated based on available data (n = ); d . mean and standard deviation of baseline a c percentage measures were estimated among those with pre-diabetes and type and type diabetes. bmi, body mass index. a c, average blood glucose level over the past months. there were statistically significant differences in characteristics of the participants recruited during the first and second biennium (table ) , indicating changes in program participant profiles and expanded program reach. compared to participants recruited during the first biennium, those recruited during the second biennium tended to be younger ( . % vs. . % aged - years), normal or overweight ( . % vs. . %), and not knowing their diabetes type ( . % vs. . %) ( table ) . in addition to considering initial recruitment, it was important to assess population representativeness over time. the program consisted of an educational session and four quarterly follow-ups to track behavioral goals and clinical outcomes. however, less than % of participants attended the first scheduled quarterly follow-up session at months, and the attendance rate for the subsequent follow-up sessions further decreased to . % at months, . % at months, and . % at months. the attendance rates at and -month follow-up sessions were higher during the second biennium than during the first biennium ( . % vs. . % at months and . % vs. . % at months). table shows the number and characteristics of overall program participants who attended and did not attend at each follow-up assessment. for all four follow-ups, retention rates were higher among those in the older age group, females, non-hispanic individuals, those with more than a high school education, and those whose primary language was spanish ( table ) . retention rates tended to be lowest for those with bmis classified as being underweight (table ) . among participants with type diabetes, those not attending a follow-up session at any given time point had significantly higher baseline a c levels than those who attended the follow-up session ( table ) . among participants with pre-diabetes or type diabetes, no statistically significant differences were observed based on baseline a c level attending a follow-up session at any given time point (table ). at the -month follow-up, the retention rate was significantly different based on participants' diabetes type ( table ). the retention rate was highest among those with pre-diabetes ( . % at months) and type diabetes ( . %), followed by those with type diabetes ( . %) and those who were unaware of their diabetes type ( . %) ( table ). however, the association between retention rates and diabetes type was not statistically significant at subsequent follow-ups (table ) . effectiveness was defined as "the impact of an intervention on important individual outcomes, including potential negative effects, and broader impact including quality of life and economic outcomes; and variability across subgroups (generalizability or heterogeneity of effect)" [ ] . this study evaluated changes in a c level among participants from baseline to each follow-up time point. in both the first and second biennium, a statistically significant reduction in a c level was observed among participants with type diabetes at the -month follow-up (b = − . , p < . in the first biennium and b = − . , p < . in the second biennium), and this a c level reduction was sustained at the -month (b = − . , p < . and b = − . , p < . ), -month (b = − . , p < . and b = − . , p < . ), and -month (b = − . , p < . and b = − . , p < . ) follow-up. for example, for participants with type diabetes who joined during the second biennium, the average a c level dropped from . % at baseline to . % at months, and this a c level reduction was sustained at subsequent follow-ups ( . % at months, . % at months, and . % at months). on average, participants with pre-diabetes had a c levels that remained controlled (< . %) from baseline to the subsequent follow-ups. for example, in the first biennium, a statistically non-significant reduction in a c level was observed among participants with pre-diabetes at all follow-ups (b = − . and p = . at months; b = − . and p = . at months; b = − . and p = . at months; and b = − . and p = . at months). participants who had type diabetes showed significantly greater reductions in a c level than those with pre-diabetes (p < . for the interaction term between time and diabetes type in both the first and second biennia) ( figure ). table . characteristics of the program participants who attended and did not attend -, -, -, and -month follow-up assessments. p-values from bivariate analyses (e.g., independent t-tests or chi-square tests) comparing the characteristics of participants who attended and did not attend the follow-up session; e . excluded participants who reported "other" primary language to prevent possibility of identifying the individuals; f . excluded participants who reported having gestational diabetes to prevent possibility of identifying the individuals; g . high missing response rates and frequency and percentage were calculated based on available data (n = at months, at months, at months, and at months); h . mean and standard deviation of baseline a c percentage measures were estimated among those with pre-diabetes, type diabetes, or type diabetes. bmi, body mass index. a c, average blood glucose level over the past months. significant reduction in a c level was observed among participants with pre-diabetes at all followups (b = − . and p = . at months; b = − . and p = . at months; b = − . and p = . at months; and b = − . and p = . at months). participants who had type diabetes showed significantly greater reductions in a c level than those with pre-diabetes (p < . for the interaction term between time and diabetes type in both the first and second biennia) (figure ). (a) (b) figure . estimated changes in a c from baseline to , , , and -month follow-up after adjusting for age, gender, ethnicity, education, language, and baseline bmi category, by diabetes type and biennia: (a) first biennium; (b) second biennium. a c, average blood glucose level over the past months. bmi, body mass index. changes in a c values over time among participants with type diabetes were also examined based on baseline a c values. estimated changes in a c by baseline a c values show similar trends over time among the participants enrolled during the first biennium ( figure a ) and second biennium (figure b ). for both biennia, there was a statistically significant modification effect of the baseline a c level on changes in a c values over time (p < . for the interaction term between time and the baseline a c level in both the first and second biennia). participants with high baseline a c values (e.g., % or higher) achieved a decline in their a c values at the -month follow-up assessments and maintained during the subsequent follow-ups. the estimated a c level decline was most pronounced for those with highest baseline a c values (e.g., % or higher). on average, participants with controlled diabetes at the baseline remained in control during the subsequent follow-ups. level on changes in a c values over time (p < . for the interaction term between time and the baseline a c level in both the first and second biennia). participants with high baseline a c values (e.g., % or higher) achieved a decline in their a c values at the -month follow-up assessments and maintained during the subsequent follow-ups. the estimated a c level decline was most pronounced for those with highest baseline a c values (e.g., % or higher). on average, participants with controlled diabetes at the baseline remained in control during the subsequent follow-ups. in a separate regression model, which included the interaction term between time and race/ethnicity, there was no statistically significant differences in changes in a c values over time among participants with pre-diabetes or type diabetes (p = . in the first biennium and p = . in the second biennium). adoption was defined as "the absolute number, proportion, and representativeness of settings and intervention agents (people who deliver the program) who are willing to initiate a program, and why" [ ] . in lieu of being able to quantify adoption, the general adoption approach was described. the coastal bend health education center (cbhec) in corpus christi served as the healthy south texas regional headquarters. the texas a&m south texas center, mcallen campus, served as a second regional hub. while planning the intervention roll-out, a hub-and-spoke model was determined to be the most effective strategy in which a central "hub" supports multiple "spokes" in communities to provide a range of services. with this approach, cbhec and the mcallen campus identified other regional partners to help recruit and deliver the program. due to the staffing requirements (e.g., needing a health professional to lead the educational workshops and sessions), the program was not delivered in all counties; rather, it was more selectively offered around the two hubs-with the spokes representing adjacent service areas. in accordance with ongoing collaborative health promotion activities in their respective local communities, both cbhec and the mcallen campus were able to call upon their extended healthcare and public health networks for program delivery assistance. regional partners that helped deliver the program represented diverse community and clinical entities including community-based organizations, federally qualified health centers, hospitals, clinics, pharmacies, school districts, academic institutions of higher education, state agencies and not-for-profit social service organizations, behavioral health organizations, and city and county government offices. partnerships varied with some organizations assuming a fuller responsibility for delivering courses on their own, some helping with overall recruitment, and some solely offering physical space for classes. implementation was defined as "the intervention agents' fidelity to the various elements of an intervention's key functions or components, including consistency of delivery as intended and the time and cost of the intervention. adaptations are also included in this re-aim element" [ ] . as an ada-recognized program, there was a need to demonstrate that ada principles of diabetes education were being followed. this involved an annual review by a designated quality control coordinator to review delivery processes, certify them as compliant, or note aspects to be corrected. program standardization across sites was facilitated by having a centralized hub for training and data reporting, in coordination with the scientific-administrative oversight functions provided by healthy south texas leadership. cbhec trained staff in program delivery using a standardized program manual and holding periodic problem-solving feedback sessions with regional program managers and implementers. adaptations to the program were discussed with the evaluation team to enhance program reach and retention. for example, program staff reported that many participants found it difficult to attend a single-day, -h workshop. therefore, other options were offered such as spreading the workshop over multiple days in two -h sessions or four -h sessions. program costs were not tracked in the first biennium, but efforts were made to retrospectively estimate actual program costs based on personnel, supply and space costs toward the end of the second biennium. the program was estimated to cost between usd and per participant, albeit with substantial variation based on how established the program was delivered at different sites, methods of recruitment, and the number of participants in each class. a major indicator of program implementation was how many participants engaged in all program activities (e.g., the initial educational workshop plus four quarterly follow-up sessions to track behavioral goals and clinical outcomes over time). as seen in table , only about % of participants attended the first quarterly follow-up session at months, and attendance rates decreased for the subsequent follow-up sessions. given that process evaluation activities were initially built into the program evaluation, program staff were made aware of this issue concerning attendance. subsequent action was taken in an attempt to bolster follow-up rates. retention rates at and -month follow-up sessions during the second biennium were significantly higher than the rates during the first biennium (i.e., . % vs. . % at months and . % vs. . % at months). maintenance was defined at the individual level as "the long-term effects of a program on outcomes after a program is completed" and at the setting level as "the extent to which a program or policy becomes institutionalized" [ ] . at the individual level, the trajectory of a c level change over time during the -month intervention period has already been reported in the effectiveness section (figures and ) . at the setting level, programs included within the healthy south texas initiative were intentionally designed to be housed within and delivered by established community partners who could draw upon their existing networks to facilitate programmatic spread and sustainability. for legislative feedback purposes, the amount of actual and in-kind dollars the health science center leveraged during the first two biennia was calculated. from private and public sources, over usd , , was identified in direct support and in-kind dollars for the healthy south texas initiative (including delivery of the diabetes education program, as well as other disease prevention and health promotion activities) by governmental and nongovernmental entities. the program is still ongoing with a legislative commitment for a third biennium (september -august ), and program delivery remains a core function of the two regional hubs. the healthy south texas diabetes education program reached and benefitted large numbers of participants in a region with documented health inequities that have perpetuated health disparities [ ] . utilizing the re-aim framework for both planning and evaluation enabled the study team to describe this diabetes program in terms of its reach, adoption, implementation, effectiveness and maintenance [ ] , and explore the unique challenges faced when applying and assessing re-aim elements in community settings [ , ] . this community-based initiative reached a large and diverse population in this region, thus supporting the external validity of the positive results observed. the hispanic population accounted for over two-thirds of the total population in the service region [ ]; hence, the ability to provide the program in spanish was a critical element that enabled the program to reach this population. an estimated , persons were served in the first two biennia across a broader range of diabetes outreach and education activities implemented by the texas a&m health science center healthy south texas initiative, in which the diabetes education program was a single component. this initiative capitalized on its understanding of the local community and organizational context, which has been deemed critical for the implementation and dissemination of other health promotion programs [ ] . this enabled the implementation sites to draw upon highly visible stakeholders and their diverse relationships with community and clinical organizations for outreach and delivery. culture, language, and access to resources are known barriers to access to care in the hispanic population with type diabetes [ ] , and collaboration with diverse local community and clinical entities enabled recruitment of culturally competent staff and facilitators to reach the hispanic population, who are likely to be an underserved population in the region. this study has generated several general take-home messages to be considered before implementing future health promotion initiatives. foremost, program planners should conduct community needs assessments and/or engage stakeholders from the communities they wish to target during initial planning, when assessing feasibility, and when deciding which components to include (e.g., considering potential cultural competency considerations and the need to tailor materials). while research studies typically need to offer incentives for participant recruitment [ ] , the healthy south texas initiative promoted program adoption by providing sites with necessary materials and/or subcontracted for services, which allowed programs to be offered free to participants who may have otherwise lacked resources to pay for such services. this was a critical component that helped alleviate or limit the participants' financial burden as a major barrier to participation, particularly among the socioeconomically disadvantaged population. when considering the generalizability of this evaluation in future settings and populations, program implementers and other key stakeholders should consider this option, where possible, as it may determine whether or not participants can access similar services in other initiatives. balancing fidelity to program implementation with the need for adaptation remains challenging in translational research [ ] . over the past two biennia, many of the recommended re-aim strategies for improving the implementation processes were implicitly followed and should serve as explicit guideposts in future studies [ , ] . for example, some sites adapted intervention delivery modes and routinized follow-up reminder contacts. other sites switched the health professional type needed to lead workshops to accommodate local needs and preferences. for example, one hub determined that in their setting a registered dietician was preferable to a registered nurse, and the ada offered some latitude when selecting the specific type of health professional used to lead workshops without jeopardizing the ada-recognized status or program fidelity. of particular note, the high attrition rates for follow-up visits raises issues about the feasibility and appropriateness of the current diabetes education program structure. the first biennium was a learning curve for the problem facilitators and implementation sites, and additional activities (e.g., reminder calls) were considered and conducted to enhance follow-up rates in the second biennium. reviewing other successful chronic disease self-management programs [ ] , the difficulties of expecting the targeted population to consistently engage in intervention sessions over a -month period were recognized. hence, one major adaptation for future programming is to consider modifications of the current diabetes education program and/or the development of a new iteration of such a program with a shorter active intervention period. long-term supports are still valuable for participant success and should still be incorporated in some capacity (e.g., by their healthcare provider, virtually, telephonically). the effectiveness of the diabetes education program demonstrated significant decreases in a c levels over time which were clinically meaningful [ ] . however, a closer look provides guidance for future targeting, which is a major concern in intervention research [ ] . while reducing a c levels is a clinical goal for persons with elevated a c levels, targeting those with a c values of and above has greater potential to be most cost effective, given these a c levels are associated with the most diabetes complications and need for costly medical care [ ] . findings from our study indicate that self-management programs adhering to ada best practices can achieve large decreases in a c levels among this population. maintenance of individual and system-level outcomes is often the most challenging re-aim element to achieve [ , , ] . it is critical to be aware of how context can influence outcomes at both levels. the practical robust implementation and sustainability model (prism) framework, which is now an integral part of the re-aim framework [ ] , helps us understand the contributory role of recipients, implementation and sustainability, infrastructure, and the external environment. one successful maintenance strategy at the individual maintenance level was to pair the diabetes education program with opportunities for class-based exercise programming throughout the year. based on feedback from program managers, participants seemed to enjoy these sessions and kept them engaged in the diabetes education program. additionally, the diabetes education program was seen as a core component of the two healthy south texas regional hubs, and organizational efforts are underway to build capacity and support for continued delivery through a network of concerned partners. in the current third biennium of state funding (september -august ), program staff are fully aware of the importance of context during these unprecedented times. thus, different options for long-term sustainability are being explored, noting that the current covid- crisis may make future state support less likely due to budgetary considerations [ ] . toward this end, strategies for defining and promoting the value proposition of this and other health education programs are being formulated. for example, fee-for-service options, seeking insurance reimbursement for recognized diabetes management programs, and/or providing the program for a modest charge to community or healthcare organizations as a community benefit are being investigated. to assist in making a value proposition based on the potential return on investments made, economic evaluation studies that can demonstrate objective value in multiple ways (e.g., monetary outcomes, measures of gains in quality of life, reductions in years of potential life lost, reductions in potentially preventable hospitalizations) are recommended. such studies are important to inform local, state, and national stakeholders about the potential return on investment for these and similar studies and should complement other evaluation activities of similar initiatives to reach a broader audience. while there were many strengths in this community-based study, there were some limitations that must be acknowledged. this is a case study of a single diabetes education program in a geographic area. hence, the findings, while promising, may not be generalizable to all community diabetes prevention and control programs or to other areas with differing population characteristics, settings, or varying levels of baseline risk (e.g., a c level). additionally, in contrast to academic-based research studies, service delivery often has priority in community-driven programs relative to data collection and management processes, which can limit the types and quality of data collected. for example, although there was a record of engagement by partnering organizations, there was no record of the extent to which organizations agreed to participate when asked. furthermore, while a large sample size was used, lack of randomization in community settings could be impacted by selection bias. the re-aim model provided a general framework for reporting the planning and evaluation of this initiative. as such, full measures on all re-aim dimensions were not collected. while this is a potential shortcoming, it is also aligned with the recognition that applying the re-aim model in "real-world" studies does not depend on the assessment of all five dimensions [ ] . despite the relatively large numbers of community residents served by the diabetes education program, the proportion reached relative to people with diabetes residing in these south texas counties was still minimal, given the high rates of adults with diabetes in the south texas region [ ] . further, the large attrition rate for follow-up sessions was considerable, which may highlight recruitment or fidelity issues and introduce self-selection and/or a healthful bias (i.e., participants with type diabetes with lower a c levels had better retention rates at all follow-up sessions). finally, data were not collected longer than months following the diabetes education program workshop; therefore, an assessment of long-term clinical control and management among participants was not possible. similarly, as the healthy south texas initiative is still ongoing, long-term program delivery and institutionalization post external funding could not be assessed. however, lessons can be learned from this initiative that advance knowledge about research translation. in line with guidelines for implementing evidence-based diabetes prevention and control programs [ ] , two successful strategies were employed to enhance reach with the ultimate goal of reducing health disparities in underserved populations. first, community health workers were integral to participant recruitment because they were seen as trusted members in the community [ ] . another successful strategy was establishing and using diabetes health champions [ ] (i.e., persons in the program who had successfully lowered their own a c levels) to serve as program promoters who could engage populations typically characterized as "hard to reach" and who were unaware of, or previously uninterested in, participating in health promotion programs [ ] . the diabetes education program provided an example of dedicated effort to meet the overall public health goal of diabetes prevention and management for all americans, especially among those experiencing a multitude of social determinants of health inequities [ ] . offering the diabetes education program through two regional hubs was a major advantage for program reach and adoption as well as launching the initiative quickly. overall, the diabetes education program, as part of the healthy south texas initiative, made a substantial impact on the target area reaching diverse and potentially at-risk populations with measured benefits as evidenced in the current study. for long-term programmatic sustainability, such programs will need to be viewed as essential to routine diabetes regional risk: mapping single and multiple chronic conditions in the united states regional differences in rural and urban mortality trends; nc rural health research program: chapel hill trends in socioeconomic status-related differences in mortality among people with chronic obstructive pulmonary disease understanding associations among race, socioeconomic status, and health: patterns and prospects healthy border : a prevention & health promotion initiative emergent public health issues in the us-mexico border region; frontiers in public health centers for diabetes control and prevention. diagnosed diabetes 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creative commons attribution (cc by) license the authors would like to thank the healthy south texas team within the texas a&m health science center for their work to deliver and evaluate this program. a special thanks is given to the diabetes education program trainers, educators, and staff in corpus christi and mcallen who are continually dedicated to improving lives in south texas. additionally, the authors would like to thank carrie l. byington, former senior vice president of the texas a&m university health science center and vice chancellor for health services of the texas a&m university system (currently executive vice president and head of uc health at the university of california) and susan ballabina, deputy vice chancellor for texas a&m agrilife for their leadership role in the healthy south texas initiative during the study period. the authors declare no conflict of interest. key: cord- - vy xkz authors: klaumann, francini; correa-fiz, florencia; franzo, giovanni; sibila, marina; núñez, josé i.; segalés, joaquim title: current knowledge on porcine circovirus (pcv- ): a novel virus with a yet unknown impact on the swine industry date: - - journal: front vet sci doi: . /fvets. . sha: doc_id: cord_uid: vy xkz porcine circovirus (pcv- ) is a recently described virus belonging to the family circoviridae. it represents the third member of genus circovirus able to infect swine, together with pcv- , considered non-pathogenic, and pcv- , one of the most economically relevant viruses for the swine worldwide industry. pcv- was originally found by metagenomics analyses in in tissues of pigs suffering from porcine dermatitis and nephropathy syndrome, reproductive failure, myocarditis and multisystemic inflammation. the lack of other common pathogens as potential infectious agents of these conditions prompted the suspicion that pcv- might etiologically be involved in disease occurrence. subsequently, viral genome was detected in apparently healthy pigs, and retrospective studies indicated that pcv- was already present in pigs by early s. in fact, current evidence suggests that pcv- is a rather widespread virus worldwide. recently, the virus dna has also been found in wild boar, expanding the scope of infection susceptibility among the suidae family; also, the potential reservoir role of this species for the domestic pig has been proposed. phylogenetic studies with available pcv- partial and complete sequences from around the world have revealed high nucleotide identity (> %), although two main groups and several subclusters have been described as well. moreover, it has been proposed the existence of a most common ancestor dated around years ago. taking into account the economic importance and the well-known effects of pcv- on the swine industry, a new member of the same family like pcv- should not be neglected. studies on epidemiology, pathogenesis, immunity and diagnosis are guaranteed in the next few years. therefore, the present review will update the current knowledge and future trends of research on pcv- . the evolution of emerging diseases is associated with factors embedded in the concept "host-agent-environment triangle" ( ) . to infect the host and cause disease, the pathogen needs to evade host defenses, which may occur through single point mutations, genome rearrangements, recombination and/or translocation ( ) . genetic uniformity generated through genetic selection of the host ( ) and the fact that demographic changes, intensification of farming, and international commerce have occurred markedly over the last decades, must be also considered as essential factors for the development of emerging diseases ( ) ( ) ( ) . as well as in humans, emerging diseases drastically affect animal populations, especially food-producing animals. livestock production in large communities (i.e., pig farms or poultry flocks) represents an excellent environment to facilitate the transmission and maintenance of huge viral populations, contributing to the pathogen evolution (through mutation, recombination and reassortment, followed by natural selection) ( ) ( ) ( ) . the intensification of livestock during the last four decades has probably been one of the main factors that contributed to the emergence of new pathogens and/or pathogen variants, leading to changes in the epidemiology and presentation of diseases ( ) . the number of viral infectious diseases in swine has significantly increased in the last years. several important worldwide distributed viruses have been reported in this period, including porcine reproductive and respiratory syndrome virus (prrsv, family arteriviridae), porcine circovirus (pcv- , family circoviridae) and porcine epidemic diarrhea virus (pedv, family coronaviridae). in addition to those worldwide widespread viruses, an important number of novel swine pathogens causing different types of diseases has been described ( , ) . although their economic impact might be variable, they are considered significant infection agents and their monitoring is nowadays performed in some parts of the world. among others, relevant examples are porcine deltacoronavirus (associated with diarrhea) ( ) , senecavirus a (causing a vesicular disease and increased preweaning mortality) ( ) , porcine sapelovirus (found in cases of polioencephalomyelitis) ( ) , porcine orthoreovirus (assumed to cause diarrhea) ( ) , atypical porcine pestivirus (cause of congenital tremors type ii) ( ) and hku -related coronavirus of bat origin (associated with a fatal swine acute diarrhea syndrome) ( ) . besides overt emerging diseases of swine, many other novel infectious agents have been detected in both healthy and diseased animals, and their importance is under discussion. this group of agents is mainly represented by torque teno sus viruses, porcine bocavirus, porcine torovirus and porcine kobuvirus, which are thought to cause subclinical infections with no defined impact on production ( , , ) . an exception may be represented by hepatitis e virus (hev); although it seems fairly innocuous for pigs, it is considered an important zoonotic agent ( , ) . recently, a novel member of the circoviridae family named porcine circovirus (pcv- ), with unknown effects on pigs, has been discovered ( , ) . porcine circovirus (pcv- ) was first described in in north carolina (usa) in a farm that experienced increased mortality and a decrease in the conception rate ( ) . sows presented clinical signs compatible with porcine dermatitis and nephropathy syndrome (pdns) and reproductive failure. in order to identify the etiological pathogen, aborted fetuses and organs from the affected sows were collected for further analyses. whilst histological results were consistent with pcv- -systemic disease, both immunohistochemistry (ihc) and quantitative pcr (qpcr) methods to detect pcv- yielded negative results. samples were also negative for prrsv and influenza a virus. homogenized tissues from sows with pdnslike lesions and three fetuses were tested through metagenomic analysis, revealing the presence of an uncharacterized virus ( ) . further analyses using rolling circle amplification (rca) followed by sanger sequencing showed a circular genome of , nucleotides. palinski et al. ( ) also performed a brief retrospective study through qpcr on serum samples from animals clinically affected by pdns-like lesions (but negative for pcv- by ihc) and pigs with porcine respiratory diseases. results revealed pcv- qpcr positivity in . and . % of the analyzed samples, respectively ( ) . interestingly, almost concomitantly, another research group from the usa reported a clinical picture pathologically characterized by multi-systemic and cardiac inflammation of unknown etiology in three pigs of different ages ranging between and week-old ( ) . several tissues from these animals were tested by next-generation sequencing (ngs) methods and pcv- genome was found. beyond ngs, in situ hybridization was performed in one out of these three pigs, confirming pcv- mrna in the myocardium (cytoplasm of myocardiocytes and inflammatory cells mainly, although to a very low frequency). based on these two initial works, the name pcv- was proposed as the third species of circoviruses affecting pigs, since pairwise analysis demonstrated significant divergence with the existing pcvs. the novel sequences showed < % of identity in the predicted whole genome and capsid protein amino acid (aa) sequence compared to the other members of the circovirus genus ( ) . taking into account the economic importance and the well-known effects of pcv- on the swine industry, a new member of the same family like pcv- should not be neglected. studies on epidemiology, pathogenesis, immunity and diagnosis are guaranteed in the next few years, but the scientific community is still in its very beginning on the knowledge of this new infectious agent. therefore, the objective of the present review is to update the current knowledge and forecast future trends on pcv- . porcine circovirus (pcv- ) belongs to the family circoviridae, genus circovirus. until , the circoviridae family was divided into two different genera named circovirus and gyrovirus ( ); however, on the basis of the viral structure and genome, a new taxonomical grouping has been recently established by the international committee on taxonomy of virus. the genus gyrovirus has been removed from the family circoviridae and reassigned into the anelloviridae family, and the new taxon cyclovirus has been included into the circoviridae family ( ) . this new genus is closely related with circovirus genus members, with some differences in the genomic structure such as the orientation of the major open reading frames (orfs). moreover, viral sequences of the genus cyclovirus have been reported in both vertebrates and invertebrates, including humans and other mammals ( ) ( ) ( ) ( ) ( ) , birds ( ) , and insects ( ) . members of the circovirus genus have been detected in vertebrates ( ) ; most recently one study reported the presence of a circovirus genome in invertebrates ( ) . one of the first circovirus discovered, psittacine beak and feather disease virus, was described in avian species ( ) and, subsequently, several reports revealed the presence of similar virions in other species such as swine ( ) , fishes ( ) , bats ( ) ( ) ( ) , chimpanzees ( ) , dogs ( ) humans ( ) , and minks ( ) . since , three species of porcine circoviruses have been formally accepted, including porcine circovirus (pcv- ), pcv- and pcv- ( , ) . structurally, circoviruses are small single-stranded dna (ssdna) viruses ( ) , characterized by a non-enveloped virion with icosahedral symmetry, and a circular genome with a diameter ranging from to nm. members of this family are constituted by capsid protein subunits organized in a dodecahedral pentamer clustered unit ( ) . pcv- has a genome size ranging from , to , nucleotides (nt) ( ) ( ) ( ) , while the circular genomes of pcv- and pcv- consist of , - , and , - , nt, respectively ( , , ( ) ( ) ( ) . porcine circoviruses contain three major orfs arranged in the strands of the replicative form (rf) ( ) . for pcv- , a total of seven putative orfs capable to encode proteins larger than kda have been predicted on both dna strands ( ) , being six of them larger than nt ( , ) . pcv- contains, besides the three major orfs, eight more predicted ones, but just orf has been characterized in more detail ( ) ( ) ( ) . pcv- contains so far three identified orfs, but only orf and orf have been characterized. the general characteristics of the three major orfs of pcvs are summarized in table . orf encodes for rep and rep ′ proteins involved in replication initiation, of and aa, respectively, in pcv- , and of and aa, respectively, for pcv- ( ). orf apparently codes for a single replicase protein in pcv- , of - aa ( , ) . orf is located on the positive strand and considered the most conserved region of the circovirus genome ( ) . the origin of replication (ori), constituted by a conserved non-anucleotide motif [(t/n)a(g/t)tattac], is located on the same strand as orf and, consequently, this frame is involved in rolling circle replication (rcr) ( ) . orf encodes the only structural protein (cap). it consists of - aa for pcv- , - aa for pcv- ( , , ) and aa for pcv- ( , ) . orf is located on the negative dna viral strand and cap protein is considered the most variable ( , , ) , and most immunogenic ( ) viral protein. nucleotide similarity of % in cap protein between pcv- and pcv- was detected through phylogenetic analyses ( ) ; moreover, the similarity in this protein is much lower ( %) among pcv- and pcv- ( ) while being - % between pcv- and pcv- ( , ) . the orf is oriented in the opposite direction of orf , also in the negative strand, which codifies for a non-structural protein with apoptotic capacity ( , ) . the orf protein consists of aa for pcv- , aa for pcv- and aa for pcv- ( , ) . the apoptotic activity of orf protein has been described both in vitro and in vivo for pcv- and pcv- ( , ), while its putative function in pcv- is still unknown. lastly, orf , also located in the negative strand, has only been described in the pcv- genome. this gene codifies for a protein of approximately aa with anti-apoptotic function ( , ) . table summarizes the nucleotide and amino acid raw distances (calculated by means of the median pairwise distances) among and within porcine circoviruses. the similarity between pcv- sequences ranges from to % throughout the analyzed years and tested countries ( , ( ) ( ) ( ) . phylogenetic analyses suggested two main groups classified as pcv- a and pcv- b and several sub-clusters ( , , ) , based on differences found between both groups in the aa sites and (s a and a v). in fact, certain antigenicity differences among groups have been proposed ( ) , although it is still too early to discuss about potential different genotypes or subgroups for pcv- . additionally, the progressive increase in sequence availability is revealing the presence of other branching patterns, which hardly fit with the "two genotype" classification. therefore, similarly to pcv- , a higher heterogeneity might be found in the future. a phylogenetic tree including full-length sequences of pcv- is depicted in figure . after the first description reported from the usa, several countries located in asia, europe and south america (figure ) have demonstrated the presence of pcv- genome in domestic pig ( , , ( ) ( ) ( ) ( ) ( ) ( ) . pcv- genome has been detected at all tested ages, including sows, mummified fetuses and stillborn ( , , ) . the frequency of viral detection found by pcr in pigs is variable according to the collected samples around the world ( table ) . a lower frequency of pcv- pcr positivity has been detected in lactating pigs when compared with nursery and fattening ones; the highest prevalence was found in animals after weaning ( , , ) . however, these studies included different pigs from fairly limited age-groups and not the same animals over time. in a very recent work performed on longitudinally sampled pigs in spain ( ), pcv- dna was found at all age-groups in four tested farms, and the frequency of infection was not clearly dominant at any age. also, pcv- has been detected at moderate to high rate in sera pools from sows in poland ( ) and thailand ( ) . pcv- genome has been detected by pcr in oral fluids and nasal swabs ( , ) as well as in feces ( , ) , semen ( ) , and colostrum ( ) . kedkovid et al. ( ) found a positive correlation between detection in serum samples and in colostrum, suggesting that the colostrum is influenced by the viremic stage of the sow. no specific studies have been performed on the virus detection in the environment, but one study indicates that the virus was found in out of sponges used for sampling pig transporting trucks after sanitation ( ) . besides domestic pigs, pcv- infects wild boar. viral dna sequences retrieved from wild boar showed more than % similarity with the available sequences from domestic pigs ( , ) . the prevalence found in tested serum samples was similar or higher than that found in domestic pigs, ranging from to . %. additionally, infection susceptibility was associated with the age in both studies; juvenile animals were statistically less often pcv- pcr positive than the older ones. in fact, a potential reservoir role of the wild boar with respect to pcv- infection has been suggested ( , ) . pcv- seems to be restricted to suidae species. however, pcv- genome has been found in out of ( . %) serum samples of dogs from china. the authors suggested that the virus might infect, therefore, non-porcine species ( ) . to date, there is no further evidence regarding susceptibility to pcv- infection in other species. pcv- has been detected in pigs with different clinical/ pathological conditions, such as respiratory, reproductive, gastrointestinal and neurological disorders; however, the virus has been also detected in apparently healthy animals ( , , ) . the conditions in which pcv- has been found are summarized in table . noteworthy, in most of these scenarios there are not complete diagnostic studies, but only the detection of the viral genome in a number of pigs affected by different clinical signs. even though the viral genome was detected, it is worthy to state that it does not imply a causative role of pcv- in the observed condition. thus, this section compiles the peer-reviewed papers, reporting pcv- dna detection in different disease scenarios. the amount of viral dna in serum samples ( - copies/ml) ( ) and tissues ( - copies/mg) ( , ) in postweaning pigs and adults was rather variable, as well as in stillborn or fetal tissues ( - copies/mg) ( , ) . in most of these cases, the number of pcv- genome copies should be considered moderate to low ( , ) . in addition, detection was possible in some instances, but the viral load was below the limit of quantification of the qpcr, which may emphasize the subclinical nature of the infection in these cases ( , ) . an association between high viral load and severity has been demonstrated for other porcine circovirus (pcv- ), especifically under pcv- -sd ( ) and pcv- -reproductive disease ( ) scenarios. however, the meaning of a given genome viral load for pcv- in healthy or diseased pigs is still to be elucidated. pcv- genome was initially retrieved from sows with clinical signs compatible with pdns in usa. in the affected farm, a decrease of . % in the conception rate was found while the sow mortality showed a . % increase ( ) . in china, pcv- was found in serum samples from sows with reproductive problems characterized by acute loss of neonatal piglets ( ) . moreover, a comparative study between healthy sows and sows with a clinical picture characterized by chronic reproductive failure (including increase in abortion and sow mortality rates) revealed that pcv- positivity was higher in affected sows ( out of , . %) than in healthy ones ( out of , . %) ( ) . viral genome has also been found in tissues from stillborn in farms experiencing reproductive failure in china ( ) ( ) ( ) and korea ( ) . pcv- dna was also detected in pigs with respiratory disorders, as already indicated in the first report of this virus ( ) . two more studies reported pcv- genome in animals from china with abdominal breathing and lesions including lung swelling and congestion ( , ) . more recently, the viral genome has been detected in fattening pigs from thailand suffering from porcine respiratory disease complex (prdc), characterized by coughing, dyspnea, fever and anorexia; the prevalence was higher in diseased animals ( %; out of ) than in healthy ones ( %; out of ) ( ). multisystemic inflammation and myocarditis were initially linked with the presence of pcv- ( ). one single study described pcv- in weaned pigs that suffered from gastrointestinal disorders (diarrhea), showing higher prevalence in pigs with clinical signs ( . %, out of ) compared to those with non-diarrhea signs ( . %; out of ) ( ). in another report, animals with congenital tremors were analyzed and pcv- was the only pathogen found in the brain, with high amount of viral dna ( ). a number of studies found pcv- in apparently healthy animals ( , , , , ), which makes much more complicated the overall interpretation of this virus as potential causative agent of disease. whilst the initially pcv- pcr positive cases were negative for three of the most important swine infectious agents (pcv- , prrsv, and porcine parvovirus, ppv) ( , , ) , subsequent studies revealed frequent co-infection with other viruses. all pathogens found in co-infections with pcv- are summarized in table . it is still too early to establish the overall picture of pcv- infection, since it is a widespread virus in healthy animals. therefore, the likelihood of disease may not depend on its presence only, but other factors may serve as illness triggering factors or up-regulate its replication under disease scenarios. the detection of the virus is currently based on molecular techniques such as conventional pcr and qpcr and its characterization by sanger sequencing or ngs. in fact, the first pcv- complete genome was identified by ngs, and subsequently sanger sequencing has been systematically applied to obtain novel pcv- sequences. several primer na, not available in the published study; * , pcv- positivity in lower frequency than diseased animals. pairs and probes have been designed for these molecular techniques ( , , ) . moreover, a duplex qpcr for the simultaneous detection of pcv- and pcv- has been also attempted ( ) . in situ hybridization, a technique used to detect viral genome on histological tissue sections, has been performed in two studies ( , ) . however, the technique is not yet completely standardized, since it is still used in minimal number of laboratories worldwide and a thorough description of the infected cell types is still missing. a minimum number of studies showed the development and validation of serological tests. two reports have published limited information about indirect enzyme-linked immunosorbent (elisa) tests using recombinant pcv- cap protein ( , ) . more recently, a pcv- specific monoclonal antibody has been produced, presumably working on formalin-fixed, paraffin-embedded tissues by means of immunohistochemistry ( ) . infection of cell cultures with pcv- tissue homogenates has been attempted in pk- ( , ) and swine testicle cells (st) ( ) without success. the cells were observed for cytopathic effects and monitored by qpcr for viral growth. however, the ct-values did not increase at each cellular passage and no cytopathic effect was observed ( , ) . therefore, there is not any pcv- isolate so far available. definitely, in order to elucidate the pcv- pathogenesis, further establishment of laboratory techniques such as viral isolation, serology, and detection of viral components in tissues is needed. in consequence, the potential association of pcv- with any clinical condition, if any, is difficult to be demonstrated due to existing technical limitations. porcine circoviruses (pcvs) are ssdna ubiquitous viruses, widespread worldwide in the domestic pig population ( ) . two species were known to infect suidae species before : pcv- , considered non-pathogenic, and pcv- , the cause of one of the most devastating porcine diseases, pcv- -sd. pcv- represents an expansion of the swine virosphere within the circoviridae family, but the up-to-date knowledge is still very limited and there is not yet any clue on its potential pathogenesis or disease causation role. it is at least curious that years ago there were serious doubts about pcv- as a cause of an overt disease characterized by severe lesions and high mortality ( ), while nowadays pcv- has been found within a number of clinical conditions and putative association has been established from the very beginning ( , ) . current literature has already reported the presence of pcv- in animals affected by different clinical pictures, although just few of them included healthy control groups ( , , , ) . in all studies, the frequency of pcv- detection in diseased animals was higher; although these results did not prove any disease causality, at least open the avenue to definitively ascertain its role in clinical/pathological manifestations. further studies on potential disease association of pcv- are needed. no data is available regarding the pathogenesis of pcv- infection. the lack of virus isolation has impeded the establishment of an infection model to date. it is known that pcv- can be found in different tissues of domestic pig and wild boar ( , , ) , indicating the systemic nature of the infection. however, the point of viral entry, primary replication, organic distribution and persistence are still unsolved issues. pcv- has been found in feces, nasal swabs, oral fluids, and trucks transporting pigs ( , , ) , which allows speculating that horizontal transmission through direct contact is probably an important route. detection of viral genome in fetuses and stillborn from farms with history of reproductive failure ( , , ) , as well as in semen and colostrum, points out also to vertical transmission as another likely route. definitively, more studies are needed to ascertain the potential excretion routes of this virus. co-infection of pcv- with both pcv- and prrsv has been reported ( , , , , ) . in fact, this was expected since both well-known pathogens are widespread in the pig population ( ) ( ) ( ) . noteworthy, it is known that both pcv- and prrsv are able to affect the immune system and, therefore, co-infections with these viruses are not unusual ( , ) . other pathogens were also detected in pcv- pcr positive samples ( , ) . very recently, pcv- has been found by ngs approach in pigs affected by periweaning failure-to-thrive syndrome in co-infection with ppv and ungulate bocaparvovirus ( ). since experimental and field studies demonstrated that co-infection with ppv increase the effect of pcv- in causing pcv- -sd ( ) , at this point it cannot be ruled out that a similar effect may occur with pcv- . further investigations are needed to determine whether pcv- might act as a secondary agent upregulating its replication once pigs are immunosuppressed or immunomodulated, or whether the frequency of co-infection is independent of the immune system affection. although pcv- genome has been detected at higher prevalence in weaned pigs ( , , ) , only one study has monitored pcv- infection longitudinally ( ) . in this study, pcv- was found in pigs at all ages with a similar frequency. this infection dynamics contrasts with that of pcv- , which infects pigs mainly between five and weeks of age, and rarely in animals at the lactation phase ( ) ( ) ( ) . this is explained by the fact that colostrum antibodies are protective against infection and then decline during the lactation and weaning phases. once maternally derived antibodies waned, an infection is followed by active seroconversion ( ) ( ) ( ) . this seroconversion usually occurs between and weeks of age and the antibodies may last until weeks of age at least ( , ( ) ( ) ( ) . regrettably, information about infection in sows, maternally derived immunity and how protective the immunity might be against pcv- is completely lacking at this moment. it is known that pcv- can be found in colostrum ( ) , implying the possibility of vertical transmission (sow to piglet) and emphasizing the potential importance of early infections. again, available information regarding these issues on pcv- is still to be generated. one study performed in samples from captured and re-captured wild boar revealed long-lasting infection (potential persistent infection), since the virus was detected during a period of at least - months in few animals ( ) . susceptibility of wild boar to pcv- was not a surprise, since this species shows susceptibility to several pathogens that affect humans and animals ( ), including pcv- ; moreover, the wild boar can also develop pcv- -sd ( ) . taking into account the potential long period of infection observed in some animals and even a higher overall prevalence in wild boar when compared with domestic pigs, such potential reservoir role deserves further investigations ( , ) . infection of pcv- in other non-suidae species is, at this point, still to be demonstrated. although pcv- dna has been found in sera from dogs in china ( ) , the lack of other detection techniques able to confirm a true infection with this virus prevents the assumption of multiple species susceptibility. another interesting aspect yet currently unknown is the potential impact of pcv- on public health. dna from pcv- and pcv- has been found in vaccines intended for use in humans ( ) , probably associated to the use of reagents from swine origin in the vaccine manufacturing. at this point, no information regarding pcv- and its role as a contaminant of human medicines do exist. on the other hand, porcine circoviruses belong to a group of microorganisms that still has not been fully addressed in terms of risk evaluation for xenotransplantation ( ) , so, pcv- should be also a priori added to such list. palinski et al. ( ) conducted a brief study in paraffin fixed tissues from to in north america and results showed a high percentage of pcr positivity in these samples, suggesting that the virus emerged before the year of its discovery. in fact, pcv- has been already demonstrated retrospectively in sweden in ( ) and spain ( ) and china in ( ) , indicating that this is not a new virus and it has been circulating during several decades in domestic pigs. moreover, pcv- has been detected in the oldest samples so far tested in these studies, suggesting that this virus could have been infecting pigs for even a longer period. however, these findings cannot be assumed as a proof of non-pathogenicity, especially when mirroring another closely-related circovirus, pcv- . although this latter virus was initially detected in association with disease by midlate s, retrospective studies showed evidence of pig infection a number of decades before ( , ( ) ( ) ( ) . in fact, in most of these investigations, evidence of pcv- infection coincided with the very first investigated year, suggesting again that pcv- might be even an older circulating virus. in addition, a retrospective study on pcv- conducted in samples of wild boar from spain during a -year period ( ) detected the virus in the first tested year ( ). overall, obtained data confirmed that pcv- is not a new virus and has been circulating for a fairly, non-determined long time in swine and wild boar populations. in fact, the most common ancestor of pcv- was estimated to be originated approximately in ( , ) . genetic characterization of pcv- is mainly done through sanger sequencing. phylogenetic analyses of pcv- genomes available from the genbank indicate they are part of different clusters. however, nucleotide identity among these sequences is really high (> %). in consequence, it seems that pcv- has remained fairly stable over the years without an independent molecular evolution according to specific areas of the world. moreover, these findings do not point out a high mutation rate as has been suggested ( , ) . if such mutation rate were high, it would have generated a higher genomic heterogeneity, which should have been detected at least in the performed retrospective studies accounting for more than years. further studies on the evolution on pcv- are crucial to solve out these controversies. the first metagenomics sequence available from pcv- revealed low identity with cap and rep genes of pcv- and pcv- and a closer identity with other circoviruses such as canine circovirus ( , ) and barbel circovirus ( ) . the circovirus genus members are able to infect a wide range of hosts, and cross-species transmission has also been reported ( ) . franzo and collaborators ( ) hypothesized the possibility of pcv- being the product of recombination related with a host jump. the analysis of genome composition of pcv- found the rep gene closely related with that of bat circoviruses and cap gene with that of avian ones ( ) . recently, novel circoviruses isolated in civets, showing higher similarity in terms of aa sequence in rep protein with pcv- , have been described ( ) . the increasing new data should be useful to clarify the relationships and origin of this virus. on the other hand fux et al. ( ) found nucleotide changes, which resulted in two aa alterations in orf /orf and orf (a v and r k), between the two proposed genotypes (pcv- a and pcv b). li et al. ( ) also suggested two groups with two individual subclades termed pcv- a- and pcv- a- . the aa site from orf , predicted to be under positive selection, was suggested to be located in a potential epitope region. the presence of possible genotypes was also suggested in other studies ( , ) . however, considering the high similarity found in partial or complete pcv- sequences (> % in most of the cases), the importance of determining genotypes or groupings at this stage seems poorly relevant. due to the sensitivity limitations of sanger sequencing, it must be emphasized the need to apply ngs technology to discover minor variants, which might unravel the presence of quasispecies undetected by the currently used technology. porcine circovirus is a recently discovered virus widespread in both domestic pigs and wild boar population. the virus can be found at all tested ages and few animals may display a persistent infection. although the virus has been found in several clinical and pathological conditions, a definitive proof of its pathogenicity is still lacking. phylogenetic information available to date indicates a low genetic variability of pcv- in comparison with other single stranded-dna viruses and indicates that the virus genome has been relatively stable across the years. fk and js did the majority of the writing and communicated with the coauthors to coordinate the document editing. js designed the outline of the manuscript. gf provided the phylogenetic analyses. fc-f, gf, ms, and jn revised the manuscript, did partial writing and approved the final version for publication. we would like to acknowledge the funding of the e-rta - - - project, from the instituto nacional de investigación y tecnologia agraria y alimentaria (spanish government). the funding from cerca programme/generalitat de catalunya to irta is also acknowledged. one world, one health: the threat of emerging swine diseases. a north american perspective natural genome-editing competences of 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liao, yuting; li, shaolin title: ct image visual quantitative evaluation and clinical classification of coronavirus disease (covid- ) date: - - journal: eur radiol doi: . /s - - - sha: doc_id: cord_uid: o bwv objectives: to explore the relationship between the imaging manifestations and clinical classification of covid- . methods: we conducted a retrospective single-center study on patients with covid- from jan. , to feb. , in zhuhai, china. patients were divided into types based on chinese guideline: mild (patients with minimal symptoms and negative ct findings), common, and severe-critical (patients with positive ct findings and different extent of clinical manifestations). ct visual quantitative evaluation was based on summing up the acute lung inflammatory lesions involving each lobe, which was scored as ( %), ( – %), ( – %), ( – %), or ( – %), respectively. the total severity score (tss) was reached by summing the five lobe scores. the consistency of two observers was evaluated. the tss was compared with the clinical classification. roc was used to test the diagnosis ability of tss for severe-critical type. results: this study included patients, males and females. there were mild ( . %), common ( . %), and severe-critical ( . %) cases, respectively. the median tss of severe-critical-type group was significantly higher than common type (p < . ). the icc value of the two observers was . ( % ci . – . ). roc analysis showed the area under the curve (auc) of tss for diagnosing severe-critical type was . . the tss cutoff of . had . % sensitivity and % specificity. conclusions: the proportion of clinical mild-type patients with covid- was relatively high; ct was not suitable for independent screening tool. the ct visual quantitative analysis has high consistency and can reflect the clinical classification of covid- . key points: • ct visual quantitative evaluation has high consistency (icc value of . ) among the observers. the median tss of severe-critical type group was significantly higher than common type (p < . ). • roc analysis showed the area under the curve (auc) of tss for diagnosing severe-critical type was . ( % ci . – . ). the tss cutoff of . had . % sensitivity and % specificity. • the proportion of confirmed covid- patients with normal chest ct was relatively high ( . %); ct was not a suitable screening modality since december , a number of cases of pneumonia with fever, cough, and dyspnea as clinical manifestations have been found in wuhan, hubei province, china [ ] . the analysis of the whole genome sequence of the respiratory samples suggests that it is a new type of betacoronavirus [ ] , which resembled severe acute respiratory syndrome coronavirus (sars-cov) [ ] . on february , , the world health organization (who) officially named it coronavirus disease . who has recently declared the outbreak a public health emergency of international concern [ ] . as of march , , , laboratory-confirmed and clinical-confirmed cases have been documented globally (i.e., the usa, vietnam, germany) [ ] [ ] [ ] [ ] , , laboratory-confirmed and clinical-confirmed cases and deaths in china as of march , [ ] . on jan. , , the first confirmed family cluster was reported in zhuhai, china, where the parents presented with unexplained pneumonia after coming from wuhan to visit their daughter who was living in zhuhai, china; afterwards, the daughter also developed respiratory symptoms and infection with sars-cov- was confirmed. as of february , the journal radiology has published several articles on the imaging features of covid- [ ] [ ] [ ] [ ] , but all of them are descriptive analyses. in february , the chinese society of radiology issued the radiologic diagnosis of pneumonia with covid- . ct plays an important role in the screening and diagnosis of covid- . the first edition of the experts [ ] divided ct manifestations into three stages: early, progressive, and severe according to the extent and features of the pulmonary abnormalities. however, it did not clarify the relationship between the extent of inflammation and the clinical presentation of the patient. in this study, we used a simple convenient method to quantify the imaging findings. we performed a retrospective, single-center study of the sars-cov- laboratory-confirmed cases with which included cases between jan. , and feb. , in zhuhai, china. a confirmed case was defined as positive by highthroughput sequencing or real-time reverse-transcriptase polymerase-chain-reaction (rrt-pcr) assay of nasal and pharyngeal swab specimens [ ] . the rrt-pcr test kits used on the patients in this study was manufactured by shanghai zhijiang biotechnology co. this study was approved by the ethics committee of the fifth affiliated hospital of sun yatsen university and the requirement for informed consent was waived since the study had no risk and would not adversely affect the subjects' rights or welfare. patient selection for this study was consecutive, and no exclusion criteria were applied. all scans were performed with the patient in the supine position during end-inspiration without intravenous contrast on two ct scanners, uct and umi scanners (united imaging). the scanning range was from the apex to lung base. all images were obtained with a standard dose scanning protocol, reconstructed at . mm slice thickness, with mm increment, mm × mm, and a sharp reconstruction kernel (b_vsharp_b). lung window setting was with a window level of − hounsfield units (hu) and window width of hu. image analysis was performed using the institutional digital database system (neusoft v . . . ). all ct images were reviewed by two radiologists with and years of experience in imaging (y.f. and w.l.). imaging was reviewed independently and final decisions reached by consensus. for disagreement between the two primary radiologist interpretations, a third experienced thoracic radiologist with years of experience (k.l.) adjudicated a final decision. no negative control cases were examined. for each of the patients, the ct scan was evaluated for the following characteristics: ( ) distribution: presence of peripheral or peribronchovascular; ( ) density: presence of ground-glass opacities, mixed ground-glass opacities, or consolidation; ( ) internal structures: presence of air bronchogram, interlobular septal thickening, cavitation; ( ) number of lobes affected by ground-glass or consolidative opacities; ( ) presence of fibrotic lesions; ( ) presence of centrilobular nodules; ( ) presence of a pleural effusion; ( ) presence of thoracic lymphadenopathy (defined as lymph node size of ≥ mm in short-axis dimension); and ( ) presence of underlying lung disease such as tuberculosis, emphysema, or interstitial lung disease were noted. ground-glass opacification was defined as hazy increased lung attenuation with preservation of bronchial and vascular margins and consolidation was defined as opacification with obscuration of margins of vessels and airway walls [ ] . two radiologists (y.f. and w.l.) reviewed all images independently blinded to the clinical information. percentage of involvement in each lobe was recorded as well as the overall lung "total severity score (tss)". each of the five lung lobes was assessed for percentage of the lobar involvement and classified as none ( %), minimal ( - %), mild ( - %), moderate ( - %), or severe ( - %), with corresponded score as , , , , or . the tss was reached by summing the five lobe scores (range from to ) [ ] . the final score of each case was decided by a third experienced thoracic radiologist (k.l.). all cases were divided into four groups: minimal, common, severe, and critical according to whether there were clinical symptoms, severity of pneumonia, respiratory failure, shock, other organ failure, etc., based on the diagnosis and treatment plan of covid- issued by national health commission ( th ed.) (in chinese) [ ] . ( ) mild type: mild clinical symptoms without pneumonia in imaging; ( ) common type: fever, respiratory tract and other symptoms with pneumonia in imaging; ( ) severe type: respiratory distress, respiratory rate ≥ times/min; in resting state, oxygen saturation ≤ %; pao /fio ≤ mmhg; ( ) critical type: respiratory failure requiring mechanical ventilation, shock and other organ failure requiring icu monitoring and treatment. statistical analysis was performed using ibm spss statistics for windows, version . (ibm corp.). continuous data conforming to normal distribution expressed by mean ± standard deviation; for those not conformed (median, p , p ) were listed. intragroup correlation coefficient (icc) was used to test the consistency of tss scores of two observers, icc values < . , . ~ . , and > . represent poor, moderate, and good repeatability, respectively. the distribution balance of involved lobes and the number of involved lobes in different clinical types were compared by chi-squared test or fisher exact test when sample sizes were small and by analysis of variance tests. wilcoxon-rank test was used for comparison of tss among different clinical types, since tss did not conform to the normal distribution. roc was used to test the differential diagnosis ability of tss in common-type group and severe-critical-type group. seventy-eight patients were included in the study. the clinical subtype classification was as follows: ( . %) had minimal, ( . %) had common, ( . %) had severe, and ( . %) had critical disease. the demographic data for all patients are shown in table . all patients were discharged after a mean hospitalized period of ± days (range - days). no patients died in this cohort. the consistency test results of ct visual quantitative analysis of two observers showed good repeatability with icc . ( % confidence interval . - . ). the distribution of pulmonary lobe involvement in different clinical types is shown in table . all lobes were involved in the severe-critical type while the lower lobes were usually involved in the common type ( / , . %). compared with the severe-critical type, the common type had a lower incidence of right upper lobe and middle lobe involvement (p = . ; p = . , respectively), and also a lower incidence of right lower lobe, left lower lobe, and left upper lobe involvement; however, there was no significant difference table . common type can involve one, two, three, and four lobes. however, due to less number of cases, there was no significant difference in the first three groups statistically. common type and severe-critical type can both involve lobes, but severe-critical type had a higher incidence than common type (p = . ). for the common type, the involved lobe number of was significantly higher than - (p = . ). the results of tss are shown in fig. . score of mild type was , while common type was - (median , p . , p . ) and severe-critical type was - (median , p , p . ). the score of severe-critical type was significantly higher than common type (p < . ). figures and were from common-type and severe-critical-type patients, respectively. roc analysis showed the area under the curve (auc) of tss for diagnosing severe-critical type was . ( %ci . - . ). the tss cutoff of . had . % sensitivity and % specificity (fig. ) . covid- is a new disease which is caused by betacoronavirus. the diameter of the virus particle is very small, about ~ nm; therefore, it is easy to reach the lung terminal structure, such as alveolar septum, alveolar wall, and interlobular septum, which causes extensive edema and lymphocyte infiltration in the lung interstitium; early alveolar exudation is not prominent, but the disease progresses rapidly [ ] . in this study, the imaging features were consistent with the previous literature reports [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] of viral pneumonia; most of the patients had ground-glass opacities and mixed ground-glass opacities; no patients demonstrated consolidation without ground-glass opacification. subpleural distribution was common. it also occurred around the bronchovascular bundle. air bronchograms and interlobular septal thickening were often present. no patients had in a recent study done by michael et al, they introduced a method to score the severity of inflammation on ct images based on summing up degree of acute lung inflammation lesions involvement of each lobe (including fig. a -year-old female had fever, cough, and sputum with a body temperature of . °c for days and admitted to the hospital on jan. , . the leukocytes and lymphocytes were decreased. she was living in zhuhai and traveled to wuhan on jan. and stayed there for days. she was healthy and nonsmoker. chest ct (images a-c) on the st day after admission demonstrated bilateral peripheral ground-glass opacities with linear opacities. tss was . the clinical type was common type. followup ct (images d, e) on the th day after onset showed peripheral shrinking consolidation with ground-grass opacities in both lungs fig. the total severity score (tss) of different clinical classifications. there were cases of light type ( %), cases of common type ( %), and cases of severe-critical type ( %). the median tss was in severecritical-type group (range - ), which was significantly higher than that of common type (median , ranged [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] ground-glass opacity or consolidation or other fuzzy interstitial opacities) [ ] . we used the same method to quantify pulmonary inflammation and correlate to the clinical classifications. there was significant difference in scores between common type and severe-critical type (p < . ). however, there was also a score overlap between the two groups, which showed that cases in the common type had a higher score, and cases in the severe-critical type had a lower score. among cases of common type, cases had fibrotic lesions which indicated that the lesions began to be repaired, and all of the patients were less than years old (range - , average . years), none of them had pulmonary complications. the specific situation of patients in a severe-critical type which had a lower score was as follows: of the patients were over years old; among them, patient was a female smoker with diabetes, aged years old, with moderate emphysema and a small amount of pleural effusion; patient was a -year-old female, with emphysema and a small amount of pleural effusion as well; the other one was -year-old female with high blood pressure; the forth case was a -year-old male without any underlying disease; however, ct images showed only progressive lesions such as ground-glass opacification and consolidation without any fibrotic lesions (fig. ). the last case was a year-old female without any underlying disease; further analysis [ ] . however, the most recent report from guangzhou had similar findings, which showed . % fig. a -year-old male was admitted to the hospital day after fever and cough with a body temperature of °c. the leukocytes were normal and lymphocytes were decreased. he was living in zhuhai and traveled to macao days before the onset of the disease and stayed in macao for week. he was healthy and nonsmoker. chest ct (images a-c) on the th day after admission demonstrated bilateral peripheral ground-glass opacities without consolidation. tss was . the clinical type was severe-critical type. follow-up ct (images d, e) on the nd day after onset showed bilateral fibrotic changes with traction bronchiectasis and ground-grass opacities confirmed patients without abnormalities on chest ct [ ] . to further explore our data, we found several characteristics. nine cases had a short time interval from onset to the latest ct examination with a range of - days, which indicated that the chest ct could be normal at the early phase. another cases had a longer time interval from onset to the latest ct scan with a range of - days. the negative findings may not relate to the shorter onset time. it remains to be further explored whether the ct negativity may relate to the degree of infection and autoimmunity. finally, the last patients had no symptoms. these patients were negative in both clinical and imaging, suggesting that some cases were potential sources of infection, which should be paid more attention to. in this study, the number of cases between groups was significantly different because too few severe-critical patients were included in this study, which decreased the reliability of statistical results. only image analysis was carried out without combining clinical information in this study; however, advanced age, underlying diseases, and pleural effusions may lead to a lower tss but severe situation. in our next study, we will include more cases, and make a comprehensive evaluation combining the clinical characteristics and laboratory examination information. the proportion of clinical mild-type patients with covid- was relatively high, screening for covid- with chest ct alone can lead to misdiagnosis in some patients, which would lead to a potential infection risk, so ct was not suitable as an independent screening tool. visual quantitative analysis based on ct images has high consistency and high diagnostic ability, which can reflect clinical classification; it is expected to accurately assess the clinical severity of covid- and guide the clinical treatment by combining with the clinical information. clinical features of patients with novel coronavirus in wuhan genomic characterization and epidemiology of novel coronavirus: implications of virus origins and receptor binding a novel coronavirus from patients with pneumonia in china importation and human-to-human transmission of a novel coronavirus in vietnam transmission of -ncov infection from an asymptomatic contact in germany first case of novel coronavirus in the united states national health commission of the people's republic of china ct imaging features of novel coronavirus ( -ncov) ct imaging of the novel coronavirus ( -ncov) pneumonia. radiology ct manifestations of two cases of novel coronavirus ( -ncov) pneumonia evolution of ct manifestations in a patient recovered from novel coronavirus ( -ncov) pneumonia in wuhan imaging diagnosis of -ncov pneumonia: expert recommendation of chinese society of radiology, the first edition fleischner society: glossary of terms for thoracic imaging notice on the issuance of a program for the diagnosis and treatment of novel coronavirus ( -ncov) infected pneumonia (trial revised fifth edition viral pneumonias in adults: radiologic and pathologic findings radiographic and ct features of viral pneumonia severe acute respiratory syndrome: temporal lung changes at thin-section ct in patients epidemiological and clinical characteristics of cases of novel coronavirus pneumonia in wuhan, china: a descriptive study clinical characteristics of coronavirus disease in china publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations acknowledgments the authors thank claudia henschke, david yankelevitz, and rowena yip from mount sinai medical center for polishing the manuscript. key: cord- -fc dn v authors: kato, kentaro; ishiwa, akiko title: the role of carbohydrates in infection strategies of enteric pathogens date: - - journal: trop med health doi: . /tmh. - sha: doc_id: cord_uid: fc dn v enteric pathogens cause considerable public health concerns worldwide including tropical regions. here, we review the roles of carbohydrates in the infection strategies of various enteric pathogens including viruses, bacteria and protozoa, which infect the epithelial lining of the human and animal intestine. at host cell entry, enteric viruses, including norovirus, recognize mainly histo-blood group antigens. at the initial step of bacterial infections, carbohydrates also function as receptors for attachment. here, we describe the function of carbohydrates in infection by salmonella enterica and several bacterial species that produce a variety of fimbrial adhesions. during invasion by enteropathogenic protozoa, apicomplexan parasites utilize sialic acids or sulfated glycans. carbohydrates serve as receptors for infection by these microbes; however, their usage of carbohydrates varies depending on the microbe. on the surface of the mucosal tissues of the gastrointestinal tract, various carbohydrate moieties are present and play a crucial role in infection, representing the site of infection or route of access for most microbes. during the infection and/or invasion process of the microbes, carbohydrates function as receptors for various microbes, but they can also function as a barrier to infection. one approach to develop effective prophylactic and therapeutic antimicrobial agents is to modify the drug structure. another approach is to modify the mode of inhibition of infection depending on the individual pathogen by using and mimicking the interactions with carbohydrates. in addition, similarities in mode of infection may also be utilized. our findings will be useful in the development of new drugs for the treatment of enteric pathogens. enteric pathogens, many of which are zoonotic, exert a major impact on public health worldwide including tropical regions. in humans and animals, the enteric pathogens, which include viruses, bacteria and protozoa, infect the intestine epithelial lining, resulting in food poisoning or diarrheal disease. when enteric pathogens enter humans or animals via the oral route, they must withstand the proteolytic conditions in the stomach before penetrating the mucus layer and accessing the underlying gut epithelium for attachment or cell invasion. adhesion of the enteric pathogens to the intestine epithelial tissue is a prerequisite for the initiation of infection. in many systems it is mediated by lectins present on the surface of the pathogen that bind to complementary carbohydrates on the surface of the host cells. carbohydrates such as heparan sulfate have been reported to play a crucial role in the entry or budding of viruses [ ] , and bacterial lectins typically act in the form of elongated submicroscopic multisubunit protein appendages, known as pili [ ] . recently, the surface proteins of apicomplexan parasites have also been reported to bind to carbohydrates on host cells [ ] . thus the initial steps of host cell recognition by enteric pathogens may incorporate common strategies. once pathogens invade the host cells, they initiate their survival mechanisms to avoid extermination by host immunity. ultimately, if infection of host cells could be inhibited, proliferation of the pathogens could be prevented and pathogenesis could be controlled. insights obtained from studies designed to address this concept will be invaluable to develop novel therapies using innovative drug design and engineered vaccine candidates to limit the infectivity of widespread enteric pathogens. here, we review the recent major advances in research on the role of carbohydrates in the infection strategies of enteric pathogenic viruses, bacteria and protozoa. we further discuss how our knowledge regarding these carbohydrates may influence prophylactic and therapeutic drug development for the treatment of diseases caused by enteric pathogens. carbohydrates function as receptors for virus entry. negatively charged carbohydrates, which are expressed on many types of cells and tissues such as sialic acid and heparan sulfate, are common viral receptors. orthomyxovirus, polyomavirus, reovirus, coronavirus, paramyxovirus and parvovirus recognize sialic acid as a receptor. adenoassociated virus, herpesvirus and flavivirus recognize heparan sulfate. on the other hand, the enteric virus norovirus recognizes histo-blood group antigens (hbgas), which are not charged. here, we focus on the association of carbohydrates with norovirus as the virus enters the host cell. norovirus, a member of the family caliciviridae, is a major cause of acute water-and food-borne gastroenteritis [ ] . norovirus infection is associated with up to % of epidemic nonbacterial acute gastroenteritis cases worldwide [ ] . noroviruses are divided into at least five genotypes, three of which (genogroups i, ii, and iv) infect humans. except for a few genotypes, all noroviruses bind to hbgas including abh antigens and lewis antigens [ , ] . in hbgas, carbohydrate core structures constitute antigenically distinct phenotypes, namely type (galβ - glcnacβ) and type (galβ - glcnacβ). h antigen (fuc-α - gal), i.e., o-type antigen, is generated by fucose transfer to a galactose residue with an α - linkage of type or type . the a antigen (galnac α - (fuc-α - )gal) and b antigen (gal α - (fuc-α - )gal) of hbgas are generated by transfer of galnac and gal, respectively, to an h structure irrespective of the carbohydrate core structure. fut and fut are α , futs that catalyze the transfer of fuc to the gal residue of type and chains, thereby resulting in the synthesis of h type and h type , respectively. hbgas are found in saliva and mucosal secretions from the intestinal epithelial cells of secretors (i.e., individuals who have the fut gene that encodes a fucosyltransferase). non-secretors, who do not express fut fucosyltransferase and consequently do not express h type or le b in the gut, are not infected after challenge with the prototype strain of norovirus, nv/ [ , ] . moreover, the association data between blood type and nv/ infection showed that, among secretor volunteers, blood groups o and a were associated with an increased risk of infection, while blood group b was associated with a decreased risk. on the other hand, epidemiological studies have shown that some norovirus strains with abh phenotypes that differ from that of nv/ can infect individuals. gii/ , which is known as a global epidemic strain, binds more hbgas than other strains, suggesting that the strength of transmission of gii/ strains is related to the broad recognition of hbgas [ ] . the recognition sites on hbgas by norovirus have been classified according to the interaction of the virus with the h, a, b, and le epitopes ( fig. ) [ , ] . hbgas are important factors for determining host specificity, although it is still unclear whether hbgas act as the primary receptor or enhance norovirus infectivity. researchers including ourselves have demonstrated that feline calicivirus (fcv), a member of the genus vesivirus, infects the upper respiratory tract by attaching to α - linked sialic acids and using junctional adhesion molecule- for internalization [ , ] . it is comparatively easy to study the life cycle of fcv because the virus replicates efficiently in cell culture without specific supplementation, whereas noroviruses are not cultivable in cell culture. carbohydrates also function as receptors for bacterial attachment at the initial step of infection. here, we describe the role of carbohydrates in bacterial infections, focusing on salmonella enterica and several assortative bacterial species that produce a variety of fimbrial adhesions (fig. ) . salmonella strains cause disease in diverse mammalian hosts. some salmonella strains have a narrow host range, such as salmonella enterica serovar typhi (s. typhi) and serovar paratyphi (s. paratyphi), which cause disease only in humans, whereas strains such as salmonella enterica serovar typhimurium (s. typhimurium) and serovar enteritidis (s. entertitidis) cause infection in numerous species including mice, poultry, pigs, sheep, cattle, horses and humans [ ] . infected orally, salmonella reach the intestinal tract and then mainly attach to the m cells of the intestinal epithelium to initiate invasion [ ] . after colonization of the intestinal epithelium, typhoid salmonella, s. typhi and s. paratyphi, invade m cells. however, s. typhi and s. paratyphi can also survive being engulfed by macrophages, which then spread throughout the body via the lymphatic and blood systems. non-typhoidal salmonella, s. typhimurium and s. enteritidis cannot survive within macrophages (fig. ) . they cause gastroenteritis in humans and animals by colonizing the intestinal epithelium and then invading and destroying the m cells and enterocytes [ ] . bacterial adherence requires both specific and nonspecific interactions. in the case of salmonella, the negative charge produced by sialic acid on the surface of the host cell is required as a non-specific adherence factor [ ] . for their specific interactions, salmonella and assor-tative bacteria possess various adhesion molecules such as a variety of bacterial fimbriae. at the initial infection step, bacterial attachment is mainly controlled by these bacterial fimbriae. individual fimbria recognize and bind to specific receptors to promote adhesion to the host cell surface [ , ] . long polar fimbriae (lpf) and plasmid-code fimbriae (pef) are categorized as type fimbriae (fig. ) . std fimbriae are categorized as π-fimbriae [ , [ ] [ ] [ ] . a previous report showed that when one of the fimbriae carried by salmonella typhimurium was deleted, only virulence for mouse was moderately altered, and that multiple fimbrial adhesins were required for full virulence [ ] . for salmonella and assortative bacteria, type fimbria is the std fimbriae are categorized as π-fimbriae. bacterial flagella are the moving apparatus of bacteria, but their components can also contribute to the binding to sugar-containing molecules. k. kato et al. best characterized [ ] . type fimbriae consist of a major component (fima) and a minor component (fimh) (fig. ) . fimh lies at the tip of type fimbriae, where it mediates binding to d-mannose-containing structures and enables the bacteria to colonize various host tissues [ ] . type fimbriae are also produced by other gram-negative bacteria, such as escherichia coli and klebsiella pneumoniae [ ] . the fimh protein of enterobacterial species including salmonella recognizes mannose-containing oligosaccharides [ ] . in the case of e. coli, previous reports have shown that fimh protein has a considerably high affinity for oligosaccharides containing manα - , such as manα - manβ - glcnac and manα - (manα - )manα - (manα - )man, which are constituents of cell surface glycoproteins [ ] . the process of bacterial adhesion to the epithelial cell surface mediated by type fimbriae (fimh) is conservative among enterobacteria. type fimbriae are highly expressed on the bacterial surface, allowing large quantities of bacteria to adhere via the fimh-mannose interaction (fig. ). type fimbriae are thin and flexible, and generally expressed at a lower level than type fimbriae. some type fimbriae are only present at low levels on the surface of bacteria, and are localized at the bacterial pole. although their expression level is low, type fimbriae frequently play an important role in bacterial infection. like type fimbriae, type fimbriae are thought to recognize carbohydrates as specific receptors, but the receptor molecules and precise functions of some type fimbriae have yet to be determined. for example, the function of the type fimbria bundle-forming pili (bfp), an important virulence factor for pathogenic e. coli strains, is not yet known (fig. ) . bfp may not be involved in initial adhesion; rather, it may participate in the formation of the bacterial colony by forming bundles that link one bacterium to another [ ] . although the function of and receptors for type fimbriae remain unclear, bacterial virulence has been shown to decrease when type fimbriae are deleted [ ] . lpf medi- salmonella and assortative bacteria express a variety of fimbriae. the minor component of type fimbriae, fimh, is present at the tip of type fimbriae, mediates binding to d-mannose-containing structures and enables bacteria to colonize various host tissues [ ] . type fimbria is highly expressed on the bacterial surface, allowing large amounts of bacteria to adhere via the fimh-mannose interaction. the various kinds of type fimbriae play an important role in bacterial infection. plasmid-encoded fimbria (pef) is required for bacterial attachment to intestinal epithelial cells. pef specifically binds to trisaccharide galβ - (fucα - )glcnac, also known as the lewis x (le x ) blood group antigen [ ] . long polar fimbria (lpf) mediates the adhesion of s. typhimurium to murine peyer's patches [ ] . extracellular matrix proteins (ecms) may act as receptors for lpf. ecms are modified with various carbohydrate moieties, and the presence of mannose inhibits the lpf-ecm interaction. mannose-containing carbohydrates may participate in bacterial adhesion via lpf [ ] . std fimbriae are categorized as π-fimbriae and are well conserved among s. enterica serotypes but absent from other related bacterial species. std fimbriae recognize and bind the h type histo-blood group oligosaccharide, the terminal fucα - galβ moiety. s. typhi and s. paratifi can survive within the macrophages after they are engulfed by phagocytosis. non-typhoidal salmonella, s. typhimurium and s. enteritidis, however, are unable to survive within macrophages. ates the adhesion of s. typhimurium to murine peyer's patches [ ] . lpf was first described in s. typhimurium, and is found in numerous pathogenic e. coli strains [ ] . although its specific receptor remains unclear, extracellular matrix proteins (ecms), which comprise an interlocking mesh of fibrous proteins and glycosaminoglycans, may act as a receptor for lpf of enterohemorrhagic e. coli o :h (fig. ). ecms are modified by various carbohydrate moieties, and the addition of mannose inhibits lpf-ecm interaction. then mannose-containing carbohydrates may participate in bacterial adhesion by lpf [ ] . in some cases, type fimbriae are encoded on plasmids. such plasmids frequently encode virulence factors for host bacteria, and are therefore called "virulence plasmids" [ ] . pef is required for bacterial attachment to intestinal epithelial cells. it specifically binds to trisaccharide galβ - (fucα - )glcnac, also known as the lewis x (le x ) blood group antigen (fig. ) [ ] . the le x antigen is defined by the presence of a terminal galβ - (fucα- )glcnac moiety on saccharide chains of glycoproteins or glycosphingolipids; in the human intestine, it is expressed mainly in crypt epithelial cells [ ] . s. typhimurium possesses pef as an adhesin that binds to a crypt-specific histo-blood group antigen that may be relevant to the pathogenesis of human infections. abundant crypt abscesses are commonly found in s. typhimurium patients, raising the possibility that the pathogen may bind to human crypt epithelium at a later stage of infection. in a situation where peyer's patches are unavailable because of an inflammatory reaction, salmonella can colonize at the crypt epithelium remaining intact and persist on the surface of the host intestinal tract [ , ] . on the other hand, some type fimbriae participate in "fimbria-mediated (pilus-mediated) conjugal transfer" of so-called "conjugative plasmids". conjugative plasmids can also be virulence plasmids if they encode not only the structural genes of the fimbriae but also other virulence factors, such as a drug resistance gene. these conjugative plasmids spread to other bacteria by horizontal transfer, and type fimbriae encoded on the plasmids play an important role in this event. for example, the r plasmid, which encodes the pilv gene and engages in the adhesion of type fimbriae, recognizes the di-saccharide moiety of bacterial surface polysaccharides (the core oligosaccharide or o-antigen unit of lipopolysaccharides, a unique structure of the bacterial cell surface) and determines the recipient bacteria of the conjugal transfer [ , ] . categorized as π-fimbriae, the std fimbriae are well conserved among s. enterica serotypes but absent from related bacterial species (fig. ) . std fimbriae recognize and bind the h type histo-blood group oligosaccharide, the terminal fucα - galβ - glcnac moiety. this structure represents the h type oligosaccharide of the o blood group antigen [ ] . the h type oligosaccharide of the o blood group antigen moiety is expressed as part of the mucin-type sugar chains of glycoproteins in the host cell. the terminal fucα - moiety of h type oligosaccharide of the o blood group antigen is essential for the recognition of std fimbriae (fig. ) . carbohydrate molecules act not only as "anchors" for pathogens but also as the determinants of host and tissue specificity. the variety of adhesion factors carried by a bacterium reflects its pathogenic profile, magnitude of virulence, host specificity, and tissue specificity. in the case of salmonella and assortative bacteria, the fimh adhesins show amino acid sequence diversity. this diversity in fimh structure results in the variation in affinity profiles. e. coli fimh shows a high affinity for aromatic αmannosides as well as manα - structures. on the other hand, the fimh of salmonella species shows a high affinity for α-mannosides and a low affinity for aromatic αmannosides [ ] . in the case of salmonella, allelic variation of fimh adhesion directs not only host cellspecific recognition but also distinctive binding to mammalian and avian receptors. this allele-specific binding profile parallels the host specificity of the respective fimh-expressing pathogen [ ] . similarly, the lewis b (le b ) blood group phenotype in combination with secretor status may hinder colonization of helicobacter pylori in certain populations [ ] . h. pylori express blood group antigen b-binding adhesion (baba), and baba binds to le b antigens. salmonella and assortative bacteria contain various adhesion factors, including several kinds of fimbriae, which contribute to bacterial virulence; however, analyses of their specific receptor moieties and functions are not yet complete [ , ] . carbohydrate moieties on the surface of pathogens are also recognized by hosts and trigger host defense mechanisms. the bacterial surface is covered with various kinds of carbohydrates. for gram-negative bacteria, including salmonella, the major carbohydrate component of the bacterial surface is lipopolysaccharide (lps). lps is categorized as a glycolipid, and is a major component of the bacterial outer membrane. because the saccharide moieties of lps differ structurally from mammalian carbohydrates, they function as targets of the host immune response. to avoid this host immune response, the lps of some bacteria, for example c. jejuni, is structurally similar to the glycosphingolipids of gangliosides [ , ] . similarly, the lps of most h. pylori strains expresses the le a and le b antigens [ ] . interestingly, the carbohydrate on the surface of the k. kato et al. host cell itself can be involved in the host defense mechanism. the salmonella flagella component flic contributes to bacterial attachment to the host cell by interacting with ganglioside molecules on the surface of the host cell, but gangliosides also act as co-receptors for salmonella enterica flic and promote flic induction of the human innate immune response [ ] . gangliosides, i.e. sialic acidcontaining glycosphingolipids, are ubiquitous components of eukaryotic cell membranes that have been identified as receptors for bacterial toxins and viruses. an in vitro assay showed that a nonflagellated mutant of s. enteritidis, constructed by disrupting the flic gene, was about -fold less invasive than the wild-type strain, but bacterial adherence was unaffected [ ] . at the attachment of salmonella enteritidis flic to the host cell surface, gangliosides thus function as receptors. on the other hand, the flagella component protein flic induces the host innate immune response by binding to toll-like receptor of the host cell, and gangliosides react as co-receptors with tlr on the flic-induced response. an in vitro assay showed that the incorporation of exogenous ganglioside gd a into the caco- cell membrane increased the effect of flic. incubation of caco- cells with a glucosylceramide synthase inhibitor reduced the innate immune response stimulated by flic [ ] . human enteropathogenic protozoas include the apicomplexans toxoplasma gondii and cryptosporidium as well as giardia and entamoeba histolytica. they are all zoonotic pathogens that invade and colonize their target tissues in the alimentary tract of the human host. they form hard cysts that resist degradation in the stomach. host-derived proteases and low ph trigger their excystation [ ] . in this section, we describe the role of carbohydrates in toxoplasma gondii invasion of intestinal epithelial cells. the ability of t. gondii to infect chinese hamster ovary (cho) cells deficient in sialic acids was reduced by . % compared to wild-type cells, indicating that sialic acid is critical for attachment and invasion of t. gondii (fig. ) [ ] . t. gondii microneme protein (tgmic ) forms a macromolecular complex with tgmic and tgmic . single deletion of the tgmic gene significantly decreases the invasion of host cells, suggesting an essential role for tgmic in host cell attachment and invasion of t. gondii [ ] . structural analysis of tgmic revealed a novel cellbinding motif called microneme adhesive repeat region (marr), which provides a specialized structure for glycan discrimination [ ] . carbohydrate microarray analyses showed that tgmic , tgmic and its homologue neospora caninum mic share a preference for α - -over α - -linked sialyl-n-acetyllactosamine sequences [ ] . p , a pan/apple domain-containing protein expressed at the apical end of the extracellular parasite, functions as a ligand in the attachment of t. gondii to chondroitin sulfate and other receptors on the host cell, facilitating invasion by the parasite (fig. ) [ ] . t. gondii display gpi-anchored surface proteins identified as surface antigen glycoprotein (sag) related sequences (srs) [ ] . sag , sag a and sag have some capacity for host cell attachment through glycan recognition (fig. ) [ , ] . sag binds to sulfated proteoglycans such as heparin, fucoidan, and dextran sulfate with high affinity [ ] . targeted disruption of sag significantly reduces host cell binding of t. gondii [ ] . e. histolytica fibronectin receptor (ehfnr) shows % homology to the intermediate subunit- of the gal/ galnac-specific lectin [ ] . electron microscopy revealed the close association of a purified ehfnr complex to adhesion plates and phagocytic invaginations. lipid rafts participate in interactions between e. histolytica and the host extracellular matrix, and it appears that raft-associated gal/ galnac lectin serves as a collagen receptor [ ] . cryptosporidium parvum surface receptors, gp and proteolytic fragments of the -kda precursor protein, gp and gp , are characterized as mucin-like and heavily o-glycosylated proteins [ ] [ ] [ ] . the gp and gp of sporozoites and merozoites have carbohydrate residues that are bound by αgalnac-specific lectins, suggesting that αgalnac residues are involved in the attachment of parasites to host cells via adherence to internal mucus. apicomplexan protozoan parasites also induce host innate immune responses via the carbohydrate molecules present on their cell surface [ ] . glycosylphosphatidylinositol (gpi) protein anchors are abundant in the membranes of tachyzoites and other apicomplexan protozoan parasites including trypanosoma, leishmania and plasmodium spp., where they can serve as ligands for innate recognition [ ] . the gpi moieties of t. cruzi and p. falciparum were found to be tlr ligands [ , ] , and t. gondii both stimulate cytokine production in macrophages and serve as tlr as well as tlr agonists. in the case of t. gondii, gpi induces tnf-α production in macrophages through the activation of the transcription factor nf-κb [ ] . carbohydrates serve as receptors for infections by viruses, bacteria and protozoa, but the usage of carbohydrates by these microbes varies depending on the microbe. at the initial infection step, these organisms do not simply utilize the electrical forces created by the positive and negative charges of the carbohydrates; rather, they make use of other systems in certain instances. one similarity shared by all three microbes regarding their interactions with carbohydrates, however, is that heparan sulfate plays an important role at entry or invasion of the host cell. blood group antigen oligosaccharides are highly expressed in the gastrointestinal epithelium [ ] . however, there are individual differences in terms of the presence of these antigens. in addition, there are individual differences in sensitivity to pathogens that recognize and bind to blood group antigens, such as norovirus and h. pylori. these individual differences in antigen expression profiles benefit the survival of the host species because the risk of an attack by a fatal virulent pathogen may be decreased to avoid extinction. a large array of glycoproteins, glycolipids and proteoglycans decorate the surface of animal cells. these glycoconjugates mediate many fundamental cellular processes, including cell-cell and cell-matrix adhesion, motility, growth and signaling [ ] [ ] [ ] . mucosal tissues represent the site of infection or route of access for most parasites, including viruses, bacteria and protozoa [ ] . on the surface of the mucosal tissues of the gastrointestinal tracts, various carbohydrate moieties are present and play a crucial role in infection. mucosal surfaces are coated with a layer of viscous mucus that ranges in thickness from μm in the stomach to μm in the intestine [ ] [ ] [ ] . mucin glycoproteins from mucus-producing cells in the epithelium or submucosal glands are the major macromolecular constituent of mucus and are responsible for the viscous properties of the mucus gel. in addition to forming a relatively impervious gel, which acts as a lubricant, a physical barrier and a trap for microbes, mucus provides a matrix for a rich array of antimicrobial molecules. underneath the mucus layer, the cells present a dense forest of highly diverse glycoproteins and glycolipids, which form the glycocalyx. membrane-anchored cell-surface mucin glycoproteins are a major constituent of the glycocalyx in all mucosal tissues. the oligosaccharide moieties of the molecules that form the glycocalyx and the mucus layer are highly diverse, and the average turnover time of the human jejunal glycocalyx is - h [ ] . consequently, both the secreted and adherent mucosal barriers are constantly renewed and can rapidly adjust to changes in the environment, for example, in response to microbial infection. epithelial mucins are a heterogenous family of large complex glycoproteins containing a dense array of olinked carbohydrates typically comprising over % of their mass. the carbohydrate structures present on mucosal surfaces vary according to cell lineage, tissue location and developmental stage [ ] . mucin glycosylation can alter in response to mucosal infection and inflammation, and this may be an important mechanism for unfavorable changes in the niche occupied by mucosal pathogens. the o-linked glycans of muchin proteins contain - residues, which occur both as linear and branched structures [ ] . in addition to the o-linked glycans, mucins contain a smaller number of n-linked oligosaccharides, which have been implicated in folding, oligomerization (muc ) and surface localization (muc ) [ ] [ ] [ ] . the terminal structures of mucin oligosaccharides are highly heterogeneous k. kato et al. and vary between and within species as well as between and even within tissues. the array of oligosaccharide structures on individual mucin molecules is also somewhat determined by stochastic events as the mucin protein moves through the golgi apparatus [ ] . the secreted mucins themselves likely function as decoys for adhesins that have been evolved by pathogens to engage the cell surface, as the mucins express many of the oligosaccharide structures found on the cell surface and are constitutively produced in large amounts, constantly washing the mucosal surfaces [ ] . proteoglycans are present on the cell surface [ ] and are also components of glycocalyx. glycosaminoglycan chains are composed of highly sulfated saccharides that give the cell surface a potent negative charge. one of the prototypical membrane proteoglycans is syndecan- , which carries conserved attachment sites for glycosaminoglycan chains [ ] . the syndecans exemplify hybrid proteoglycans because they contain mixtures of the two major types of glycosaminoglycan chains, heparan sulfate and chondroitin sulfate. the other major family of membrane proteoglycans is the glypicans, which contain gpi anchors in a tissue-specific and temporally regulated manner. their presence in the basolateral membranes of polarized cells varies [ ] . glycolipids are also a component of the cell membrane. a large variety of glycolipids is present on the surface of animal cells. the carbohydrate moieties vary, and each glycolipid may exhibit a special function, as an annular lipid, surface receptor marker or matrix lipid. for brain and neuronal cells, gangliosides (sialic acid-bearing glycolipids) are the major cell surface determinants [ ] . glycolipids function as the receptor for various biologic factors and also as the receptor for various pathogens. they are present at the undermost part of the glycocalyx. pathogens can recognize the glycolipids, directly bind to the cell membrane, and invade the host cell. glycolipids also function as receptors for certain effector molecules, such as bacterial toxins, produced by pathogens and directly react with the host cell. for example, cholera toxin binds to ganglioside gm [ ] . thus, for pathogens living in the outer mucus layer, it is difficult to make contact with the surface of normal epithelial cells because of the huge amount of mucin that functions as a "decoy" or "physical barrier". mucosal pathogens have, therefore, developed mechanisms to subvert these defense mechanisms of the mucosal layer. on the other hand, intestinal m cells, specifically designed to capture and present microbes to the underlying lymphoid tissue, can be regarded as a "hole" in the mucin barrier. the dome epithelium lacks goblet cells and therefore does not produce gel-forming mucins. their apical cell surface has only sparse microvilli and an apparently thin glycocalyx [ , ] . m cells are specialized epithelial antigen-transporting cells that constitute a minor proportion ( %~ % in humans and mice) of the follicle-associated epithelium that covers the lymphoid follicles of organized gut-associated lymphoid tissue such as peyer's patches [ ] [ ] [ ] [ ] . glycoprotein (gp ) was identified as an m cell-specific molecule [ ] . the gp expressed on m cells functions as a bacterial uptake receptor [ ] . gp recognizes fimh, a major component of the type fimbriae, which binds to certain glycoproteins on mammalian cells in a mannosedependent manner [ ] . consequently, even though m cells constitute only a very small percentage of mucosal epithelial cells, they are the major point of attachment and/or entry used by numerous mucosal pathogens including bacteria (e.g., s. typhimurium, shigella flexneri, yersinia enterocolitica and vibrio cholerae), viruses (e.g., reovirus, hiv- and polio virus) and parasites (e.g., cryptosporidia) [ , , ] . during cell-pathogen interactions (i.e., infection and/or invasion), carbohydrates function as receptors for various pathogens. on the other hand, carbohydrates (glycoconjugates) can also function as a barrier to infection. on the surface of mucosal tissue, the glycocalyx physically prevents microbes from accessing the cell membrane. some glycoconjugates, a component of the glycocalyx, contain carbohydrate structures that are recognized by pathogens. mucins often contain oligosaccharide moieties that correspond to the receptor for various pathogens. on the surface of the mucosal layer, microbes binds to these receptor moieties and are captured at the mucus layer, which consequently blocks the infection. moreover, when secretory mucins containing receptor carbohydrate structures "trap" pathogens, the pathogens are also carried away. m cells are specialized epithelial antigentransporting cells scattered in the follicle-associated epithelium that covers the gut lymphoid follicles such as peyer's patches. m cells can efficiently engulf particles as large as bacteria; however, the mucus layer of m cells and the surrounding area is relatively thin. glycoconjugates such as gp are expressed on the surface of m cells and function as receptors for bacterial attachment [ ] . in the case of the host-parasite interaction, the various kinds of glycoconjugates sometimes function as receptors for the invading pathogens, but they can also function as barriers tropical medicine and health vol. no. , and traps for the host defense system. in recent years, the damage caused by enteric pathogens, especially norovirus and salmonella, has expanded through the food chain [ , , ] . these pathogens cause food poisoning in humans and gastrointestinal diseases in animals all over the world. even today, they are often responsible for large-scale outbreaks of food poisoning. therefore, the prevention and treatment of infections caused by these pathogens is essential. in this review, we discussed the interaction between host cells and microbes such as viruses, bacteria and protozoa that involve carbohydrates such as sialic acids, heparan sulfate, and the carbohydrate moieties of abh and lewis antigens, mannose components, ecms and le x . the development and use of drugs that target these carbohydrates is anticipated, even though the microbes vary widely and have different modes of infection. accordingly, when an anti-microbial drug is developed on the basis of the interaction between a microbe and a carbohydrate, host cell modification of the drug's structure and/or inhibition of the mode of infection will need to be individualized while still taking advantage of the similarities between interactions. moreover, the host gastrointestinal tract cell surface, which is the object of microbial infection, is composed of glycoproteins, glycolipids, and proteoglycans. these molecules are potential targets for carbohydrate drugs used in the treatment of infectious diseases. oseltamivir and zanamivir are neuraminidase inhibitors that competitively inhibit the activity of the viral neuraminidase on the sialic acid that is found on glycoproteins on the surface of host cells [ ] . by blocking the activity of this enzyme, they prevent new viral particles being released from infected cells. there are various kinds of polysaccharides on the surface of bacteria. lipoteichoic acid (lta), a type of glycolipid, is a component of the bacterial cell wall of grampositive bacteria. studies have shown that lta stimulates the immune system [ , ] . recently, lta has been studied for use as a novel kind of biologically active substance. recently, sulfated polysaccharides have been analyzed as drug candidates for protozoan infectious diseases [ , , ] . according to our data, the sulfated positions in the carbohydrates can be critical for the inhibitory quality [ ] . collectively, these studies highlight the possibility that carbohydrate drugs may be developed for the prophylaxis and treatment of parasitic infectious diseases. the results of our studies highlight the possibilities for countermeasures against malaria and toxoplasmosis [ , ] . heparan sulfate glycosaminoglycans as primary cell surface receptors for herpes simplex virus salmonella enterica serovar typhimurium requires the lpf, pef, and tafi fimbriae for biofilm formation on hep- tissue culture cells and chicken intestinal epithelium analyses of interactions between heparin and the apical surface proteins of plasmodium falciparum norovirus disease: changing epidemiology and host susceptibility factors human susceptibility and resistance to norwalk virus infection noroviruses distinguish between type and type histo-blood group antigens for binding norovirus and histoblood group antigens: demonstration of a wide spectrum of strain specificities and classification of two major binding groups among multiple binding patterns junctional adhesion molecule is a functional receptor for feline calicivirus alpha , -linked sialic acid acts as a receptor for feline calicivirus the role of m cells in salmonella infection sialic acid is required for nonspecific adherence of salmonella enterica ssp. enterica serovar typhi on caco- cells contribution of fimbrial operons to attachment to and invasion of epithelial cell lines by salmonella typhimurium binding specificity of salmonella plasmid-encoded fimbriae assessed by glycomics salmonella enterica serotype typhimurium std fimbriae bind terminal alpha( , )fucose residues in the cecal mucosa salmonella enterica serovar typhi uses type ivb pili to enter human intestinal epithelial cells multiple fimbrial adhesins are required for full virulence of salmonella typhimurium in mice salmonella enterica serovar typhimurium binds to hela cells via fim-mediated reversible adhesion and irreversible type three secretion system -mediated docking direct evidence that the fimh protein is the mannose-specific adhesin of escherichia coli type fimbriae carbohydrates as future anti-adhesion drugs for infectious diseases role of intimin and bundle-forming pili in enteropathogenic escherichia coli adhesion to pediatric intestinal tissue in vitro the lpf fimbrial operon mediates adhesion of salmonella typhimurium to murine peyer's patches initial adherence of epec, ehec and vtec to host cells long polar fimbriae of enterohemorrhagic escherichia coli o :h bind to extracellular matrix proteins pathogenomics of the virulence plasmids of escherichia coli thin pilus pilv adhesins of plasmid r recognize specific structures of the lipopolysaccharide molecules of recipient cells pilv adhesins of plasmid r thin pili specifically bind to the lipopolysaccharides of recipient cells fimh alleles direct preferential binding of salmonella to distinct mammalian cells or to avian cells role of lewis a and lewis b blood group antigens in helicobacter pylori infection chemical structures of the core regions of campylobacter jejuni serotypes o: , o: , o: , and o: lipopolysaccharides expression of the human cell surface glycoconjugates lewis x and lewis y by helicobacter pylori isolates is related to caga status gangliosides act as co-receptors for salmonella enteritidis flic and promote flic induction of human beta-defensin- expression in caco- cells inactivation of the flagellin gene of salmonella enterica serotype enteritidis strongly reduces invasion into differentiated caco- cells active invasion and/or encapsulation? a reappraisal of host-cell parasitism by cryptosporidium host cell surface sialic acid residues are involved on the process of penetration of toxoplasma gondii into mammalian cells synergistic role of micronemal proteins in toxoplasma gondii virulence atomic resolution insight into host cell recognition by toxoplasma gondii members of a novel protein family containing microneme adhesive repeat domains act as sialic acid-binding lectins during host cell invasion by apicomplexan parasites a novel pan/apple domain-containing protein from toxoplasma gondii: characterization and receptor identification the surface of toxoplasma: more and less targeted disruption of the glycosylphosphatidylinositolanchored surface antigen sag gene in toxoplasma gondii decreases host cell adhesion and drastically reduces virulence in mice toxoplasma gondii: the role of parasite surface and secreted proteins in host cell invasion the surface anti- gen sag mediates the attachment of toxoplasma gondii to cell-surface proteoglycans physical, structural, and functional properties of the beta integrin-like fibronectin receptor (beta ehfnr) in entamoeba histolytica entamoeba histolytica: lipid rafts are involved in adhesion of trophozoites to host extracellular matrix components a novel multidomain mucin-like glycoprotein of cryptosporidium parvum mediates invasion mediation of cryptosporidium parvum infection in vitro by mucin-like glycoproteins defined by a neutralizing monoclonal antibody cloning and sequence analysis of a highly polymorphic cryptosporidium parvum gene encoding a -kilodalton glycoprotein and characterization of its -and -kilodalton zoite surface antigen products toll-like receptor recognition of toxoplasma gondii signaling of immune system cells by glycosylphosphatidylinositol (gpi) anchor and related structures derived from parasitic protozoa impaired production of proinflammatory cytokines and host resistance to acute infection with trypanosoma cruzi in mice lacking functional myeloid differentiation factor induction of proinflammatory responses in macrophages by the glycosylphosphatidylinositols of plasmodium falciparum: cell signaling receptors, glycosylphosphatidylinositol (gpi) structural requirement, and regulation of gpi activity activation of tlr and tlr by glycosylphosphatidylinositols derived from toxoplasma gondii tissue distribution of histo-blood group antigens genetic analysis of proteoglycan structure, function and metabolism microbial adherence to and invasion through proteoglycans biological roles of oligosaccharides: all of the theories are correct mucins in the mucosal barrier to infection the adherent gastrointestinal mucus gel layer: thickness and physical state in vivo a novel method for the visualization of the in situ mucus layer in rat and man colonic mucin: methods of measuring mucus thickness functional morphology of the mucosa of the small intestine n-glycosylation is required for the surface localization of muc mucin characterization of the oligosaccharides associated with the human ovarian tumor marker ca roles of calreticulin and calnexin during mucin synthesis in ls and ht /a human colonic adenocarcinoma cells kinetic modeling confirms the biosynthesis of mucin core (beta-gal( - ) alpha-galnac-o-ser/thr) o-glycan structures are modulated by neighboring glycosylation effects biology of the syndecans: a family of transmembrane heparan sulfate proteoglycans integral membrane heparan sulfate proteoglycans glycolipids at the cell surface and their biological functions studies on the molecular mechanism of anion transport across the red blood cell membrane the composition and function of m cell apical membranes: implications for microbial pathogenesis m cells and the pathogenesis of mucosal and systemic infections epithelial m cells: differentiation and function glycoprotein (gp ): grabbing the fimh bacteria into m cells for mucosal immunity uptake and transport of intestinal macromolecules and microorganisms by m cells in peyer's patches -a personal and historical perspective structure and functon of intestinal mucosal eptihelium uptake through glycoprotein of fimh(+) bacteria by m cells initiates mucosal immune response bacterial adhesion and entry into host cells entry of microbes into the host: using m cells to break the mucosal barrier cellular routes of invasion by enteropathogens salmonella and produce: survival in the plant environment and implications in food safety neuraminidase inhibitors for influenza b virus infection: efficacy and resistance lipotechoic acid in lactobacilli: d-alanine makes the difference structure-function relationship of cytokine induction by lipoteichoic acid from staphylococcus aureus effects of dextran sulfates on the acute infection and growth stages of toxoplasma gondii gellan sulfate inhibits plasmodium falciparum growth and invasion of red blood cells in vitro this study was supported by grants-in-aid for young scientists, and scientific research on innovative areas ( ) from the ministry of education, culture, science, sports, and technology (mext) and for research on global health issues from the ministry of health, labour and welfare of japan, the program for promotion of basic and applied researches for innovations in bio-oriented industry (brain), the science and technology research promotion program for agriculture, forestry, fisheries and food industry, the naito foundation, and the program to disseminate tenure tracking system from the japan science and technology agency (jst). we thank mr. tatsuya iwanaga for his help with the illustrations. key: cord- -fl l b authors: daryabor, gholamreza; atashzar, mohamad reza; kabelitz, dieter; meri, seppo; kalantar, kurosh title: the effects of type diabetes mellitus on organ metabolism and the immune system date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: fl l b metabolic abnormalities such as dyslipidemia, hyperinsulinemia, or insulin resistance and obesity play key roles in the induction and progression of type diabetes mellitus (t dm). the field of immunometabolism implies a bidirectional link between the immune system and metabolism, in which inflammation plays an essential role in the promotion of metabolic abnormalities (e.g., obesity and t dm), and metabolic factors, in turn, regulate immune cell functions. obesity as the main inducer of a systemic low-level inflammation is a main susceptibility factor for t dm. obesity-related immune cell infiltration, inflammation, and increased oxidative stress promote metabolic impairments in the insulin-sensitive tissues and finally, insulin resistance, organ failure, and premature aging occur. hyperglycemia and the subsequent inflammation are the main causes of micro- and macroangiopathies in the circulatory system. they also promote the gut microbiota dysbiosis, increased intestinal permeability, and fatty liver disease. the impaired immune system together with metabolic imbalance also increases the susceptibility of patients to several pathogenic agents such as the severe acute respiratory syndrome coronavirus (sars-cov- ). thus, the need for a proper immunization protocol among such patients is granted. the focus of the current review is to explore metabolic and immunological abnormalities affecting several organs of t dm patients and explain the mechanisms, whereby diabetic patients become more susceptible to infectious diseases. the metabolic syndrome is defined by the presence of metabolic abnormalities such as obesity, dyslipidemia, insulin resistance, and subsequent hyperinsulinemia in an individual ( ) . dyslipidemia, the main characteristic of metabolic syndrome, is defined by decreased serum levels of high-density lipoproteins (hdls) but increased levels of cholesterol, free fatty acids (ffas), triglycerides (tg), vldl, small dense ldl (sdldl), and oxidized ldl (ox-ldl) ( table ) ( ) . individuals with the metabolic syndrome are much more likely to develop type diabetes mellitus (t dm), cardiovascular diseases (cvds), and fatty liver disease ( ) ( ) ( ) . t dm, the most common form of diabetes (∼ %), is characterized by a systemic inflammatory disease accompanied by insulin resistance (ir) or decreased metabolic response to insulin in several tissues, including the adipose tissue, liver, and skeletal muscle, as well as by reduced insulin synthesis by pancreatic beta cells ( , ) . studies on immunometabolism have indicated that the metabolic states and immunological processes are inherently interconnected ( ) . in this scenario, metabolites derived from the host or microbiota regulate immunological responses during health and disease ( ) . accordingly, in obese individuals, expanded adipose tissue at different locations, by initiating and perpetuating the inflammation, induces a chronic low-level inflammatory state that promotes ir ( ). every organ system in human body can be affected by diabetes, but the extent of organ involvement depends largely on the severity and duration of the disease (figure and table ). during the progression of diabetes, hyperglycemia promotes mitochondrial dysfunction and induces the formation of reactive oxygen species (ros) that cause oxidative stress in several tissues such as blood vessels and pancreatic beta cells ( ) ( ) ( ) . accumulating damage to the mitochondria, as well as several macromolecules, including proteins, lipids, and nucleic acids by ros promotes the process of aging ( ) . as a result, pancreatic β cells that require functional mitochondria to maintain insulin synthesis fail to generate high enough levels of insulin ( , ) . in the absence of compensatory mechanisms, stress-responsive intracellular signaling molecules are activated and cellular damage occurs. elevated intracellular levels of ros and subsequent oxidative stress play an important role in the pro-atherosclerotic consequences of diabetes and the development vascular complications ( , ) . moreover, the non-enzymatic covalent attachment of glucose and its toxic derivatives [e.g., glyoxal, methylglyoxal (mgo), and deoxyglucosone] to the biological macromolecules such as nucleic acids, lipids, and proteins leads to the formation of advanced glycation end products (ages) ( , ) . accumulated ages block the insulin signaling pathway and promote inflammation ( , ) . in addition, the attachment of ages to their receptors [e.g., cd , galectin- , scavenger receptors types i (sr-a ), and ii (sr-a )] on the surfaces of immune cells in the circulation and tissues activates the expression of pro-inflammatory cytokines and increases free radical generation ( ) . furthermore, due to the chronic exposure of cells to high glucose levels in untreated t dm patients, glucose toxicity might occur in several organs. this will figure | effects of t dm on body organs. t dm is an inflammatory state that affects circulatory system, gastrointestinal tract, pancreatic beta cells, liver, and skeletal muscles and makes them dysfunctional. nfald, non-alcoholic fatty liver disease; nash, non-alcoholic steatohepatitis; er, endoplasmic reticulum. eventually lead to nephropathy, cardiomyopathy, neuropathy, and retinopathy. gut microbiome dysbiosis is another important factor that can facilitate the induction and progression of metabolic diseases such as t dm ( ) . the gut microbiome dysbiosis, by altering the barrier functions of intestine and the host metabolic status, promotes the insulin resistance in diabetic patients ( ) . diabetes also impairs the immune system and increases the susceptibility of patients to serious and prolonged infections ( ) . this is likely to be the case with the severe acute respiratory syndrome coronavirus (sars-cov- ), as well ( , ) . in the current paper we will review recent research to explore the impairment of body organs in t dm patients and explain how diabetic patients become more susceptible to certain infectious diseases. vascular homeostasis is an important function of the endothelium. under homeostatic conditions, the ecs maintain the integrity of blood vessels, modulate blood flow, deliver nutrients to the underlying tissues, regulate fibrinolysis and coagulation, control platelet adherence and patrol the trafficking of leukocytes (figure a ) ( ) . normal ecs also internalize high-density lipoproteins (hdls) and its main protein part apolipoprotein a-i (apoa-i) in a receptor-mediated manner to activate endothelial cell nitric oxide (enos) synthase and promote anti-inflammatory and antiapoptotic mechanisms ( figure b ) ( ) . hdl receptors on the surfaces of ecs include: the atp-binding cassette (abc) transporters a and g , the scavenger receptor (sr)-b and the ecto-f -atpase ( ) . according to the epidemiological studies, diabetes mellitus is considered as one of the main risk factors for cvd (figure ) ( ) . from the beginning of t dm, the functions of ecs are impaired, which is the main cause of disease-related side-effects ( ) . ecs can initiate and perpetuate the inflammatory milieu during the pathogenesis of diabetes. due to the negative impacts of hyperglycemia and subsequent oxidative stress, cvds are more common among diabetic patients ( ) . it has been observed that incubation of human aortal endothelial cells (haecs) with a medium containing high glucose concentrations (hg, mm) increases the intracellular levels of mgo and glycated proteins that in turn activate the unfolded protein response (upr) and trigger inflammatory and prothrombotic pathways ( ) . glycated apoa-i, which is formed during hyperglycemia, modifies its structure, decreases its lipid-binding ability, prevents cholesterol efflux from macrophages and impairs its anti-inflammatory function ( , ) . vaisar et al. have shown that hdls from diabetic patients have a reduced capacity to trigger enos production and suppress tumor necrosis factor-α (tnf-α)mediated inflammatory responses within ecs ( ) . diseases such as t dm that induce high levels of vascular injury are accompanied by an elevated number of circulating endothelial cells (cecs) ( ) . t dm-related risk factors such as dyslipidemia, hyperglycemia, and hyperinsulinemia as well as other conditions (e.g., inadequate physical activity, smoking, and high blood pressure) facilitate the formation of atherosclerotic plaques/lesions ( ) . dyslipidemia, due to the elevated flux of ffa from insulin-resistant tissues and spillover from entry (c) blood vessels in t dm patients. during the progression of the disease, red blood cells become glycated, while activated ecs synthesize elevated levels of adhesion molecules and chemokines that facilitate monocytes recruitment, adhesion, and transmigration across the endothelium toward the subendothelial region. monocytes are then differentiated into macrophages and eventually, by excess lipid uptake, generate foam cells. subsequently, further immune cell infiltration into the atherosclerotic lesion occurs, where their inflammatory cytokines promote platelet activation, ec apoptosis, and increased generation of ros and ox-ldl. (d) interactions between oxldl and its receptor aggravate ros generation, nf-κb activation and inflammation. ec, endothelial cell; rbc, red blood cell; plt, platelet; hdl, high-density lipoprotein; ox-ldl, oxidized low-density lipoprotein; ros, reactive oxygen species; enos, endothelial nitric oxide synthase; no, nitric oxide; lox- , lectin-type oxidized ldl receptor . into adipocytes, is considered as an important risk factor for developing cvd among diabetic patients. this is because dyslipidemia promotes inflammation, endothelial dysfunction, and platelet hyperactivation ( , ) . during the progression of atherosclerosis, lipids, immune cells, and extracellular matrix accumulate in the arterial intima or subendothelial regions ( figure c ) ( ) . advanced plaques can impede blood flow and cause tissue ischemia or might become disrupted and generate a thrombus that stops the blood flow of important organs. vascular complications of diabetes engage either tiny or large blood vessels (micro-and macroangiopathy, respectively). microangiopathies, which can be seen in the kidneys, vasa nervorum and eye tissues, cause nephropathy, neuropathy, and retinopathy. macroangiopathies, by inducing atherosclerosis in the coronary, carotid, and peripheral arteries, increase the risk of myocardial infarction (mi), stroke and peripheral artery disease (pad). macrovascular complications due to ec dysfunction are considered as an important cause of mortality and morbidity among diabetic patients ( ) . oxidative stress has an essential role in the induction of vascular complications during the course of diabetes ( ) . ec dysfunction (e.g., delayed replication, dysregulated cell cycling, and apoptosis), as well as enhanced ox-ldl formation are some consequences of oxidative stress. it has been well-established that sdldl and ox-ldl have an enhanced atherogenic ability and are more useful biomarkers than total ldl for predicting cvd ( , ) . sdldl particles have a smaller size than other ldl particles. thus, sdldl particles are more easily oxidized, and their atherogenic potential is enhanced. during oxidative stress, levels of ox-ldl increase by the excess action of reactive oxygen species (ros) ( ) . subsequently, ox-ldl interaction with scavenger receptors, including cd , sr-a /cd , sr-b , and lectin-like ox-ldl receptor- (lox- ) on the surface of ecs activates the nadph oxidase that in turn increases the expression of ros and activates the transcription factor nf-αb ( ) . afterwards, the expression of lox- , adhesion molecules (e.g., selectins and integrins) and the secretion of pro-inflammatory cytokines and chemokines are increased, while no synthesis is decreased in ecs ( figure d ) ( ) ( ) ( ) . ec-derived chemokines bind to their cognate receptors on the surfaces of monocytes and recruit them toward the inflamed endothelium. following this, selectin-based rolling and integrin-based attachment of monocytes to the ecs cause their migration toward the subendothelial region, where they develop into lipid-laden macrophages or foam cells later on ( ) . the scavenger receptor lox- plays an important role in the uptake of ox-ldl during atherogenesis. it is strongly expressed on the surfaces of ecs, but has an inducible pattern of expression on the surface of macrophages and smooth muscle cells ( ) . the accelerated uptake of ox-ldl by macrophages accounts for their transformation into foam cells, the initial hallmark of atherosclerosis ( , ) . besides, diabetes leads to both quantitative and qualitative defects in circulating angiogenic progenitor cells (capcs) that take part in the repair of injured endothelium ( ) . it has been shown that humans or mice with decreased numbers of cd + cd + cd + cd dim sca- + flk- + capcs have an increased prevalence of t dm, elevated hba c levels and aggravated cvd risk scores ( , ) . in diabetic patients, despite elevated serum levels of proangiogenic molecules, like angiopoietin- / , epo, and vegf-a, angiogenesis is impaired. this is mainly due to the decreased expression levels of vegfr and cxcr on the surfaces of capcs, which makes them unresponsive to the angiogenic factors ( , ) . it has also been shown that circulating proangiogenic granulocytes composed of eosinophils and neutrophils are also impaired in diabetic patients ( ) . besides, elevated levels of ages in t dm cause ec dysfunction and vascular inflammation ( ) . ren et al. have shown that incubation of human coronary artery endothelial cells (hcaecs) with ages causes decreased expression (at both mrna and protein levels) and enzymatic activity of enos, increased levels of ros, diminished mitochondrial membrane potential and declined activity of catalase and superoxide dismutase in treated cells ( ) . another study by lan et al. has shown that ages in the pancreas decrease ec viability and induce their apoptosis in an nfκb signaling-related manner ( ) . however, apigenin ( ′ , , trihydroxyflavone) can protect ecs against oxidative stress and subsequent inflammatory reactions mediated by ages ( ) . apigenin binds to methylglyoxal (mgo) and forms a complex that inhibits age formation. chettab et al. have shown that the expression of icam- as well as the production of il- , are significantly increased in huvecs cultured in hg medium compared to cells cultured in normal glucose (ng, . mm) conditions ( ). bammert et al. found out that incubation of huvecs with hg media promotes the generation of endothelial microparticles (emps) that, when added to normally cultured huvecs, downregulate the expression of anti-apoptotic microrna mir-let a, but enhance the synthesis of active caspase- and cause cell apoptosis ( ) . several micrornas, including mir- , mir- a, mir- , mir- a, mir- , mir- , mir- a, mir- , and mir-let d, regulate vascular homeostasis. it has been shown that the expressions of mir- a and mir- are significantly reduced in circulating mps isolated from diabetic patients compared with normal individuals. this could be involved in making diabetic individuals more susceptible to coronary heart disease ( ) . moreover, hg media upregulate the expression of nadph oxidase that will induce the generation of ros. this leads to subsequent apoptosis of the huvecs through a ros-dependent caspase- pathway ( ). su et al. have demonstrated that argirein medication, by inactivating nadph oxidase, can prevent endothelial cell apoptosis in a rat model of t dm and hence attenuate vascular dysfunction ( ) . hg further increases the permeability of the huvecs in a protein kinase c (pkc)-dependent manner ( , ) . hassanpour et al. showed that incubation of endothelial progenitor cells with the serum of t dm patients inhibits their migration toward bfgf, increases their expression of vegfr- , but reduces their expression of vegfr- and induces their apoptosis ( ) . however, humanin (hn), a mitochondriumderived peptide, is cytoprotective against apoptosis during pathological conditions, such as diabetes mellitus ( ) . it has been demonstrated that simultaneous incubation of h c cells, a line of rat cardiac myoblasts, with h o and hn decreases the intracellular levels of ros, preserve mitochondrial function/structure and decline cellular apoptosis ( ) . wang et al. have indicated that the treatment of huvecs with hn before their incubation with hg medium increases the expression of enos, while decreasing the expression of endothelin (et- ), vcam- , tnf-α, il- β, and e-selectin in a krüppellike factor (klf )-dependent manner. such changes in the expression of integrins prevent the attachment of monocytes to huvecs ( ) . accordingly, hn might be used to prevent the development of hyperglycemia-associated ec dysfunction in t dm. ec activation and expression of adhesion molecules also facilitate activation and adhesion of platelets. this will increase the risk of thrombosis and promote the development of thrombotic angiopathy, typical for diabetic patients. platelets are tiny anucleated cellular fragments generated from megakaryocytes in the bone marrow. they circulate in the blood for ∼ - days and play essential roles in hemostasis and in controlling vascular integrity ( ) . circulating inactive platelets move in the proximity of vessel walls (figure a ) and rapidly get activated in response to vascular injury. at the end of their life, platelets are cleared from circulation with the action of the liver and spleen-resident macrophages. platelets have an essential role in the initiation and progression of inflammation. platelet hyperactivation that occurs during inflammatory states (e.g., t dm) facilitates the pathogenesis of cvds ( figure c ) ( , ) . it has been shown that elevated levels of resistin, an adipokine, in diabetic patients enhances oxidative stress, promotes endothelial dysfunction and facilitates platelet activation ( ) . activated platelets with an increased mean volume [mean platelet volume (mpv)] secrete microparticles (mps) and soluble adhesion molecules (e.g., sp-selectin and scd l) that in turn activate endothelial and immune cells ( ) ( ) ( ) . higher levels of platelet-derived mps, which correlate positively with fasting blood sugar and glycated hemoglobin, have been shown in newly diagnosed t dm patients compared to healthy individuals ( ) . in t dm patients thrombotic microangiopathies can lead to the development of cvds ( ) . platelets in the patients adhere to ecs and aggregate more rapidly than in healthy individuals thereby increasing the risk of thrombosis. in a mouse model of t dm, zhu et al. have shown that ages interact with cd , a member of the type scavenger receptor family, on the surfaces of murine platelets to activate them and induce a prothrombotic state ( ) . elevated levels of the p y receptor on the surface of platelets in t dm expose diabetic patients to a prothrombotic condition. this receptor has an essential role in platelet activation ( ) . zhou et al. have shown that long non-coding rna (lncrna) metallothionein pseudogene (mt p ), which is markedly upregulated in megakaryocytes of t dm patients, enhances the expression of p y receptor in platelets ( ) . they indicated that this is due to the inhibitory action of mt p on mir- . virtually all parts of the human digestive system, including the gastrointestinal tract, pancreas, and the liver are affected by diabetes. the git is populated with a myriad of microorganisms, including principally bacteria but also archaea, viruses, fungi, and protozoans that dynamically influence the health status and homeostasis of the host. the physiological functions of the git resident microbes improve gut integrity, protect against microbial pathogens and regulate immune responses ( ) . mucosal barriers, such as intestinal epithelial cells (iecs) and the mucus layer, spatially isolate the host immune system and gut microbiota to prevent unnecessary immune activation and intestinal inflammation. they also facilitate the uptake of nutrients through receptors and transporters. however, hyperglycemia, in a glut -dependent manner, can influence the mucus and alter the integrity of adherence and tight junctions between intestinal epithelial cells of diabetic mice. this will enhance the permeability of the intestinal barrier leading to so called "leaky gut." subsequently, hyperglycemia may facilitate the dispersal of an enteric infection into a systemic infection (figure ) ( ) . interestingly, the reversal of hyperglycemia, conditional deletion of glut from the iecs and inhibition of glucose metabolism will fix the barrier dysfunction and prevent the spread of bacteria ( ). xu et al. have shown that faecalibacterium prausnitzii, one of the most frequent commensal bacteria in normal individuals with essential roles in gut homeostasis, generates anti-inflammatory molecules that enhance the expression of tight junctions and improve intestinal integrity during diabetes ( ) . however, in some cases, gut microbiota dysbiosis or altered microbial composition of the intestines could induce t dm and lead to its progression ( ) . of interest, the widely used antidiabetic drug metformin can improve barrier integrity and restore the healthy microbiota composition of the gut in diabetic patients ( ) . the intestinal commensal bacterium akkermansia muciniphila can also act as a sentinel to reduce microbial translocation across the gut and prevent the subsequent inflammation in patients with t dm ( ) . hyperglycemia can further decrease the intracellular levels of glutathione (gsh) but increase inos activity and no production in the iecs ( ). zhao et al. have found out that hyperglycemia in a pkcα-dependent manner inhibits the ubiquitination, internalization and degradation of the divalent metal transporter (dmt ) present on the microvillar membranes of iecs. subsequently, intestinal iron uptake is enhanced and accumulated iron ions aggravate diabetes-related complications and increase mortality ( , ) . the pancreas consists of the exocrine and endocrine compartments. the endocrine part is made of different cell types, including α, β, δ, and ε cells that secrete glucagon, insulin, somatostatin, and ghrelin hormones, respectively. these cells are aggregated into specialized structures called islets of langerhans, which play an important role in controlling blood glucose levels through the secretion of insulin and glucagon. in t dm, despite normal levels of β-cell replication and islet formation, β-cell apoptosis is increased so that the number of cells declines by ∼ % (figure ) ( ) . during the progression of t dm, the insulin-resistant state forces β-cells to compensate for the lack of insulin by elevating its synthesis to restore the normal blood glucose level. however, in severe diabetic patients, β-cell exhaustion, and subsequent persistent hyperglycemia occur ( ) . furthermore, chronic elevated serum levels of free fatty acids, seen in obesity and t dm, induce lipotoxicity in beta-cells and suppress their insulin secretion ability ( ) . to alleviate chronic inflammation, overcome insulin resistance (ir) and to prevent β-cell apoptosis, stem cells or stem cell derivatives such as insulin-producing cells (ipcs) and exosomes have been suggested ( ) ( ) ( ) ( ) . their effects are believed to be mainly due to their anti-inflammatory activities. secretagogin (scgn) is predominantly expressed by pancreatic β-cells protecting their normal functions. scgn also acts as an insulin binding protein to make it more stable, avoid its aggregation, improve its functions and enhance its secretion ( , ) . in t dm patients, due to the islet cell dysfunction and endoplasmic reticulum (er) stress, serum levels of scgn are elevated reflecting stress and dysfunctional islet cells ( ) . moreover, in patients with t dm, islet amyloid polypeptide (iapp or amylin), a peptide hormone and one of the main secretory products of pancreatic β-cells, tends to deposit in the islets of langerhans, form insoluble fibrils and impair secretory functions of β-cells ( ) . iapp is costored with insulin in the secretory granules of pancreatic β cells. in steady-state conditions it regulates food intake, insulin secretion, and glucose metabolism ( ). ribeiro et al. have noted that pancreatic extracellular vesicles (evs) from healthy individuals, but not from t dm patients, directly bind to iapps and prevent amyloid formation within the pancreatic islets ( ) . the authors showed that the altered protein-lipid composition of the evs is the main reason for this discrepancy ( ) . however, chatterjee et al. have shown that β-cells from t dm patients have a dysfunctional proteasome complex that fails to degrade pancreatic iapp, whereby amyloid formation is induced ( ) . furthermore, in t dm patients, lipids accelerate the formation of fibrillary iapp, which aggravates islet cell damage ( ) . dhar et al. have demonstrated that chronic use of mgo in sprague-dawley rats increases the expression of nf-αb, mgo-derived ages and their receptors in pancreatic β cells. mgo can also induce apoptosis of islet β cells, increase fasting plasma glucose levels and impair glucose tolerance ( ) . in t dm patients the plasma level of mgo directly correlates with fasting blood sugar and hba c levels ( ) . bo et al. further showed that mgo in a dose-based manner impairs insulin secretion of pancreatic β-cell lines min and ins- through increased generation of ros and by induction of mitochondrial dysfunction ( ). robertson et al. have found out that elevated levels of ros in pancreatic β-cells inhibit the pancreas duodenum homeobox- (pdx- ) transcription factor that is needed for insulin synthesis ( ) . it has been shown that chronic use of mgo in animals could induce t dm, while simultaneous use of alagebrium, which breaks age compounds, attenuates the disease ( ) . it has also been reported that during the course of diabetes dedifferentiation and conversion of β-cells into αand δ-"like" cells occurs ( ) . in conclusion, the pancreatic β cell function is progressively reduced during the progression of t dm. the liver is by far the most important metabolic organ with essential roles in regulating homeostasis and mediating glucose and lipid metabolism. metabolic activities of the tissue are precisely controlled by the actions of metabolic substrates, including free fatty acids (ffas) and hormones ( ) . t dm patients usually suffer from a chronic liver condition called non-alcoholic fatty liver disease (nafld). it is characterized by steatosis that means ectopic fat storage in hepatocytes and subsequent insulin resistance (figure ) ( ) . lipid accumulation in hepatocytes leads to impaired biogenesis of mir- that facilitates insulin signaling and prevents lipogenesis ( ) . several factors such as obesity, increased serum levels of fatty acids, and insulin resistance can increase the risk of fatty liver disease. p y receptor, through the induction of the c-jun n-terminal kinase (jnk) and prevention of insulin signaling, can promote insulin resistance in hepatocytes in t dm ( ) . in some cases, nafld may progress into an aggressive form of inflammatory fatty liver disease called non-alcoholic steatohepatitis (nash), which might cause liver cirrhosis and organ failure ( ). dang et al. have indicated that exosomes released from the adipose tissues of obese mice due to the smaller mir- - p content can promote insulin resistance in the murine hepatocyte cell line aml (alpha mouse liver ) ( ) . the adipokine visfatin that is released from the adipose tissue of obese individuals has also been shown to activate the pro-inflammatory stat signaling pathway and nf-κb in the human liver cell line hepg and promote their insulin resistant state ( ) . nevertheless, the hepatocyte growth factor (hgf) can alleviate the insulin resistance of hepatocytes and control their triglyceride and cholesterol contents ( ) . skeletal muscle (sm) is the main tissue that releases glucose after insulin stimulation. hence, insulin resistance in sm has a pivotal role in the metabolic dysregulation of t dm. insulin resistance in sm is the primary defect of t dm that facilitates the progression of fatty liver disease, deposition of fat in the liver (figure ) ( ) . skeletal muscle from diabetic patients expresses less genes related to insulin signaling and metabolic pathways, but more apoptosis and immune-related genes ( ) . this inflammatory milieu is mainly due to the proinflammatory actions of obesity-related adipose tissue mediators, which are released into the circulation and promote inflammation within the sm ( ). furthermore, obesity causes intermyocellular and perimuscular adipose tissue expansion that acts like adipose tissue depots to enhance sm inflammation ( ) . it has been shown that human skeletal muscle cells (hsmc), isolated from diabetic patients, after a -h culture generate significantly more tnf-α, il- , il- , il- , monocyte chemotactic protein (mcp)- , growth-related oncogene (gro)-α, and follistatin compared to non-diabetic individuals ( ) . this altered secretion of myokines (e.g., cytokines secreted by sms) is an intrinsic feature of sm during the progression of t dm. in sm, glut- , which is quickly translocated to the cell surface, facilitates glucose uptake in response to insulin hormone as well as muscle contraction. pinto-junior et al. have shown that the use of age-albumin in rats increases the expression of the inflammatory molecule nf-κb within the sm. nf-κb binds to the promoter of the glut- gene and suppresses its expression (at both mrna and protein levels) ( ) . accordingly, glut- levels on the surfaces of sm decrease and subsequently, whole-body ir develops. the immune system is generally classified into two main arms, innate and adaptive (or acquired) immunity. adaptive immunity is mediated by b cells, which produce antibodies and t cells, which are classified into cd + helper cells and cytotoxic cd + cells. a considerable literature has discussed the dysfunctional immune responses in diabetic patients ( table ) ( ) ( ) ( ) ( ) ( ) ( ) . abnormal immune cell activation and subsequent inflammatory environment has an essential role in the progression of t dm ( ) . in this regard, chronic inflammation due mainly to the activation of the myeloid cell lineage (e.g., macrophages and neutrophils), is directly related to the induction of ir ( , ). their numbers are elevated, are larger and more granular, express diminished levels of antioxidant genes but elevated levels of pro-apoptotic and pro-inflammatory genes. complement system attachment of c-type lectin proteins to mannose residues is decreased, lectin pathway is impaired, cd activity is reduced, mac deposition in vascular walls is increased. dendritic cells (dcs) their numbers and activity are reduced. their cholesterol efflux is decreased, generate foam cells, have dysfunctional efferocytosis. are activated, constitutively release nets, produce high levels of mpo, ros, and calprotectin (s a /a ), are more susceptible to apoptosis, their migration, phagocytosis and microbial killing are impaired. nk cells their numbers are increased but are usually dysfunctional, express high levels of glut but decreased levels of nkg d and nkp , have reduced degranulation capacity, are more susceptible to apoptosis. nkt cells their numbers are increased, produce high levels of ifn-γ, il- , and il- , express high levels of nkp , nkg d, and nkp but low levels of nkg a and b. innate lymphoid cells (ilcs) ilc s are increased and produce high levels of ifn-γ. humoral immunity (b cells) germinal centers are reduced, ab production and isotype switching is defective, abs become glycated, abs fail to activate complement. functions of osteoclasts, which are bone-resident innate immune cells ( ) . this may affect bone structure and delay bone healing. defects in the innate, as well as adaptive immunity, are supposed to be the main cause of diabetic individuals' susceptibility to infections ( ) . furthermore, some microorganisms, especially bacteria, in hyperglycemic conditions are better nourished and become more virulent, while also having a better milieu to cause infections. complement system the complement system is a first-line defense mechanism against invading microorganisms. it acts via different but interconnected classical, alternative, and lectin pathways ( ) . ilyas et al. have shown that under high glucose conditions, the attachment of ctype lectin proteins to high-mannose containing glycoproteins is substantially decreased in a dose-dependent manner. these carbohydrate-binding proteins include mannose-binding lectin (mbl), surfactant protein d (sp-d), dendritic cell-specific intercellular adhesion molecule- -grabbing non-integrin (dc-sign, cd ), and dc-sign-related (dc-signr) protein ( ) . reduced binding of mbl in the presence of high levels of sugar causes a significant reduction in the lectin pathway activity, but does not influence classical or alternative pathway activity ( ) . nevertheless, barkai et al. did not find significant differences in the function of classical or mbl pathways between t dm and healthy individuals ( ) . however, significantly decreased activity of ficolin- -mediated lectin and alternative pathways, as well as decreased levels of c d and soluble complement c b- (sc b- ) were seen in diabetic patients with escherichia coli-mediated urinary tract infections ( ) . this may be linked to a reduced ability of diabetics to protect themselves against bacterial infections. the lipopolysaccharides of certain gram-negative bacteria, like salmonella serotype o , as well as the cell walls of fungi, are rich in mannose. possibly, because of this, in addition to additional provision of nutrients, an increased prevalence of fungal infections is seen in t dm patients ( , ) . patel et al. found a significantly higher prevalence of oral candida carriage in diabetic patients compared to healthy controls ( ) . they found that candida albicans was the most commonly isolated species followed by c. tropicalis, but uncommon species such as c. lusitaniae and c. lipolytica were also isolated ( ) . another study by jhugroo et al. showed that c. albicans is the predominant yeast isolated from oral mucosal lesions of diabetic patients, followed by. c. tropicalis and c. krusei dendritic cells (dcs) are a heterogeneous population of specialized and professional antigen-presenting cells (apcs) that create a crucial link between the innate and adaptive immune responses ( , ) . some studies have shown that the numbers of dcs are reduced in both type and diabetes ( , ). seifarth et al. have found that t dm patients with poor metabolic control have decreased numbers of both myeloid and plasmacytoid dcs compared with healthy controls. this could make them more susceptible to opportunistic infections ( ) . in the case of good blood glucose control, the reduction in dc numbers was less prominent but still significant, especially for myeloid dc (mdc ) cells ( ) . another study by blank et al. demonstrated that women with t dm and poor glycemic control (hba c ≥ %) have fewer numbers of circulating plasmacytoid dcs (pdcs) compared to diabetic women with good glycemic control (hba c < %) or to healthy women ( ). montani et al. have recently shown that hyperglycemic medium and hyperglycemic sera derived from t dm patients prevent the maturation of monocytes into effective dcs and their activation in vitro ( ) . interestingly, quercetin, a flavonoid with antiinflammatory and antioxidant characteristics, prevented such effects ( ) . macrophages are important immune cells that play critical roles through all stages of the pathogenesis of t dmrelated atherosclerosis ( ). swirski et al. have shown a significantly elevated number of pro-inflammatory monocytes in the circulation of apoe −/− mice, an animal model of atherosclerosis, compared to control mice ( ) . modifications of the lipoproteins in the arterial walls of diabetic individuals make them pro-inflammatory and activate the overlying endothelium. in response, monocytes are recruited into the subendothelial region, differentiate into macrophages and internalize the accumulated lipoproteins. finally, cholesterol-laden foam cells are generated. they promote inflammation and progression of the disease through the synthesis and secretion of cytokines, chemokines, ros, and matrix metalloproteinases (mmps) (figure c ) ( ) . foam cells lose their migratory potential, die by apoptosis and generate a necrotic core within the atherosclerotic plaque ( ) . it has been demonstrated that the use of mesenchymal stem cells in apoe −/− mice reduces the numbers of monocytes/macrophages at the site of inflammation, decreases lipid deposition and diminishes plaque size ( ). ma et al. have studied the effects of long-term hyperglycemia in diabetic mice and found out that compared to non-diabetic control mice, the numbers of f / + macrophages isolated from spleen (spms), as well as from peritoneal exudates (pems) of diabetic mice are significantly decreased ( ) . subsequently, sun et al. showed that stimulation of pems from diabetic mice in vitro with ifn-γ and lipopolysaccharide (lps) significantly decreased the expression of intercellular adhesion molecule (icam- or cd ), cd , tnf-α, and il- , while it increased the production of nitric oxide (no) ( ) . they further showed that stimulation of pems isolated from diabetic mice with il- caused an enhanced arginase activity ( ) . kousathana et al. have demonstrated that circulating monocytes isolated from diabetic patients produce higher levels il- , while having an impaired activation of the nlrp inflammasome and subsequently reduced il- β production ( ) . however, they showed that proper glycemic control would restore such modifications. poor inflammatory responses in circulating monocytes, as well as in macrophages, are responsible for elevated susceptibility to infections and their severity in patients with t dm. macrophages play a critical role in tissue repair. early in wound healing, they are pro-inflammatory to clear pathogens and debris but later, they resolve inflammation and promote tissue repair. in pathological conditions, failure to transform from pro-inflammatory to the anti-inflammatory proliferative phase can cause chronic inflammation in the affected tissue ( ). have shown that an impaired wound healing process in animals with t dm is due to high levels of nlrp inflammasome activity, which promotes the generation of il- β and il- in macrophages ( , ) . efficient skin wound healing process is mediated by the up-regulation of the peroxisome proliferatoractivated receptor (ppar)-γ in macrophages that convert their pro-inflammatory phenotype into healing-related. pparγ suppresses cytokine production by macrophages and hence is upregulated in inflamed tissue-resident macrophages. however, in t dm, pparγ expression is down-regulated in skin-resident macrophages that enhance the activity of nlrp- inflammasome and cause chronic inflammation. using myeloid-specific pparγ −/− mice, it has been shown that the absence of ppar-γ in macrophages is sufficient to delay the healing process and extend tissue inflammation ( ) . in t dm patients, chronic hyperglycemia and hyperlipidemia trigger the secretion of a damage-associated s a molecule (calgranulin a) from pancreatic islets that in turn increase macrophage infiltration ( ) . westwell-roper et al. have shown that iapp aggregates in t dm patients polarize islet-resident macrophages toward the m -like f / + cd b + cd c + phenotype that produces pro-inflammatory cytokines, including tnf-α, il- β, and il- . furthermore, m cells promote islet inflammation, cause β-cell malfunction and apoptosis ( ) . in t dm, excess phagocytosis of apoptotic β-cells by macrophages induces their lysosomal permeabilization, generation of ros, inflammasome activation, and pro-inflammatory cytokines secretion ( ) . collectively, these observations reveal that the functions and plasticity of macrophages are compromised during the progression of t dm. neutrophils are the most prevalent circulating leukocytes and one of the main components of innate immunity. they are recruited to the sites of infection through chemotaxis following complement activation, most importantly by c a. activated neutrophils bind via their surface receptors to induced ligands on the surfaces of inflamed endothelial cells to migrate to tissues. there they phagocytose and kill invading microbes with lysosomal enzymes, antimicrobial peptides and by the generation of ros ( ). neutrophils from patients with t dm, but not from healthy individuals, are activated and produce elevated levels of ros. so, it could increase the risk of random organ injury ( ) . in diabetic patients, the plasma levels of homocysteine are elevated, which is mainly due to its impaired clearance rate ( ) . this will induce neutrophils to constitutively release neutrophil extracellular traps (nets) that can cause vascular damage and delays in wound healing ( , ) . it has been shown that the circulating level of hydrogen sulfide (h s) is significantly reduced in fasting blood of patients with t dm compared with healthy individuals as well as in streptozotocin-induced diabetic rats compared with controls ( ) . h s is produced from cysteine by the action of several enzymes. it acts as a regulator of cell signaling and homeostasis ( ) . it is essential to maintain balanced levels of antioxidants and protect tissues from oxidative stress ( ) . the use of h s or the endogenous l-cysteine in vitro blocks the production of il- and monocyte chemoattractant protein- (mcp- ) in the human u monocyte cell line incubated in high-glucose medium ( ) . yang et al. have shown that h s treatment decreases netosis and enhances the healing process of diabetic wounds by preventing ros-dependent erk / and p activation ( ) . it has been shown that the levels of net components, including histones, elastase and proteinase- , are elevated in the sera from patients with diabetic foot ulcers ( ) . wang et al. have recently indicated that hg dramatically enhances nadph oxidasedependent net generation in diabetic rats and humans. it was proposed that this could have a role in the induction of diabetic retinopathy ( ) . indeed, patients with t dm have elevated plasma levels of mgo, which can induce the production of proinflammatory cytokines like tnf-α, il- , and il- by neutrophils and make them more susceptible to apoptosis ( ) . myeloperoxidase (mpo), which is abundantly produced by neutrophils, but only to a small extent by monocytes and macrophages, might be useful as an early biomarker of inflammation in diabetic individuals ( ) . binding of mpo to endothelial cells increases its half-life. thereby, more proinflammatory oxidant hypochloric acid (hclo) is generated that extends the damage to blood vessels ( ) . in t dm patients, neutrophil activities, including migration, phagocytosis and microbial killing are impaired. this makes diabetic individuals more susceptible to infections ( ) . it has been welldocumented that neutrophils isolated in animal models of t dm have an impaired tlr signaling pathway. this is reflected as a diminished cytokine and chemokine production, possibly as a consequence of reduced phosphorylation of nfκb and iκbα ( ) . the half-life of these neutrophils as well as their in vivo migration and myeloperoxidase activity are decreased. during hyperglycemia, neutrophils produce calprotectin (s a /a ), which interacts with the receptor for advanced glycation end products (rage) on the surface of hepatic kupffer cells and promotes the synthesis of il- ( ). subsequently, il- stimulates hepatocytes to increase the generation of thrombopoietin that in turn attaches to its receptor on the surfaces of bone marrow precursor cells and megakaryocytes to enhance their proliferation and expansion. this results in reticulated thrombocytosis, which means elevated megakaryocyte activity and thrombopoiesis. interestingly, diabetes-related thrombocytosis and subsequent atherothrombosis can be reduced by lowering blood glucose, depleting kupffer cells or neutrophils or by preventing the binding of s a /a to rage using paquinimod ( ) . thom et al. have shown that the incubation of human and murine neutrophils with hg medium would cause their cytoskeletal and membrane instability. this will induce the generation of . to µm diameter microparticles and activate the nlrp inflammasome ( ) . microparticles, which are potently pro-inflammatory, are found in the circulation of healthy individuals, but their generation is increased during cell activation in several diseases, including t dm and cardiovascular diseases ( , ) . furthermore, serum levels of soluble fasl (sfasl) are increased in patients with t dm thereby activating neutrophils and aggravating the inflammatory milieu ( , ) . the proinflammatory roles of sfasl are mediated through increased amounts or activity of nfκb, il- β, caspase- , cd b/cd , and ros ( ) . caspase- activation prevents the sfasl-dependent apoptosis of neutrophils and inhibits their expression of fas and caspase- ( ) . accordingly, hyperglycemia disturbs the normal functions of neutrophils and increases the susceptibility to infections by pathogenic microorganisms. the expression level of nkg d is negatively correlated with hba c levels implying that chronic hyperglycemia would cause nk cell dysfunction ( ) . also, hyperglycemia increases the expression of unfolded protein response (upr) genes in nk cells and induces their apoptosis ( ) . nkt cells express simultaneously markers of both t cells (tcr and cd ) and nk cells [cd , cd , cd (nkg d), and cd (nkp )]. nkt cell subsets produce a broad range of cytokines, including gm-csf, ifn-γ, tnf-α, il- , il- , il- , il- , il- , il- , il- , and il- ( ) . they recognize lipids and glycolipids presented by cd d molecules. phoksawat et al. have shown that the frequency of cd + cd + cd null cd + nkg d hi nkt cells, which produce high levels of il- , are increased in diabetic patients and their numbers are directly correlated with hba c levels ( , ) . lv et al. have recently shown that the numbers of cd + cd + nkt cells are higher in diabetic patients compared to healthy individuals ( ) . they further showed that such cells are mostly cd + , produce elevated levels of ifn-γ and il- and express high levels of nkp , nkg d, and nkp but low levels of inhibitory receptors nkg a and b ( ) . the co-culture of these cells with huvecs significantly decreased their proliferation and migration abilities that were mainly il- dependent ( ) . taken together these studies show that diabetic individuals appear to have elevated levels of inflammationpromoting nkt cells. ilcs are critical effectors of innate immunity that produce both regulatory and pro-inflammatory cytokines to promote tissue repair, immunity, and inflammation ( ) . mature ilcs lack the tcrs. based on their cell surface markers, cytokine production as well as expression of transcription factors the ilcs are classified into types , , and ( ) . these correspond to the different types of cd + t helper cells: th , th , and th , respectively. ifn-γ is the cytokine signature of ilc s, while type cytokines (e.g., il- and il- ) are mainly produced by ilc s and the main product of ilc s are il- and il- . regarding transcription factors, t-bet is mainly expressed by ilc s, gata and rorα are mostly expressed by ilc s and rorγt is predominantly expressed by ilc ( ) . in t dm, the numbers of circulating as well as adipose tissue-resident ilc s are increased compared with normal individuals ( , ) . the frequency of circulating ilc s is positively correlated with fasting plasma glucose (fpg), hba c, homeostasis model assessment for insulin resistance (homa-ir), serum-free fatty acids (ffas) and adipose tissue insulin resistance index (adipo-ir) ( , ) . it has also been shown that patients with increased numbers of ilc have an elevated risk of developing t dm ( ) . a study by wang et al. indicated that adipose tissue-resident ilc s, via the production of ifn-γ, promote tissue fibrosis and induce diabetes in obese individuals ( ) . liu et al. have demonstrated that the numbers of ilc s as well as serum cytokine levels of il- , il- , and il- are significantly elevated in diabetic kidney disease patients and have a positive correlation with disease severity ( ) . they further demonstrated that ilc s, through the tgf-β signaling pathway, are involved in renal fibrosis seen in diabetic kidney disease ( ) . however, galle-treger et al. indicated that the engagement of the glucocorticoid-induced tumor necrosis factor receptor (gitr/or tnfrsf ) on the surface of activated ilc s promotes their secretion of il- and il- , ameliorates glucose homeostasis, protects against the onset of and improves established insulin resistance ( ) . the protective role of ilc s during acute metabolic stress has also been well-documented by dalmas et al. ( ) . humoral immunity (b cells) elevated levels of blood glucose generate covalent sugar adducts with several proteins through non-enzymatic glycation. this can impair humoral immunity in many ways, e.g., by modifying the structure and functions of immunoglobulins (igs) ( ) ( ) ( ) ( ) ( ) ( ) . such modifications in the structure of igs can be determined using matrix-assisted laser desorption ionization (maldi) mass spectrometry ( , ) . the molecular mass of igs in diabetic patients is higher than in normal subjects ( ) . this can lead to reduced efficiency of vaccines that stimulate humoral immunity in these patients. it has been shown that immunization with influenza (flu) vaccines in diabetic patients induces normal or even elevated levels of flu-specific antibodies compared with normal individuals ( ) ( ) ( ) ( ) . however, the ability of the dysfunctional glycated antibodies to neutralize viruses is impaired, which will increase the susceptibility to infections. farnsworth et al. have shown that in t dm, class switch defects in the assembly of antibody genes are also present ( ) . in a model system, mice with t dm have decreased amounts of specific anti-staphylococcus aureus antibodies (total as well as igg), which will increase the risk of infection and morbidity of diabetic mice. however, the levels of igm were elevated, but inefficient in protecting against infection, possibly because of their inability to directly promote phagocytosis. in another study, farnsworth et al. have demonstrated that defects in humoral immunity, as shown by decreased levels of total igg and anti-staphylococcus aureus antibody, aggravate foot infections in a murine model of t dm ( ) . this was due to a reduced germinal center induction and decreased numbers of t and blymphocytes within the germinal centers. this causes failures in antibody generation and class-switch recombination ( ) . mathews et al. have shown that the protective levels of antibodies against streptococcus pneumoniae surface protein a are lower in diabetic patients compared to non-diabetic individuals. these antibodies also have a reduced potential to trigger complement activation on the surface of pneumococci, whereby phagocytosis of the bacteria becomes compromised ( ) . they showed that hyperglycemia reduces both the antibody titers as well as the ability to deposit complement on the bacteria. the abovementioned changes in the ability to protect against s. aureus and s. pneumoniae are important, because these bacteria belong to the most common infection-causing pathogens in diabetic patients. another major group is constituted by gram-negative bacteria that commonly cause e.g., urinary tract infections. many studies have shown that t-cell functions are impaired in individuals with t dm ( ) ( ) ( ) ( ) . elevated levels of activated cd + cd + t helper cells, cytotoxic t-cells, and th cells have been observed in obese diabetic patients compared to nonobese ones ( , ) . nevertheless, pbmcs isolated from obese diabetic patients produced smaller amounts of il- , il- , and tnf-α after stimulation with phytohemagglutinin (pha) ( ) . martinez et al. indicated that diabetic patients have reduced pathogen-specific memory th responses as well as decreased numbers of cd + t cells in response to stimulation with streptococcus pneumoniae ( ) . th cells are critical for the recruitment of neutrophils to the infection site and improve the phagocytosis of invading bacteria and yeast ( ) . moura et al. have shown that diabetic patients, particularly those with foot ulcers, have reduced levels of naive t-cells, but an elevated number of effector t cells and a reduction in the tcr-vβ repertoire diversity ( ) . the observed changes are mainly due to an abnormal amount of inflammatory cytokines (e.g., ifn-γ and tnf-α) produced during infection and to subsequent robust stimulation of t-cells. leung et al. have reported that ischemic tissues of t dm patients contain elevated numbers of tnf-α and ifn-γ producing th cells but diminished numbers of regulatory t cells (tregs), which suppress angiogenesis and decrease vascular density ( ) . the high rate of infectious diseases in t dm patients might also be linked to a reduction in the mitochondrial dna function that causes downstream lymphocyte dysfunction and subsequently increased susceptibility to infection ( ) ( ) ( ) ( ) . in support, we have recently shown that the numbers of ifn-γ producing cells against cytomegalovirus (cmv), epstein-barr virus (ebv), and influenza virus are fewer in t dm patients compared to normal controls ( ) . kumar et al. have also investigated the functions of cd + t cells and nk cells in the whole blood of t dm patients infected with mycobacterium tuberculosis (m.tb). compared to controls, the patients exhibited a reduction in cytokine production (ifn-γ, il- , il- a/f, and tnf-α) and decreased expression of cytotoxic molecules (perforin, granzyme b, and cd a) ( , ) . these studies conclude that the functions of both cd + and cd + t-cell are defective in t dm patients. t dm is usually associated with an elevated risk of asymptomatic bacteriuria, urinary tract infections (utis), pyelonephritis and non-sexually transmitted genital infections, such as balanitis and vulvovaginal infections ( ) ( ) ( ) . the incidence of infections with a complicated course is significantly higher in diabetic patients compared to healthy controls ( table ) . it seems that it is principally defects in the innate immune responses of diabetic individuals that are responsible for the increased susceptibility and prevalence of infections ( , ( , ) cd + tcells mycobacterium tuberculosis ( , ) susceptible to the causative pathogen of lyme disease, borrelia burgdorferi ( ) . the disease is mainly due to the ability of the bacteria to escape complement opsonization and attack, which leads to an impaired uptake and killing of bacteria by neutrophils ( ) . neutrophil dysfunction also increases the susceptibility of diabetic animals to staphylococcus aureus ( ) ( ) . during the progression of t dm in human subjects, the basal phenotype of macrophages is altered so their capacity to control mycobacterium tuberculosis is diminished ( ) . martinez et al. have indicated that alveolar macrophages isolated from diabetic mice express decreased levels of macrophage receptor with collagenous structure (marco) and cd that are engaged in the recognition of trehalose , '-dimycolate, a bacterial cell wall component ( ) . diabetes increases the severity of tuberculosis (tb) and enhances the risk of progression to the active form in latent infections ( , ) . diabetic tb patients have elevated frequencies of th and th cells as well as increased serum levels of inflammatory cytokines, including ifn-γ, tnf-α, il- β, il- , il- , il- a, and il- but decreased levels of il- compared to non-diabetic tb patients. this can contribute to dysfunctional immune responses and poor immune control of a tb infection ( ) . a positive correlation between the serum levels of ifn-γ, tnf-α, il- , and il- a with hb-a c levels was also observed. this indicates an association between impaired control of diabetes and the proinflammatory milieu. tripathi et al. have demonstrated that serum levels of il- were significantly decreased in tb-infected t dm mice and humans compared to non-diabetic tb-infected mice and humans ( ) . they revealed that the treatment of tb-infected diabetic mice with recombinant il- or ilc s (cellular source of il- ) increased the survival of mice, prevented the accumulation of neutrophils near alveoli, diminished the generation of neutrophil elastase (ela ) and prevented epithelial cell damage ( ) . tan et al. have shown that b. pseudomallei and m. tuberculosisinfected pbmcs of diabetic patients fail to produce il- . this leads to a decreased ifn-γ production, poor bacterial killing and elevated intracellular bacterial loads ( ) . an impaired il- production is mainly due to decreased intracellular glutathione (gsh) concentrations within the infected cells of diabetic individuals ( ) . such a combination of an inflammatory microenvironment and dysfunctional immune responses enhances the bacterial load and can subsequently amplify lung injury and fibrosis in diabetic tb patients. chellan et al. have further shown that infections caused by enterococcus faecalis, staphylococcus aureus, and pseudomonas aeruginosa are more prevalent in the wounds of diabetic patients ( ) . t dm patients are more susceptible to utis caused by antibioticresistant escherichia coli, proteus spp., klebsiella spp., coagulasenegative staphylococci, enterobacter spp., and enterococci ( , ) . diabetic patients are also more susceptible to helicobacter pylori (h. pylori) infections ( ). cui et al. have recently reported that t dm patients have an increased risk of infection with kaposi's sarcoma-associated herpesvirus (kshv or hhv- ) ( ) . they further showed that the viral load and antibody titers are positively correlated with blood glucose levels ( ) . diabetic patients also have been shown to have an increased risk of infection with the severe acute respiratory syndrome coronavirus (sars-cov) ( ( ) . the influenza virus that usually causes self-limiting infections can induce severe forms of the disease in diabetic patients ( , ) . following the h n influenza pandemic, diabetic individuals suffered from more severe infections compared to non-diabetic people ( , ) . diabetic patients have also a higher prevalence of chronic cytomegalovirus (cmv), herpes simplex virus (especially hsv- ), and varicellazoster virus infections ( ) ( ) ( ) . accordingly, it seems that the immune response against viruses is impaired in diabetics, and these patients need more care during viral infections. coronavirus virions are enveloped positive-strand rna spherical viruses with a diameter of ∼ nm characterized by spike proteins projecting from their surface and with an unusual large rna genome ( ) . the spike (s) protein of the virus binds to its receptor on the surface of cells by which intracellular proteases are induced ( ) ( ) ( ) . subsequently, the s protein priming and cleavage occurs that allow viral fusion to the plasma membrane and entrance of viral genome into the cells ( ) . sars-cov and sars-cov- use angiotensin-converting enzyme (ace ) as their receptor while mers-cov uses dipeptidyl peptidase- (dpp ) to enter the cells ( , ) . ace is strongly expressed in blood vessels, pancreas, intestine, brain, lungs, heart, and testis ( ) . interestingly, nasal epithelial cells, especially goblet, and ciliated cells express the highest levels of ace and the intracellular protease transmembrane serine protease (tmprss ) that facilitates the entrance of the sars-cov- ( ) . furthermore, the expression of ace is significantly up-regulated in diabetic patients and those treated with ace inhibitors ( ) . coronaviruses cause respiratory, enteric and central nervous system (cns) diseases in various animal species except rats and mice ( ) . most coronavirus infections are mild, but major outbreaks of deadly pneumonia have been caused by sars-cov, mers-cov, and sars-cov- in , , and - , respectively ( ) . on march , , the world health organization (who) announced the pandemic of sars-cov- , the etiologic agent of coronavirus disease- (covid- ) ( ) . the novel coronavirus pandemic, which has emanated from wuhan, china, promotes symptoms similar to those caused by the sars-cov outbreak in . the viral pandemic, which has put the world on alert, has caused over . × confirmed human cases and at least × deaths throughout the world (https://www.worldometers.info/coronavirus/) by june , . most of the infected people experience only mild to moderate respiratory disease and recover soon without the need for special treatment. however, aged individuals and those with health problems, including diabetes, obesity, cardiovascular disease (cvd), hypertension, immune deficiency, and chronic respiratory disease are more likely to develop serious illness (https://www.who.int/health-topics/coronavirus#tab= tab_ ). patients death is mainly due to the acute respiratory distress syndrome, disseminated intravascular coagulation, hemorrhage, coagulopathy, acute organ (e.g., kidney, heart, liver) injury, multi-organ failure, and secondary bacterial infections ( ) . elevated levels of adipose-tissue derived adipokines, interferon, and tnf-α in diabetic patients may impair immune-responses against sars-cov- ( , ) . it has been shown that diabetic patients have impaired clearance of sars-cov- from their circulation ( ) . accordingly, diabetic patients due to the diminished viral clearance, impaired t cell function, and accompanied cardiovascular disease are more susceptible to the coronaviruses infection and subsequent cytokine release syndrome (crs) ( , ) . in support, elevated levels of il- β, il- , il- , il- , il- , il- , ifn-γ, interferon gamma-induced protein (ip- ), granulocyte colony-stimulating factor (g-csf), macrophage inflammatory protein α (mip α), serum ferritin, fibrinogen, plasminogen, c-reactive protein (crp), and d-dimer have been observed in patients with covid- ( , , , ) . covid- patients, especially those requiring intensive care unit (icu) have decreased total lymphocytes (lymphopenia), t cells (both cd + and cd +), b cells, and nk cells ( , ) . it should be noted that most of the surviving t cells in such patients have an exhausted phenotype ( ) . consequently, disease severity is mainly because of the host immune response to viral infection. current evidence about the relationship between pathophysiological mechanisms of diabetes and covid- are limited and further research is still needed. patients with t dm have an elevated risk of infection with plasmodium falciparum ( ) , toxoplasma gondii ( ), opisthorchis viverrini ( ), strongyloides stercoralis ( ), cryptosporidium parvum ( ), blastocystis hominis ( ), ascaris lumbricoides ( , , ) , and giardia lamblia ( ) . interestingly, diabetic patients who were treated with metformin had less p. falciparum infections compared to untreated patients ( ) . omaña-molina et al. have shown that in a mouse model of t dm the animals have an increased susceptibility to granulomatous amoebic encephalitis (gae) caused by trophozoites of acanthamoeba culbertsoni ( ) . the possible reasons for the increased risk of diabetics for parasitic infections are metabolic abnormalities and immune dysregulation. chellan et al. have shown a higher prevalence of fungal infections in the wounds of diabetic patients ( ) . the prevalence correlated with the levels of hba c. the most widely observed fungal isolates were c. albicans, candida parapsilosis, c. tropicalis, trichosporon asahii, and aspergillus species. some of them were resistant to antifungal medications ( ) . al mubarak et al. have also demonstrated that diabetic patients with periodontitis are more susceptible to infection with c. albicans, c. dubliniensis, c. tropicalis, and c. glabrata ( ) . the incidence of candidiasis was significantly increased in patients over the age of with hba c > ( ). it has also been shown that diabetic patients are more susceptible to utis caused by c. albicans ( ) . hyperglycemia impairs the normal functions of the circulatory system, gastrointestinal tract, pancreatic beta cells, liver as well as of skeletal muscles to boost systemic insulin resistance. a hyperglycemic environment also leads to immune cells dysfunction. it increases intestinal permeability, which subsequently enhances the risk of infections in t dm patients. accordingly, further research is still needed to find missing links between impaired physiological/immunological mechanisms and increased susceptibility to infections in t dm patients. the information would be important for better therapy and the design of much more effective vaccination strategies in diabetic patients. gd 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the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -oao pzy authors: hayward, joshua a; tachedjian, mary; cui, jie; field, hume; holmes, edward c; wang, lin-fa; tachedjian, gilda title: identification of diverse full-length endogenous betaretroviruses in megabats and microbats date: - - journal: retrovirology doi: . / - - - sha: doc_id: cord_uid: oao pzy background: betaretroviruses infect a wide range of species including primates, rodents, ruminants, and marsupials. they exist in both endogenous and exogenous forms and are implicated in animal diseases such as lung cancer in sheep, and in human disease, with members of the human endogenous retrovirus-k (herv-k) group of endogenous betaretroviruses (βervs) associated with human cancers and autoimmune diseases. to improve our understanding of betaretroviruses in an evolutionarily distinct host species, we characterized βervs present in the genomes and transcriptomes of mega- and microbats, which are an important reservoir of emerging viruses. results: a diverse range of full-length βervs were discovered in mega- and microbat genomes and transcriptomes including the first identified intact endogenous retrovirus in a bat. our analysis revealed that the genus betaretrovirus can be divided into eight distinct sub-groups with evidence of cross-species transmission. betaretroviruses are revealed to be a complex retrovirus group, within which one sub-group has evolved from complex to simple genomic organization through the acquisition of an env gene from the genus gammaretrovirus. molecular dating suggests that bats have contended with betaretroviral infections for over million years. conclusions: our study reveals that a diverse range of betaretroviruses have circulated in bats for most of their evolutionary history, and cluster with extant betaretroviruses of divergent mammalian lineages suggesting that their distribution may be largely unrestricted by host species barriers. the presence of βervs with the ability to transcribe active viral elements in a major animal reservoir for viral pathogens has potential implications for public health. retroviruses (family retroviridae) are a diverse and widely distributed family of rna viruses distinguished by their use of a viral rna-dependent dna polymerase (reverse transcriptase; rt) and ability to integrate into the genomes of their cellular hosts [ ] . in addition to the existence of infectious viral particles that are horizontally transmitted between hosts (exogenous retroviruses), the capacity of retroviruses to integrate into the host germline also generates vertically transmissible endogenous retroviruses (ervs) [ , ] . ervs may or may not be capable of producing infectious viral particles, and germline integration over the course of multiple generations typically leads to the accumulation of mutations that render them defective and non-functional [ ] . the retroviral family is composed of seven genera: alpharetrovirus, betaretrovirus, gammaretrovirus, deltaretrovirus, epsilonretrovirus, lentivirus, and spumavirus [ ] . the genomic organization of retroviruses is classified as either 'simple' or 'complex' , with simple retroviruses encoding the structural polyproteins gag and env, and the functional polyproteins pro and pol [ ] . complex retroviruses encode additional accessory and regulatory proteins with diverse functions that typically establish and maintain virus replication and pathogenesis [ ] . the core elements of all retroviruses are flanked by a pair of typically untranslated nucleotide regions at their ′ and ′ ends. in the provirus, formed by integration of the viral cdna into the host cell chromosome, these regions are referred to as 'long terminal repeats' (ltr) [ ] . exogenous retroviruses of zoonotic origin have been associated with disease in humans, the most notable being human immunodeficiency virus (hiv) [ ] . other retroviruses such as human foamy virus (hfv) and human t-cell leukemia virus (htlv) are known to be capable of infecting humans [ , ] . the retroviruses most recently associated with human disease are betaretroviruses. the up-regulation of gene products derived from the human endogenous retrovirus-k (herv-k) group of betaretroviruses has been linked to a diverse range of cancers such as those of the breast, ovaries, and prostate alongside other significant human maladies [ , ] . the genus betaretrovirus consists of the type b and type d groups of exogenous and endogenous retroviruses and the herv-k group of endogenous retroviruses. among the exogenous, infectious members of the genus are the type b mouse mammary tumour virus (mmtv), the type d jaagsiekte sheep retrovirus (jsrv), which causes pulmonary carcinoma in sheep, and the type d mason-pfizer monkey virus (mpmv) which causes wasting and immunosuppression in new-born rhesus monkeys [ ] [ ] [ ] . all betaretroviruses utilize variants of the lysine trna primer binding site (pbs) and encode a deoxyuridine triphosphatase (dutpase), within their pro gene which functions as a nucleocapsid-dutpase fusion protein [ ] [ ] [ ] . type b and type d betaretroviruses differ in several respects including their complement of accessory factors, virion morphology, strategies for rna nuclear export, and the length of their ltr regions. type b betaretroviruses contain spherical viral cores and have ltrs of~ , nucleotides while type d contain cylindrical viral cores and have ltrs of~ nucleotides. the prototypical type b betaretrovirus, mmtv, encodes the accessory proteins regulator of export of mmtv mrna (rem) and negative acting factor (naf ), which have roles in viral mrna export, protein synthesis and gene expression [ ] [ ] [ ] , in addition to the virulence factor, superantigen (sag) [ ] . the type d retrovirus jsrv has been shown to encode the trans-acting factor rej which has a role in protein synthesis and may assist rna nuclear export [ ] . while no distinct oncogenes or sag-like virulence-associated proteins are known to be encoded by type d betaretroviruses, the env protein of jsrv is associated with oncogenesis [ , ] . there are two major strategies employed by betaretroviruses to export unspliced or partially spliced viral rna from the nucleus that use distinct export pathways. complex betaretroviruses such as mmtv employ a hiv rev-like accessory protein encoded within the env gene that binds and facilitates export of intron containing retroviral rna by recruitment of the cellular karyopherin export factor, chromosome region maintenance /exportin (crm /xpo ) [ , ] . simple betaretroviruses such as mpmv contain a constitutive transport element (cte) within the nucleotide sequence at the ′ end of the retroviral genome that recruits a cellular binding factor, tap (nuclear rna export factor ; nxf ) which mediates nuclear export [ , ] . importantly, ervs provide a unique opportunity to study the evolutionary history of this family of viruses as they are essentially genetic 'fossils' of past retroviral infections [ , ] . as such, their existence serves as an indication of the potential host range of a given retroviral lineage and may be interpreted as evidence for the possible existence of exogenous retroviruses that have yet to be isolated. indeed, previous studies have reported a number of endogenous betaretroviruses (βervs) in species for which no exogenous betaretrovirus has yet been identified. these include mammalian species as diverse as primates, horses, rats, lemurs, and an australian marsupial, the common brushtail possum [ ] [ ] [ ] . there are over , known species of bats (order chiroptera), accounting for approximately % of all mammalian species [ ] . bats are relatively divergent from other mammals, having branched off from the perissodactyla (containing horses) approximately million years ago (mya) [ ] . they are divided into two major groups: megabats (suborder megachiroptera) which are mainly fruit-eating, and microbats (suborder microchiroptera), small insectivores that navigate by means of echolocation [ ] . notably, bats harbour over viral species from a diverse range of virus families including the paramyxoviridae, coronaviridae, herpesviridae, rhabdoviridae, arenaviridae, togaviridae, flaviviridae, orthomyxoviridae, reoviridae, bunyaviridae, filoviridae, and picornaviridae [ ] . bats, belonging to the mammalian superorder laurasiatheria, are a major viral reservoir that is evolutionarily distinct from another major viral reservoir, rodents, which together with primates belong to the superorder euarchontoglires [ , ] . bats have recently gained attention as they have been implicated in numerous newly emerging diseases of humans caused by viruses such as sars-coronavirus, hendra virus, nipah virus, and the ebola virus [ ] [ ] [ ] . this track record of zoonotic transmission of previously unknown viral pathogens from bats to humans has prompted calls for a proactive approach to future emerging diseases originating in bats [ ] . to this end a natural history survey of bats has begun, and we have recently reported the discovery of diversified defective endogenous gammaretroviruses in both mega-and microbats [ , ] . previous studies of βervs have tended to focus on isolated viruses, although a report on the βervs of murid hosts indicated that the genus betaretrovirus might possess a diverse and previously unrecognized range of sub-types extending beyond the classical type b/type d paradigm [ ] . using transcriptome and genome analyses of the megabats pteropus alecto (black flying fox) and pteropus vampyrus (large flying fox), and the microbats myotis lucifugus (little brown bat), rhinolophus megaphyllus (eastern horseshoe bat), and rhinolophus ferrumequinum (greater horseshoe bat), we herein examine βervs present in a diverse range of bat species. in conjunction with phylogenetic analyses, we incorporated the diversity of genomic organizations and the use of specific lysine trna pbs to identify eight distinct groups of betaretroviruses. to determine if bats contained and expressed a full suite of integrated endogenous betaretroviral genes we generated and analyzed transcriptome databases of p. alecto, r. megaphyllus, and r. ferrumequinum. gag, pol, and env protein sequences were translated from the genomes of extant betaretroviruses: mmtv, jsrv, mpmv, squirrel monkey retrovirus (smr), and simian retrovirus (srv). local tblastn searches were conducted to determine if the transcriptomes contained nucleotide sequences that, when translated into any of their six reading frames, contained significant protein sequence similarity to the betaretoviral protein query sequences. because the variation in length between different transcripts causes difficulty when interpreting relatedness if similarity is expressed as a percentage identity, the significance of the similarity levels observed was determined on the basis of the e-value (probability of random sequence identity) of the blast hits. each transcriptome was found to contain mrna sequences with notable similarity (e-values < × - ) to the betaretroviral proteins gag, pol, and env, with the exception of the r. ferrumequinum transcriptome in which no betaretroviral gag-like transcripts were identified (table ) . reciprocal blastx searches of the transcript hits with the lowest e-values (i.e. the top hits presented in table ) against the ncbi non-redundant protein database returned predominantly betaretroviral hits. the majority of the mrna sequences identified within the bat transcriptomes were partial, not being of sufficient length to reveal an entire gag, pol, or env gene sequence. as a point of reference, the nucleotide sequence lengths of mpmv gag, pol, and env are , , , , and , , respectively, while the majority of the transcripts identified in the blast analyses were < , . the p. alecto transcriptome was found to contain two retroviral transcripts , and , nucleotides in length which overlap each other by , bases with % sequence identity. the extent of overlap and perfect identity indicated that the two sequences likely represented a fulllength retroviral genomic sequence > , bases in length that was later determined through phylogenetic analysis to be a βerv. this full-length p. alecto βerv genomic transcript was named paerv-βa (pteropus alecto endogenous retrovirus -betaretrovirus a) ( figure ). in addition to paerv-βa, different transcripts covering the length of one distinct betaretroviral pol transcript (papol- ) most closely related to jsrv pol and one env transcript (paenv- ) similar to type c gammaretrovirus and mpmv-like type d betaretrovirus env were identified in the p. alecto transcriptome. a single transcript (rfenv- ) covering the length of a gammaretrovirus-like env gene most similar to rd env was identified in the r. ferrumequinum transcriptome. these transcripts were incorporated into the subsequent phylogenetic analyses. the paerv-βa sequence was found to begin nucleotides upstream of the gag start methionine and contains all of the expected core retroviral genes along with the betaretroviral dutpase domain ( figure ). all of the genes were found to be defective as they each contained frameshift mutations. in addition, the pol and env genes contained premature stop codon mutations. identification of a nucleotide polypurine tract (ppt) allowed the delineation of the beginning of the unique ′ (u ) region. conserved retroviral active site motifs were present in the protease (dxg), reverse transcriptase (ddd), and integrase (dde) domains. the major homology region (mhr; nucleotide coordinates , - , ) and zinc fingers (nucleotide coordinates , - , and , - , ) conserved in gag were also present. two additional orfs were identified; the first overlaps the ′ end of the pro gene while the second overlaps the u region. however, protein translations of the orfs compared to the publicly accessible protein family (pfam) database revealed no known protein domains. in addition, blastp analysis of the translations against the ncbi non-redundant protein database yielded no hits. later identification of closely related p. vampyrus βervs (pverv-βj and pverv-βk) indicated that the orf overlapping the u region was not legitimate. given the successful identification of betaretrovirus-like nucleotide sequences in the transcriptomes, we sought to mine the publicly available genomes of p. vampyrus and m. lucifugus for full-length endogenous betaretroviruses. the aforementioned extant betaretoviral protein sequences together with the retroviral mrna sequences identified in the bat transcriptomes were used to conduct tblastn and tblastx searches on the p. vampyrus and m. lucifugus genomes. these searches revealed a number of hits in the genomes that contained betaretroviral gag, pol, and env genes. full-length ervs were delineated by the identification of retroviral gag, pol, and env sequences positioned next to each other and located between a pair of ltrs. in total, we identified full-length βervs in p. vampyrus and six in m. lucifugus (table ). these bat βervs contain all of the expected core elements and the betaretrovirus-specific dutpase domain. as retroviruses were previously categorized based on the specific trna that anneals to their pbs required for initiation of reverse transcription, we determined the specific trna used by all identified bat βervs through nucleotide alignment with known mammalian lysine trna sequences (additional file : figure s ). the pbs was intact and could be identified in the majority of the bat βervs, and all but one (mlerv-βe) was found to harbour a pbs complementary to either trna lysine , (lys , ) or trna lysine (lys ) typical of betaretroviruses. reciprocal blastp searches confirmed that the gag, pol, and env of these full-length ervs were more similar to known betaretroviral proteins than those of other retroviral genera with pol sequence similarities ranging from % to % (additional file : table s ). all of the bat βervs possessed ltrs of - nucleotides in length, as expected for type d betaretroviruses with the exception of pverv-βb with ltr length typical of type b betaretrovirues ( bp) ( table ). each bat βerv was found to contain a ppt immediately upstream of their ′ ltr regions. we analyzed each pro and pol figure a schematic representation of paerv-βa. two transcripts were identified in the p. alecto illumina sequenced transcriptome that overlapped by , nt with % sequence identity which were used to assemble the paerv-βa genomic sequence. indicated are the retroviral genes gag, pro, pol, and env, which have been rendered defective by random mutation since integration. also shown are the key enzymatic active sites of the viral protease (d×g), reverse transcriptase (ddd), and integrase (dde); the betaretroviral dutpase domain in pro; two unique open reading frames (orfs); the polypurine tract (ppt); and the (unique ') (u ) region. orf* does not appear to be genuine, but rather has arisen as a result of an insertion mutation that has disrupted a stop codon. gene and identified the expected enzymatic active site motifs in the retroviral protease (d×g), reverse transcriptase (ddd), and integrase (dde) domains. the gag gene of each βerv contained the expected mhr and zinc-knuckles. while the m. lucifugus genome sequencing coverage was relatively high ( × coverage), the p. vampyrus genome has only been sequenced to . x coverage. the nature of a low-coverage genome such as this means that within the assembled 'scaffolds' there occasionally exist stretches of nucleotides of ambiguous identity. in this regard, several of the bat βervs reported herein contain short 'non-sequenced regions' (nsr) ( table ) . as a result, the pbs present in pverv-βa and the mhr of pverv-βb could not be identified as they contained nsrs overlapping those elements. to confirm that each βerv was the product of a retroviral integration event, the four-nucleotide repeats known as genomic target site duplication (tsd) sequences that flank the proviruses were identified (additional file : table s ). tsds were identified for all proviral βervs with the exception of pverv-βd and f whose ′ ltrs were masked by nsr, the genome size is given for the proviral version of the βervs. § the genome size of paerv-βa is uncertain as the known sequence begins nt upstream of the gag gene and does not include the (unique ') region. b the core retroviral genes gag, pro, pol, and env that contain frameshift or premature stop mutations are described as 'defective' , those that contain neither of these are described as 'intact' in bold font. c the pro open reading frame (orf) of each βerv was found to encode a betaretroviral dutpase protein domain. d the number of orfs that do not code for the core genes and are nucleotides or greater in length. e the length of the long terminal repeats (ltrs). * for those βervs whose ′ and ′ ltr lengths differ, the value of the ′ ltr is given. f the specific lysine (lys) trna complementary to the primer binding site (pbs) for each βerv is given. † the specific identity of the pbs of mlerv-βe is uncertain. nsr: non-sequenced region. pverv-βk whose ′ ltr appears to be truncated, and pverv-βb which is the sole βerv to have intact and unambiguous ltrs yet no identifiable tsds. to determine if closely related clusters of βervs were generated as a result of post-integration chromosomal duplication events, we compared their flanking chromosomal dna through a blastn analysis (additional file : table s ). one pair of bat βervs (pverv-βk and pverv-βj) was found to have homology in the chromosomal regions immediately up-and downstream of the proviruses. pverv-βk and pverv-βj appear to have arisen as a result of a duplication of a single integrated provirus. the truncation of the ′ ltr of pverv-βk suggests a chromosomal duplication event. next, we examined the phylogenetic relationships of the bat βervs identified in our analysis of the bat genomes and transcriptomes (table ) . accordingly, the gag, pol, and env of the full-length bat βervs were aligned with those of known exogenous and endogenous betaretroviruses and phylogenetic trees were estimated for each ( figure ). in all three trees a great diversity of bat βervs was observed, with individual βervs clustering with members of the type d (e.g. mpmv and jsrv), type b (e.g. mmtv), and herv-k groups. the close relationship between viral sequences derived from transcriptomes and some endogenous viral sequences mined from bat genomes suggests that at least some of the bat βervs have the ability to transcribe. notably, a number of bat βervs (pverv-βj, k and paerv-βa), together with several exogenous betaretroviruses, were found to possess env sequences that formed a cluster so highly divergent, and more closely related to gammaretroviruses, as to require omission from the initial betaretroviral env tree bootstrap values are denoted as ** > %; * > % and < %. the trees are midpoint rooted for purposes of clarity only. βerv proteins of p. vampyrus and p. alecto are highlighted in red text. βervs of m. lucifugus are highlighted in blue text. the clades within the gag and pol trees highlighted with a grey background (γ-env) contain betaretroviruses whose env sequence is not sufficiently closely related to the env of other betaretroviruses to be included in the env tree. ( figure c ). finally, we also found some evidence for within-genome recombination (e.g. mlerv-βc, d and e) as reflected in the phylogenetic incongruence between the gag and pol and env trees. our reciprocal tblastx searches indicated that the p. alecto erv (paerv-βa) and two of the p. vampyrus ervs (pverv-βj and k) encoded env sequences that were more similar to gammaretroviral env, while still possessing gag and pol sequences that closely resembled those of known betaretroviruses (see above). to confirm this observation we undertook a phylogenetic analysis of the env sequences of known gammaretroviruses and betaretroviruses, together with the newly identified βerv env sequences ( figure ). this analysis confirmed previous observations [ , ] that the env sequences of some extant type d betaretroviruses, namely mpmv, smr and simian retrovirus serotypes and (srv and srv ), cluster with gammaretroviral env, as do those of pverv-βj, k, paerv-βa, paenv- (env sequence derived from p. alecto), and rfenv- (env sequence derived from r. ferrumequinum). other type d retroviruses such as jsrv and the enzoonotic nasal tumor viruses (entv) of sheep and goats did not fall into this cluster. this indicates that a recombination event has occurred, in which a sub-lineage of type d betaretroviruses acquired a gammaretroviral env gene. our analysis of the full-length bat βervs revealed an unexpected diversity of genomic organizations, as a number were found to contain unique orfs. some of these orfs were in alternative reading frames within the core element domains and others were either upstream of gag, or downstream of env. furthermore, the differential use of trna lys , and trna lys was not found to be restricted to either type b or type d betaretroviruses. rather, it appears that a switch between the two has occurred multiple times throughout the history of the genus. this diversity of genomic organization was used in conjunction with the phylogenetic analyses of gag, pol, and env (with prime consideration given to the highly conserved pol phylogeny) and the trna usage to identify eight distinct groups within the betaretrovirus genus ( figure ). the eight betaretroviral subgroups that we propose are distinguished from each other by major evolutionary differences such as deep phylogenetic divergence with strong bootstrap support (> % of trees resolving the clade), significant mutations in key genetic features such as a switch to the use of a different pbs, or the presence of retroviral genes from a different genus. group i (represented by herv-k ) consists of the herv-k group of endogenous betaretroviruses which contain a pbs similar to trna lys , and have a deep phylogenetic divergence from other betaretroviruses. no known exogenous betaretroviruses or bat βervs currently reside in group i. group ii (represented by mlerv-βa) consists of a phylogenetic cluster of endogenous bat βervs that branched off from group i early in betaretroviral history. three bat βervs are included in this group. the pbs of mlerv-βa and mlerv-βb are complementary to trna lys , and lys , respectively, while the trna usage of pverv-βa is unknown as a nucleotide nsr overlaps its pbs. mlerv-βa contains a large , nucleotide insertion within its ′ ltr that contains a codon orf. this insertion presumably arose post-integration and the nature of this genetic element is unknown. a pfam domain search and blastp analysis of the translation of the orf against the ncbi non-redundant protein sequence database did not identify any known protein domains or similarity to any known protein. group iii (represented by mlerv-βc) consists of microbat ervs that possess a phylogenetically divergent pol (bootstrap support > %) and a pbs complementary to trna lys . within this group is mlerv-βc, the first fully intact bat βerv to be identified, and which raises the possibility that exogenous members of group iii may yet exist as undiscovered infectious betaretroviruses. group iv (represented by mlerv-βe) appears to have diverged as a part of the type b betaretroviral lineage. however, the precise phylogenetic position of group iv's sole member, mlerv-βe, is not supported by high bootstrap support in any of the trees. furthermore the precise identity of its pbs is uncertain. the pbs does not appear to be specifically complementary to either lys , or lys trna, but rather it appears to be complementary to an alternative mammalian lysine trna. there are presently no known extra copies of mlerv-βe within the m. lucifugus genome. mlerv-βe is distinguished by its possession of a unique orf upstream of gag. this orf begins within the ′ ltr and terminates three nucleotides upstream of the gag start methionine, within the same reading frame. orfs upstream of gag may be relevant to gag expression considering that murine gammaretroviruses encode an alternative n-terminally extended version of gag, glyco-gag, that has a role in the promotion of viral replication [ , ] . no promoter elements or tata boxes were predicted to exist upstream of the orf, however a tata box is predicted within the orf coupled with a possible start methionine downstream, encoding a potential amino acid protein. group v (represented by pverv-βb) consists of archetypically structured type b betaretroviruses (mmtv-like) that contain long ltrs (~ , bases). it is possible that the extension of the ′ ltr has facilitated the emergence of orfs in this location as in the case of mmtv's sag gene. in this regard, pverv-βb has an orf within its ′ ltr. this orf is codons in length, much shorter than mmtv's sag protein, which is amino acids long. while it is possible that the orf was longer at integration and has simply been interrupted by stop codon mutations since that time, a tblastn analysis of mmtv's sag protein against the ′ ltr of pverv-βb did not reveal any significant sequence similarity. also in this group is eqerv, an endogenous horse betaretrovirus, which does not contain a sag gene or sag-like orf within its ′ ltr [ ] . group vi (represented by pverv-βd) consists of jsrv-like type d betaretroviruses that contain short ltrs (~ bases) and env protein sequences that do not phylogenetically cluster with those of the gammaretrovirus genus. members of this group harbour a pbs complementary to trna lys , and may or may not contain additional orfs within their core element domains, as is the case for jsrv and entv's orf-x located within pol, and pverv-βd, which has an orf overlapping the ′ end of the env gene. group vii (represented by pverv-βf) consists wholly of bat βervs. group vii members are phylogenetically type d-like and are primarily distinguished by a pbs complementary to trna lys as opposed to trna lys , which is the expected pbs complementarity for type d betaretroviruses. also, several bat βervs in this group possess a unique orf upstream of gag that is distinct from that of group iv's mlerv-βe. this orf begins within the ′ ltr and terminates nucleotides upstream of the gag start codon. promoter elements and tata boxes are predicted to exist upstream of this orf. as there were differences in the start position of this orf in the various group vii bat βervs (pverv-βe -i), likely due to random mutation since integration, a nucleotide alignment of the region was generated (additional file : figure s ). the alignment demonstrated that the consensus orf contained a possible start methionine that would code for a amino acid protein. one member of this group, pverv-βe, is almost fully intact as it does not appear to contain any frameshift mutations and only a single premature stop codon within the pro gene. group viii (represented by pverv-βj) consists of mpmv-like type d betaretroviruses. the distinguishing feature of this group is the possession of an encoded env polyprotein that phylogenetically clusters with those of gammaretroviruses rather than those of other betaretroviruses. the bat βervs in this group have an additional feature which is an orf beginning bases downstream of the env stop codon and terminating bases into the ′ ltr. this is exemplified in pverv-βj. a nucleotide sequence alignment of the extreme ′ region (additional file : figure s ) of the closely related pverv-βj, k, and paerv-βa generated a consensus sequence that contained this orf and revealed that the equivalent orf sequences in pverv-βk and paerv-βa are respectively interrupted by a frameshifting deletion mutation and stop mutation. this orf contains a possible start methionine that would generate a amino acid protein. this alignment also indicated that the alternative orf* in paerv-βa (figure ) was likely to be an artifact as the u region contained an eight nucleotide insertion that disrupts a stop codon which, if the insertion did occur after integration, has generated an artificial orf. the paerv-βa genome was derived from illumina based transcriptome sequencing while the pverv-βj and pverv-βk genomes were derived through wholegenome shotgun/sanger sequencing. accordingly, each method can be used to orthogonally verify the other. a full alignment of the three proviruses (additional file : figure s ; demonstrating . % nucleotide identity between pverv-βj and pverv-βk and . % between pverv-βk and paerv-βa) supports the veracity of these proviral sequences and provides further evidence that the group viii βervs are likely derived from a single integration event. the unique orfs identified in the bat βervs of all groups were subjected to a blastp analysis against the ncbi non-redundant protein database and pfam domain search. however, no blast hits or known protein domains were identified. to determine if the groupings we had assigned were congruent with known functional differences between retroviruses with respect to betaretroviral rna nuclear export strategies, we analyzed the bat βervs, alongside known exogenous and endogenous betaretroviruses, for evidence of motifs indicative of the major export strategies (additional file : table s ). to this end we employed a computational analysis to search for the presence of nuclear localization signals (nls) and nuclear export signals (nes) common to the retroviral rev-like proteins used in the archetypal rev/rev-responsive element (rre) equivalent export mechanism. we also searched for the presence of tap-binding elements (tbe) within and downstream of the env gene, which would imply the utilization of the cte export pathway, and for direct nucleotide repeats (dr) and inverted nucleotide repeats (ir) that might suggest the formation of stem-hairpin-loop structures known to be associated with the cte [ ] . while a number of βervs were predicted to contain either an nls or an nes, only mlerv-βb and pverv-βb were found to contain both. these βervs broadly cluster with herv-k and mmtv, which respectively encode the rev-like proteins rec and rem, and the presence of both nls and nes points to the possibility that they encode rev-like proteins and make use of the crm nuclear rna export pathway. the majority of the βervs in group vii were found to contain tbe, indicating that the original exogenous forms of these retroviruses likely utilized the nuclear export pathway accessed by the cte. we used an analysis of the ltrs to estimate the time since integration of the bat βervs. this analysis evaluated the extent of the difference between the nucleotide sequences of the ′ and ′ ltrs of each βerv, which are expected to be identical at the time of integration. the number of nucleotide differences between the ′ and ′ ltr is assumed to be proportional to the time since integration, although this may be compromised by such factors as gene conversion [ ] . under this assumption, all βervs integrated into the genomes of the ancestors of modern bats within a wide time range of between . and . million years ago (mya), and hence long after the divergence of bats from other mammalian lineages (table ) . this, in turn, suggests that (i) that the original exogenous forms of these βervs targeted ancient bats, and (ii) there has been a continual integration of betaretroviruses into bat genomes during their evolutionary history. we coupled our analysis of the genomic features of the bat βervs with the phylogenetic patterns observed in the gag, pol, and env trees (with primacy given to the phylogeny of the highly conserved polymerase sequences) to generate a hypothetical series of events that may have led to the current state of diversity in the genus betaretrovirus ( figure ). our analysis indicates that while the ancient progenitor betaretrovirus likely made use of a trna lys pbs, its specific identity is uncertain. groups i and ii appear to have branched off together early in betaretroviral history. this has led, in the case of the herv-k betaretroviruses, to the emergence of distinct genetic elements such as the np and rec proteins, whose current endogenized forms have possible roles in tumorgenesis [ , ] . group iii's phylogenetic position places its point of divergence after the split of groups i and ii but prior to the split between the type b and type d lineages. the divergence between type d and type b βervs seems to have occurred as a result of their differential use of trna lys , and trna lys , respectively. within the type b lineage are groups iv and v which, although possibly splitting after the divergence of type b and type d, differ in the length of their ltrs, their trna usage, and their additional genetic elements. within the type d lineage an early event appears to have been a recombination between a betaretrovirus and a gammaretrovirus, which has caused a divergence between jsrv-like and mpmv-like type d betaretroviruses. in this split, group viii appears to have diverged from groups vi and vii through the acquisition of a gammaretroviral env gene. group vii later diverged from group vi by a switch from the use of trna lys , to trna lys and differentiation of their additional orfs. we searched for the expression of betaretroviral genes in the transcriptomes of the megabat p. alecto and the microbats r. megaphyllus and r. ferrumequinum. through this analysis we determined that betaretroviral genes were being transcribed into mrna within each species and we identified that a full-length genomic transcript of a betaretrovirus (paerv-βa) was being expressed in p. alecto. as each of the genes of paerv-βa were found to contain mutations that likely rendered them non-functional, it seems reasonable to conclude that the transcript was expressed from a defective βerv rather than a functional exogenous betaretrovirus. it is important to note that we cannot exclude the possibility that the reported paerv-βa transcript was derived from multiple similar sequences during transcriptome assembly and due to recombination between similar transcripts during cdna synthesis or pcr as published [ ] . our analysis of the genomes of the megabat p. vampyrus and the microbat m. lucifugus revealed that they contain a genetically diverse range of full-length βervs. in the case of m. lucifugus this included an intact βerv (mlerv-βc) that did not contain any mutations that would clearly render the gene products non-functional. however, it should be noted that as revealed by the ltr analysis, nucleotide substitutions have occurred in the mlerv-βc sequence. while the critical enzymatic active site motifs are intact, whether or not the nucleotide substitutions that have occurred in the coding domains would have a detrimental effect on the functionality of the gene products is not known. in analyzing the genetic content of the full-length βervs for the presence of orfs, aside from those coding for the core genes, we set a minimum cut-off of [ ] . nd: not dated; these βervs could not be dated using this method. pverv-βd and pverv-βf contained non-sequenced regions within their ′ ltr, while pverv-βg and pverv-βk contained bulk deletions within their ′ ltrs. codons to limit the amount of incidental non-coding orfs that would be identified. however, many retroviral accessory and regulatory genes, such as rec and np of herv-k and vpr and tat of hiv- , are shorter than codons and are often encoded over the span of two exons. despite the high minimum cut-off, it is striking that the bat βervs possessed a diverse array of additional orfs. while we cannot confirm that these are indeed protein coding domains, much less speculate on their function, the existence of similar elements is not without precedent among the betaretroviruses. one example is the 'orf x' of jsrv, the function of which is unknown but it has been found to be broadly conserved amongst jsrv isolates [ ] . several of the orfs we identified overlap the proviral ltrs, which consist of typically untranslated regions. this is also not unprecedented, with a prime example being the sag gene of the betaretrovirus mmtv, which is situated entirely within the u region of the ′ ltr. the presence of unique orfs in βervs may indicate the evolution of novel retroviral genes whose products have regulatory or accessory functions required for the retroviral life-cycle and/or pathogenesis. in addition to the βervs reported in this study we noted the presence in both mega-and microbats of betaretrovirus-like retroelements that resemble βervs but lack the env gene; these were not investigated further (data not shown). we reported each βerv as a distinct entity. nevertheless it is reasonable that some of their number, particularly the βervs within each of groups vii and viii, represent a common progenitor infectious betaretrovirus that has undergone duplication events via retrotransposition or recombination since an original, single integration event. for example, the integration time of pverv-βj coupled with its similarity to paerv-βa and pverv-βk may mean that these βervs originated from a single integration into the genome of the common ancestor of p. vampyrus and p. alecto and that at least a single duplication event has occurred within p. vampyrus (or the common ancestor). however, it is also arguable that multiple integrations of closely related infectious retroviruses separated from each other by perhaps a small number of infectivity cycles occurred. we attempted to address this question by a comparative analysis of the flanking genomic dna located immediately up-and downstream of the proviruses and by identifying the tsd that border each provirus and arise as a by-product of the integration mechanism [ ] . unique tsd indicate distinct integration events. in the case of the group viii βervs a tsd for pverv-βk could not be identified as its ′ ltr appears to be truncated. this may indicate that it is a copy of pverv-βj that has arisen through a chromosomal duplication event. this appears to be confirmed by the identification of genomic dna bordering pverv-βj that is homologous to genomic dna flanking pverv-βk. as paerv-βa is a genomic transcript it does not contain tsd. in the case of group vii βervs all of the identifiable tsd differ from one another, indicating separate integration events. additionally, no flanking genomic dna homology was identified amongst the members of the group. notably, both the phylogenetic and ltr analyses revealed a great diversity of βervs in bat genomes. our molecular clock dating suggested that the earliest viral incorporation event occurred at approximately mya which is older than the separation of the megabats and microbats studied (around mya) [ ] . in addition, it is clear that some of the βervs present in bat genomes were vertically transmitted from their ancestors; e.g. mlerv-βa and pverv-βa are grouped together and are of similar age having been integrated approximately mya. however, it is also the case that many of the bat βervs formed via independent viral invasion and incorporation as they have different phylogenetic positions as well as different estimated ages of integration. in addition to their genomic diversity, we observed that a number of phylogenetic clusters within the genus differed in their more fundamental aspects. specifically, the use of trna lys , or trna lys was not restricted to the divide between type b and type d betaretroviruses, and a clade that was distinct in both gag and pol trees possessed a gammaretroviral env gene. this prompted us to define eight sub-groups (group i-viii) within the genus that accounted for these fundamental differences in the context of phylogenetic divergences at the amino acid level of the core polyproteins. our ltr analysis also revealed that bats have been infected with betaretroviruses for most of their evolutionary history. this supports the notion that bats are a potential reservoir for infectious betaretroviruses. a previous study reported a short, partial retroviral sequence (cperv-β , ac ) in the genome of the microbat carollia perspicillata (seba's short-tailed bat) [ ] . however, this sequence contained large deletions, was missing the entire pro and pol genes, and only fragments of the gag and env genes remained. the partial env of cperv-β most closely matched the env of the betaretrovirus smr and on that basis it was reported as a betaretroviral sequence. in this study, we report a series of complete βervs in mega-and microbat genomes representing the breadth of the genus betaretrovirus. although cperv-β does contain a lysine trna-specific pbs, without a pol gene to phylogenetically differentiate it or the presence of the characteristically betaretroviral dutpase domain within pro, it cannot be known with certainty whether it is a group viii betaretrovirus or a gammaretrovirus. the study by ballie et al. [ ] and a recent study by anai et al. [ ] both noted the similarity between the env of type c gammaretroviruses and some type d betaretroviruses which was attributed to a likely recombination event. we have shown that the betaretroviruses, which possess a gammaretrovirus-like env, form a single clade in both gag and pol phylogenies. this indicates that a single recombination event produced these group viii betaretroviruses. furthermore, the typical mammalian gammaretroviral use of trna proline and glycine-specific pbs and the absence of dutpase domains from their pro genes [ ] can be used to infer that the nature of the recombination event was the insertion of a type c gammaretroviral env gene into a type d betaretrovirus. previous studies also determined a recombinatorial origin for the type d env [ , ] . however, this conclusion was reached prior to the sequencing of the genome of jsrv [ ] , which does not possess a gammaretrovirus-like env, and its subsequent classification as a type d retrovirus. as such, it was hypothesized that it was this recombination event that gave rise to the type d lineage of betaretroviruses [ , ] . our analysis aimed to provide a clarification of the differences between, within, and outside of the type b and d groups of betaretroviruses. accordingly, we suggest that the fundamental feature giving rise to the division between the type b and d lineages may have been the use of different primer binding sites, not the possession or not of a type c env gene, which appears to be a more recent and more significant lineage divergence within the type d group. ballie et al. [ ] described seven groups within the genus betaretrovirus. these groupings were made solely on the basis of pol gene nucleotide sequence similarity. while manually determining amino acid sequences from genes that contain frameshift mutations is difficult, when the manual reconstruction is closely informed by the alignment of each translated frame against known betaretroviral polymerases, amino acid sequence reconstruction is a viable option. as such, our phylogenetic analyses differ from those undertaken previously in that they are based on amino acid sequence alignments, and our groupings are based on differences in the fundamental genomic features in addition to phylogenetic clustering. tristem [ ] reported on the identification and classification of the highly diverse endogenous retroviruses present in the human genome (hervs) and suggested that trna pbs specificity, in addition to the polymerase phylogeny of endogenous retroviruses, should inform their classification. this is because even if the ervs of a given species cluster together in phylogenies, the use of different trna pbs may be evidence of separate origins. indeed, that study made the assumption that hervs with alternative pbs homologies were derived from cross-species transmissions. with this in mind, we analyzed the pbs sequences of the identified βervs and used this information to aid and inform the delineation of our grouping scheme. mammalian cells restrict the export of intron containing mrna from the nucleus to the cytoplasm, and betaretroviruses have been found to utilize two different mechanisms to circumvent this restriction and export unspliced genomic rna and singly-spliced env mrna. the type b betaretrovirus mmtv, and the herv-k endogenous retroviruses are known to use rem and rec, respectively, which are hiv rev-like export proteins, that possess equivalent mechanisms of action [ , [ ] [ ] [ ] . the type d betaretroviruses mpmv and srv make use of the cis-acting cte, which in the absence of a retroviral accessory protein, recruits cellular proteins to effect nuclear export of intron containing viral rna [ , ] . this apparent dichotomy has been complicated by recent lines of investigation that have found that i) mmtv likely possesses a second, rem-independent mechanism for the export of singly-splice env mrna [ ] ; and ii) the type d betaretrovirus jsrv contains both a cte and a rev-like protein, rej, which while found to possess a primary function related to gag synthesis, also enhances rna export in some cell types [ , ] . this indicates that betaretroviruses may make use of multiple export mechanisms, possibly providing some measure of redundancy to promote productive replication in different contexts. we conducted a computational analysis to predict the presence of rna export motifs that would indicate which mechanism was utilized by each βerv. we found that bat βervs, clustering with betaretroviruses known to utilize the crm export pathway, typically contained one or both of the nls and nes motifs, suggesting that they too encode a rev-like protein. it was not surprising that some βervs were predicted to contain one motif but not the other, as random mutation since integration is expected to interfere with sequence-based motif prediction. it is also possible that the nes of some betaretroviral rev-like proteins (such as is the case for herv-k rec) are encoded at the exon boundary and/or within a frame different to that used by env, making the prediction of nes from the env protein sequence challenging. a number of βervs in group vii were found to contain retroviral tap-binding motifs, defined as published [ ] , implicating their use of the cte:tap export pathway. the presence of putative nls and nes in some group vii βervs suggests that rev-like elements may also be present. as rev-like proteins are encoded within the env gene, the recombination event that replaced the betaretroviral env with a gammaretroviral env and gave rise to group viii would have caused the incidental loss of any encoded rev-like protein. such a lineage would only have remained viable if it either possessed an alternative mechanism for export, or never made use of a rev/rre equivalent export mechanism in the first place. that rev-like proteins are widely distributed amongst the betaretroviruses suggests that it is not unreasonable that the progenitor of group viii did possess a rev-like protein. this possibility is supported by the existence of the rej protein of jsrv, as jsrv clusters alongside group viii in the type d lineage. in addition, several bat βervs in groups vi and vii contain putative nls and nes motifs, suggesting that members of these groups contain rev-like elements. if group viii did lose a rev-like protein upon acquisition of a gammaretroviral env, then two explanations for the lineage's survival are apparent: i) the recombination event was confined to env and the betaretroviral cte possessed by mpmv and srv, which is located immediately downstream of env, already existed as a redundant export mechanism and remained after the event, or ii) the recombination event included the nucleotide sequence downstream of the env gene, and a putative cte-like element was acquired in the process. with regard to the second possibility it is important to note that the mrna nuclear export mechanism of gammaretroviruses has not been elucidated and the proposal of a cte-like element remains hypothetical. however, this notion is supported by the observation that accessory proteins have not been reported for gammaretroviruses, expression of unspliced and singly-spliced viral mrna would require nuclear export, and that a cte-like cisacting nuclear export element would necessarily be located in singly-spliced env mrna. in either event, our analysis leads to the surprising implication that the betaretroviruses are part of a fundamentally complex retroviral genus and that one lineage, group viii, has evolved through gene replacement into a simple retrovirus sub-group that does not possess any distinct accessory proteins or virulence factors. using the phylogenetic analysis of retroviral pol sequences we proposed a pathway through which the genus betaretrovirus may have evolved from its progenitor. this hypothetical evolutionary history paints an interesting picture of a broad and diverse retroviral genus whose distribution may be largely unrestricted by host species barriers. the βerv members of a number of groups are represented in hosts who are distantly related, such as group viii, which contains host species from bats, primates, rodents, and marsupials. this suggests that cross-species transmission of betaretroviruses is a likely and common occurrence, such that betaretroviruses may be particularly adept at evading host defences. this possibility is intriguing, particularly in light of the wide array of additional orfs found within the genus that hint at the existence of as yet undiscovered betaretroviral accessory and virulence factors; these could, for example, act as countermeasures to circumvent the action of host intracellular restriction factors that are known to act as barriers to cross-species transmission [ ] . the wide distribution of diverse βervs in bats and rodents suggests that these two largest groups of mammals play a major role as both hosts and cross-species transmitters for betaretroviruses. bats and rodents are globally distributed, appearing on all continents with the exception of antarctica [ , ] . as such it appears reasonable to postulate that they have both played a large role in the global spread and evolution of betaretroviruses. we have demonstrated the presence of a range of βervs in mega-and microbats that possess a diversity that cannot be confined to the classical type b/type d division. among their number we identified an intact βerv that may be capable of producing infectious virions, and our ltr analysis indicates that betaretroviruses have been circulating in bat populations throughout their evolution and likely still do. our evidence that bats have carried a range of exogenous infectious betaretroviruses and that cross-species transmission has been commonplace has important implications for disease emergence. indeed, the reported association between the betaretrovirus mmtv and human breast cancer and primary biliary cirrhosis may mean that betaretroviral zoonosis is already causing disease in humans [ ] [ ] [ ] . urban expansion into the natural habitats of bats is gradually increasing the amount of overlap between bat and human environments, and with it the amount of contact between bats and humans [ ] . in many countries the practice of hunting bats as a source of consumable bushmeat is common [ ] . these circumstances provide the opportunity for retroviral transmission between bats and humans. we propose that the transmission of a betaretroviral infection from bats into humans is possible. as such, it is imperative to continue to survey those viruses present in bats. approval for the use of bat tissue was granted by the australian animal health laboratories animal ethics committee (protocol aec ) and by the animal ethics committee of east china normal university (approval number ). p. alecto transcriptome datasets were generated from the non-stimulated thymus tissue of a healthy male juvenile bat and the pooled total rna obtained from mitogen-stimulated spleen, white blood cells, and lymph node and the unstimulated thymus and bone marrow obtained from one pregnant female and one adult male as described previously [ ] . the p. alecto transcriptome is accessible through the ncbi sequence read archive (http://www.ncbi.nlm.nih.gov/traces/sra/) [sra: srp ]. the r. ferrumequinum transcriptome was generated using whole brain tissue as published [ ] . the p. alecto and r. ferrumequinum transcriptomes were sequenced using the illumina next-generation sequencing (ngs) platform as described previously [ , ] . the p. alecto transcriptome was assembled using velvet, oases, and mira software packages as described previously [ ] . the r. ferrumequinum transcriptome was assembled using the brujin graph and soapdenovo software packages as described previously [ ] . the generation of the r. megaphyllus transcriptome was conducted as follows: four wild bats, (one female and male) were caught in the booloumba creek caves in queensland, australia in november and tissues from brain, kidney, large and small intestines, liver, lung, spleen, heart, skin, bone and reproductive organs were pooled and stored in rnalater (ambion). total rna was isolated from the pooled bat tissues using the qiagen rneasy kit. dna was prepared from purified total rna ( . μg per cdna reaction) using the evrogen mint cdna synthesis kit (cat # sk ) but with a modified oligodt adapter primer containing the recognition sequence for gsui ( ′ agcagtggtatcaacg cagagt ctggag(t) vn). the cdna was normalized with a duplex specific nuclease (dsn) using a modification of the protocol described in the evrogen trimmer cdna normalization kit (cat # nk ). after the second limited pcr amplification ( cycles) with the m primer, pcr buffer, primers and enzyme were removed using the machery nagel nucleospin ii kit. dna was then digested overnight with gsui to remove the ′ polya tail adapter sequence so as to remove stretches of homopolymer ts and as which can effect the sequencing run due to cross-talk (homopolymer flash). five micrograms of normalized amplified double stranded cdna was purified using the machery nagel nucleopsin kit with the selective removal of the gsui digested base pair (bp) ′polya adapter sequence using a modification of the binding conditions. library preparation for roche sequencing for the gs flx platform was performed by the australian genome research facility ltd, st lucia, queensland with sequence output of mb, , single-end reads with an average read length of bp. clc genomics workbench version . . (clc bio, aarhus, denmark) was used to trim reads based on quality and to remove the evrogen normalization primer sequence, subsequent , reads were de novo assembled using clc genomics workbench default settings and blast databases were prepared using either de novo assembled or trimmed unassembled reads. the gag, pol, and env genes of each genome sequence were translated into protein sequences using the clc main workbench . (clc bio). to identify the transcripts of interest we used the tblastn function of the clc main workbench incorporating the following parameters: blosum matrix, word size = , e-values < × - , gap costs of existence , extension , and low complexity filtered. to confirm that the transcripts identified were more similar to betaretroviruses than other retroviral genera we performed a reciprocal blast analysis of each transcript against the ncbi non-redundant protein database (http://blast.ncbi.nlm. nih.gov/blast.cgi) using the blastx function of the clc main workbench with the following parameters: blosum matrix, word size = , e-values < × - , gap costs of existence , extension , low complexity filtered, and limit by entrez query = viruses. annotated sequences of the full-length betaretroviral sequences included in the phylogenetic analyses (paerv-βa, papol- , paenv- , and rfenv- ) are included as additional file . we generated the genomic sequence of paerv-βa using two transcripts identified in the p. alecto transcriptome during the initial blast analysis which were aligned using the clc main workbench and trimmed by and nucleotides at the ′ and ′ extremities of their overlapping region, respectively. to determine the presence of full-length βervs in mega-and microbats we retrieved the genomes of m p. vampyrus and m. lucifugus from the ensembl database (http://www.ensembl.org/index.html). we searched for genomic sequences with similarity to the aforementioned extant betaretroviral proteins by conducting a tblastn analysis of the genomes using the clc main workbench with the following parameters: blosum matrix, word size = , e-values < × - , gap costs of existence , extension , and low complexity filtered. we searched for genomic sequences with similarity to the betaretroviral transcripts identified in the bat transcriptomes by conducting a tblastx analysis of the genomes using the clc main workbench with the following parameters: blosum matrix, word size = , e-values < × - , low complexity filtered. to sort fulllength from fragmented βervs and various other retroelements within the blast output, a script was created using microsoft office excel (microsoft corporation, redmond, usa) that compared the blast data for the gag, pol, and env analyses and identified scaffolds that emerged as a hit in each. the long terminal repeats (ltrs) which were used to delineate the full-length βervs were identified by subjecting each identified gene scaffold to a blastn analysis in which the entire sequence was aligned with itself to identify repeated sequences using the following parameters: word size = , match score = , mismatch score = − , gap costs of existence , extension , and low complexity filtered. transcription promoter elements within the ′ ltrs of the βervs were predicted using the online promoter predictor tool nnpp . [ ] (http://www.fruitfly.org/ seq_tools/promoter.html). tata boxes were predicted using the hamming-clustering method through the online hctata tool [ ] (http://zeus .itb.cnr.it/~webgene/ wwwhc_tata.html). poly(a) signal sites were predicted using the hamming-clustering method through the online hcpolya tool [ ] (http://zeus .itb.cnr.it/~webgene/ wwwhc_polya.html). primer binding sites were identified by an alignment of the genomic nucleotide sequence between the ′ ltr and the beginning of the gag gene of each βerv against the university of strasbourg's online trna database [ ] (http://trna.bioinf.uni-leipzig. de/dataoutput/search) using the associated blast tool (default parameters). open reading frames (orfs) were identified within each βerv using the clc main workbench. the dutpase protein domains and nucleocapsid zinc knuckles were identified by subjecting the translated gag and pro genes to a protein family (pfam) domain search [ ] through the clc main workbench using the publicly accessible pfam database (http://pfam. sanger.ac.uk/). the conserved major homology region (mhr) of gag and enzymatic active sites of the retroviral protease (dxg), reverse transcriptase (ddd), and integrase (dde) were identified through a protein sequence alignment, using the create alignment function of the clc main workbench, between the gag, pro, and pol of each bat βerv against those of the aforementioned extant betaretroviruses. prediction of rna export elements nls and nes were predicted by analyzing the env, or if known, the rev-like protein sequence of each betaretrovirus. nls were predicted using the online tool cnls mapper [ ] (http://nls-mapper.iab.keio.ac.jp/ cgi-bin/nls_mapper_form.cgi) with a prediction score threshold of . . nes were predicted using the online tool netnes . [ ] (http://www.cbs.dtu.dk/services/ netnes/). the strength of each nes prediction within the env/rev-like protein is defined as strong if the scores for the neural network model and hidden markov model, together with the overall nes score, are above the algorithm-assigned threshold. the strength is weak if one of the scores is below the threshold. no nes is predicted for proteins in which more than one score is below the threshold. tbe, dr, and ir were identified by subjecting the nucleotide sequence within and downstream of env ending at the poly(a) signal site within the ′ ltr of each betaretrovirus to a blastn analysis in which the sequence was aligned against itself to identify repetitive elements using the following parameters: word size = , match score = , mismatch score = − , gap costs of existence , extension , and low complexity not filtered. all nucleotide and protein alignments were conducted using the create alignment function of the clc main workbench except where stated otherwise. to determine the evolutionary relationships among the different bat betaretroviruses we inferred the phylogenetic relationships among the gag, pol and env amino acid sequences. all of the reference sequences were downloaded from ncbi (additional file : table s ) and aligned with bat sequences using muscle [ ] . we employed the gblocks program [ ] to remove regions of high sequence diversity and hence uncertain alignment. phylogenetic relationships were then inferred using the maximum likelihood (ml) method available in phyml . , employing spr (subtree pruning and regrafting) branch-swapping [ ] and incorporating , bootstrap replications to determine the robustness of each node. the prottest . program [ ] was used to select the best-fit model of amino acid substitution, which was found to be lg+i+Г for all data sets. a time-scale for βerv evolution was established as described previously [ ] and employing the bayesian markov chain monte carlo method (mcmc) available in the beast v . package [ ] . we first acquired the genomic substitution rates (r) for mega-and microbats. for this, divergence times of mega-and microbats were taken from the fossil record [ ] and used to calibrate date estimates for the rest of the species tree, assuming an uncorrelated lognormal relaxed molecular clock. all phylogenetic trees were inferred using the gtr substitution model and the yule speciation prior, and the beast analyses were run until all relevant parameters converged, with % of the mcmc chains discarded as burn-in. the estimated substitution rates were then used to calculate the age of each βerv using the following formula: t=(d/r)/ , where t is the invasion time of each βerv (million years), d is the number of differences per site among the both ′ and ′ ltrs, and r is the genomic substitution rate (substitutions per site per year). the genbank accession numbers of the retroviruses used in this study are listed in additional file : table s . additional file : figure s . alignment of extant and bat betaretroviral primer binding sites (pbs). the pbs of bat endogenous betaretroviruses and those of known extant and exogenous betaretroviruses are aligned and grouped according to the specific lysine trna complementary to the pbs. *the pbs complementarity of mlerv-βe is uncertain. figure s . alignment of the orf present in the group vii endogenous betaretroviruses (βervs) of bats. the region from the beginning of the ′ ltr to the beginning of the gag gene of each group vii bat βerv was aligned and a consensus sequence generated. the annotations belong to the consensus sequence and depict the ′ ltr, predicted promoter element and tata boxes, the pbs complementary to trna lys (lys pbs), and an open reading frame (orf). figure s . annotated alignment of the group viii endogenous betaretroviruses (βervs) of bats. the region from the end of the env gene to the ′ long terminal repeat (ltr) of each group viii bat βerv was aligned and a consensus sequence generated. the annotations belong to the consensus sequence and depict an open reading frame (orf), the beginning of the ′ ltr, and mutations in paerv-βa and pverv-βk that influence the presence of orfs. additional file : table s . comparison of βerv polymerase sequences to those of known betaretroviruses. table s . identification of the target site duplications (tsd) flanking endogenous betaretroviruses. table s . comparison of the ′ and ′ flanking regions of phylogenetically clustered βervs. table s . analysis of betaretroviral rna export motifs. table s . genbank accession numbers and ensembl database locations of the retroviruses used in this study. historical introduction to the general properties of retroviruses studies of endogenous retroviruses reveal a continuing evolutionary saga retroviral virions and genomes converging strategies in expression of human complex retroviruses origin of hiv- in the chimpanzee pan troglodytes troglodytes persistent zoonotic infection of a human 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and goats identification and characterization of novel human endogenous retrovirus families by phylogenetic screening of the human genome mapping project database identification of a rev-related protein by analysis of spliced transcripts of the human endogenous retroviruses htdv/herv-k rec (formerly corf) function requires interaction with a complex, folded rna structure within its responsive element rather than binding to a discrete specific binding site indik s: identification of the remresponsive element of mouse mammary tumor virus jaagsiekte sheep retrovirus encodes a regulatory factor, rej, required for synthesis of gag protein antiretroviral restriction factors rodent evolution: back to the root mouse mammary tumor virus-like sequences in human breast cancer human mammary tumor virus in inflammatory breast cancer cloning the human betaretrovirus proviral genome from patients with primary biliary cirrhosis distribution and activity of bats at local and landscape scales within a rural-urban gradient bats as bushmeat: a global review the immune gene repertoire of an important viral reservoir, the australian black flying fox application of a time-delay neural network to promoter annotation in the drosophila melanogaster genome hamming-clustering method for signals prediction in ′ and ′ regions of eukaryotic genes trnadb : compilation of trna sequences and trna genes the pfam protein families database systematic identification of cell cycle-dependent yeast nucleocytoplasmic shuttling proteins by prediction of composite motifs analysis and prediction of leucine-rich nuclear export signals muscle: multiple sequence alignment with high accuracy and high throughput improvement of phylogenies after removing divergent and ambiguously aligned blocks from protein sequence alignments new algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of phyml . prottest: selection of best-fit models of protein evolution bayesian phylogenetics with beauti and the beast . submit your next manuscript to biomed central and take full advantage of: • convenient online submission • thorough peer review • no space constraints or color figure charges • immediate publication on acceptance • inclusion in pubmed, cas, scopus and google scholar • research which is freely available for redistribution additional file : figure s . unannotated alignment of the full proviral genomes of the group viii endogenous betaretroviruses (βervs) of bats.additional file : annotated sequences of paerv-βa, papol- , paenv- , and rfenv- . the authors declare that they have no competing interest.authors' contributions mt, jah, jc, and gt conceived the study. jah, jc and mt performed the analyses, mt generated the r. megaphyllus transcriptome, hf collected bats from which tissue was obtained to generate the transcriptome data. all authors contributed to the writing of the paper. all authors have read and approved the submission of the manuscript. key: cord- - u i qmc authors: almomani, huda y.; pascual, carlos rodriguez; al-azzam, sayer i.; ahmadi, keivan title: randomised controlled trial of pharmacist-led patient counselling in controlling hypoglycaemic attacks in older adults with type diabetes mellitus (rose-adam): a study protocol date: - - journal: res social adm pharm doi: . /j.sapharm. . . sha: doc_id: cord_uid: u i qmc introduction: hypoglycaemia is one of the most serious adverse effects of diabetes treatment. older adults are at the highest risk to develop hypoglycaemia. several studies have established the important positive role of educational interventions on achieving glycaemic control and other clinical outcomes, however, there is still a lack in studies that evaluate the impact of such type of interventions on hypoglycaemia risk in elderly patients with type diabetes. the purpose of this research is to evaluate the effectiveness of pharmacist-led patient counselling on reducing hypoglycaemic attacks in older adults with type diabetes mellitus. methods: and analysis: this study is an open-label, parallel controlled randomised trial, which will be conducted in the outpatient clinics at the largest referral hospital in the north of jordan. participants who are elderly (age ≥ years), diagnosed with type diabetes mellitus, and taking insulin, sulfonylurea, or any three anti-diabetic medications will be randomly assigned to intervention (sugar handshake) and control (usual care) groups. the sugar handshake participants will have an interactive, individualised, medications-focused counselling session reinforced with a pictogram and a phone call at week six of enrolment. the primary outcome measure is the frequency of total hypoglycaemic events within weeks of follow up. secondary outcomes include the frequency of asymptomatic, symptomatic, and severe hypoglycaemic events, hypoglycaemia incidence, and time to the first hypoglycaemic attack. we will also conduct a nested qualitative study for process evaluation. ethics and dissemination: the human research ethics committee of the university of lincoln and the institutional review board of king abdullah university hospital approved this protocol. the findings of this study will be presented in international conferences and published in a peer-reviewed journal. trial registration number: the study protocol has been registered with clinicaltrials.gov, nct . background hypoglycaemia is the major limiting factor in diabetes management. hypoglycaemia has been found to be associated with cardiovascular events such as myocardial infarction, arrhythmias and cardiovascular mortality as well as cerebral complications such as dementia. - . additionally, hypoglycaemia can impact patients' quality of life. patients with moderate or worse symptoms of hypoglycaemia are less satisfied with their treatment and report poorer adherence to their medications. the great burden of hypoglycaemia is largely presented by the considerable health care cost resulting from hospitalisations, ambulance services, emergency department visits, and absenteeism from work. , older adults are the most susceptible age group to develop hypoglycaemia and to experience hypoglycaemia-related complications. the correlation between ageing and hypoglycaemia in type ii diabetes is multifactorial. [ ] [ ] [ ] [ ] [ ] factors such as physiological changes in elderly that would affect the pharmacokinetics profile of anti-diabetic drugs, comorbidities that affect heart and kidneys, nutrition changes and cognitive impairment that could affect concordance and compliance with treatment regimen [ ] [ ] [ ] [ ] [ ] . as the population of older adults is increasing, globally; it is expected to see an increase in the prevalence of t dm in older adults. , in jordan, the prevalence of diabetes in people aged years and over increased significantly over ten years period. it is estimated that the number of elderly persons in jordan will be three times higher in than it was in , consequently more older adults will be at risk of developing type ii diabetes and diabetes-related complications. there are several studies on the association between hypoglycaemia and patient characteristics such as patients' perspectives and attitudes towards diabetes management skills, self-monitoring of blood glucose, and non-adherence to anti-diabetic medications in jordanian population. - however, there is a dearth of data on the interventions that could potentially prevent hypoglycaemia in such patients, especially the older population. therefore, it is imperative to develop diabetes-related care strategies targeted to this broad population of patients to cope with the growing figures in the future. in the context of diabetes, pharmacist-led care interventions appear to have a pivotal role in glycaemic control, improving self-care activities, medication adherence, improving quality of life, and reducing related complications. - pharmacist-led care interventions can be individualised to each patient to achieve glycaemic control. , , however, a few randomised controlled trials (rcts) have investigated the impact of such interventions on hypoglycaemia in adults diagnosed with t dm. although elderly people are considered heterogenous group with different characteristics from younger adults, none of these trials has explored the effect of educational interventions in this age category. jordanian pharmacy education equips the pharmacists with robust clinical knowledge and clinical skills to work with other healthcare professionals to provide optimal quality care to the patients . therefore, the aim of this study is to evaluate the effectiveness of pharmacist-led patient counselling on preventing hypoglycaemia in older adults with t dm. educational interventions are considered to be "complex" interventions compared with classic examples of drug interventions in rcts. that is an educational interventions' success or failure could be attributed to a myriad of factors besides the interventions' effectiveness. the factors such as the delivery of the intervention, understanding of the intervention by the patients and implementation of intervention would decide the fate of an educational intervention. for this reason, a process evaluation is valuable to identify whether an intervention works and how, barriers for its implementation, and how to improve it in the future. undertaking qualitative studies to evaluate the interventions during the implementation stage helps in modifying the ongoing interventions as well as the study design to make them more feasible and effective. the study will be a single-centre, two groups, : parallel, open-label, pragmatic randomised controlled trial with a nested process evaluation embedded; and will be conducted in two clinics at a referral tertiary hospital in jordan. participants will be randomly assigned to either the intervention group hereinafter referred to as the "sugar handshake" group or the control group. figure illustrates the detailed study flow chart. hypoglycaemic episodes pose some challenges as the frequency of hypoglycaemic events is the primary factor in calculating the sample size. , we referred to the most relevant study that used a similar outcome measure, methodology and intervention to our research to calculate the needed sample size. the previous study found that the mean total number of hypoglycaemic attacks in the control and the intervention groups were . ± . and . ± . per patient in weeks, respectively. we used the reported frequency of hypoglycaemic attacks in both groups to calculate the minimum required sample size. therefore, we need to recruit at least patients to achieve a significance level of . and a study power of %. accounting for % to compensate attrition rate and missing data, the final sample size wished to be recruited is participants ( in each group). we will recruit older adults who are years and above, diagnosed with t dm and being prescribed sulfonylurea, insulin, or any three anti-diabetic medications. exclusion criteria include patients unable to understand instructions or to give consent, diagnosed with haemolytic anaemia or haemoglobinopathies, on palliative care for cancer, with advanced- stage or end-stage diseases who are terminally ill, diagnosed with psychosis or severe depression, or with life expectancy < months, impaired mental capacity, unwilling to take home glucose measurements or to use the glucose meter, or unwilling to return for follow up. patients who have a partner or a first-degree relative who has been enrolled in the study, are excluded as well. we will use two recruitment methods to reach potentially eligible patients: an advertisement placed in the reception room where patients wait for their appointments, and through direct identification of potentially eligible patients at the recruitment sites. ha will be responsible for recruiting participants in the endocrinology and diabetic foot care clinics meanwhile the research assistant (ra) will recruit from the cardiology clinics. ha and the ra will explain the trial purpose and processes and provide participant information sheets (supplementary file ) to the interested patients. they will also confirm the eligibility of patients who are willing to participate and will ask them to sign a written consent form we will randomly assign participants to the intervention and control groups on a : basis. the random sequence will be generated using the website (www.randomization.com) to generate the randomisation schedule. envelopes will be used to conceal the allocation and will be opened by the researcher sequentially at the time of each participant's enrolment. the study envelopes will contain the study name, the participants' codes, the group to which the participants are randomised. the randomisation sequence and the study envelopes will be prepared by a third independent party who will not be involved in the study. the envelopes will be closed and opaque and will be given to the researcher and the ra who are involved in conducting the study. the envelopes that will contain the group allocation will be kept in a locked cabinet in the hospital. blinding since this is an open-label study, the patients and the data collectors will not be blinded to the assigned group. eligible patients will be informed about the purpose of the study and the study-related activities before they sign the consent form. proper measures will be taken along the trial duration to minimise performance and ascertainment bias that may result from unblinding participants. all participants will be unblinded to the study groups and to the real purpose of the trial at the follow-up visit that would mark the end of the trial. additionally, participants who are assigned to the control group will receive the sugar handshake intervention at the follow- up visit. intervention group (sugar handshake) the educational intervention, the sugar handshake, is designed to promote behavioural change to prevent hypoglycaemia. we applied the principles of the behaviour wheel theory (bcw)to design the intervention. our educational intervention would enhance the physical and psychological capabilities of the patients through improving their knowledge and skills in managing and preventing hypoglycaemia. the intervention would also lead to the behavioural change by a conducive environment to promote behaviour change by addressing the physical and cultural needs of the patients. we have structured the reporting of the intervention in line with the tidier (template for intervention description and replication) checklist and guide. participants assigned to the sugar handshake group will receive individualised counselling regarding hypoglycaemia in addition to the usual care provided at the trial sites. the intervention is designed by ha who is a pharmacist with prior work experience in patient counselling and pharmacist-related clinical services. ha has trained the ra on delivering the intervention. the intervention is delivered in two steps i.e., a face-to-face conversation at the enrolment visit followed by a phone call six weeks later. step one: face-to-face conversation at the enrolment visit the sugar handshake intervention will cover comprehensive strategies to prevent and handle hypoglycaemia with instructions related to anti-diabetic medications and managing drug-related problems. table step two: phone call at the th week: participants will receive a -minute follow up call at week six of enrolment; so that the first step of the intervention would be reinforced as well as participants' queries/questions would be answered. participants will also be asked about the number and timing of having hypoglycaemic attacks during the first six weeks in the trial to re-consider modifying the intervention components. participants in this group will be offered guidance on hypoglycaemia diagnosis and the proper use of the glucose meters in addition to the usual care. they will also be provided with instructions on hypoglycaemia treatment. as participants in both groups will receive the same information regarding hypoglycaemia recognition, they will have similar ability to recognise hypoglycaemic attacks. at week of enrolment, participants will receive a phone call to remind them of using the glucose meters and documenting hypoglycaemic attacks. of special note, participants who complete the trial duration will receive the intervention at the debrief visit and after returning the hypoglycaemia diaries. all participants in both groups will be given glucose meters and test strips with a demonstration on proper use to measure their blood glucose levels at morning before breakfast daily for consecutive weeks. they will also be handed diaries and instructed to additionally, it is impractical for dm patients to measure their bg levels frequently to diagnose hypoglycaemia. hence, it is imperative to account for both symptomatic and asymptomatic types of hypoglycaemia. we will use diaries to measure types of hypoglycaemia and we will ask participants to fill in the diary on a daily basis for weeks. participants will be asked to fill in the diary with the date of each day and the fasting blood glucose reading. additionally, they will be asked to tick on the boxes for every time they experience severe hypoglycaemia, symptoms of hypoglycaemia at the time of fasting blood glucose measurement, and symptoms of hypoglycaemia during the rest of the day. participants will document a symptomatic attack if the symptoms resolve after receiving the corrective actions. the rate of each type of hypoglycaemia will be measured according to the hypoglycaemia diaries filled by the participants. severe hypoglycaemia will be measured directly according worked and what didn't, and how the study could be improved in future research. we will collect qualitative data using semi-structured interviews from a handful of participants in each study group. we have prepared the interview guide (table ) based on the objectives of the study and the mrc domains for process evaluation. table : interview schedule you have previously read in the participant information sheet that we are conducting a phone interview as a part of this study and you accepted to participate in it. therefore, i would like to ask you some questions about your experience with the study processes that have been provided to you at the inclusion visit. the information will help us in improving several aspects of the study. the interview should take about minutes. are you available to respond to the questions at this time? . i would like to start by asking: how do you describe your participation in the study so far? if needed, the interviewers may explain the question by the follow-up question: how do you rate your participation in the study from good to poor and why? . what has worked for you from the study processes that you were asked to do? -why do you think they have? what hasn't worked for you from the study processes that you were asked to do? -why do you think they haven't? . from your perspective, how the study could be improved? please consider any aspects of the study that you think could be improved. per-protocol analysis will be conducted including participants who are compliant to % or more of the study protocol. , the missingness in the primary outcome will be handled under the assumption of "missing at random" rather than "missing completely at random", because we are expecting that the probability of missing data depends on observed covariates or outcomes rather than unobserved data. therefore, the method chosen to handle missing data in the outcomes is multiple imputations. , descriptive analysis will be used across randomised groups and quantitative analysis will be conducted in rstudio version . . ( - - ). continuous variables will be presented as means, standard deviation, median and interquartile range, meanwhile, categorical variables will be presented as frequencies and percentages. the randomised groups will be examined and compared for all variables. categorical variables will be evaluated using the chi-square test. continuous variables will be tested for normality and based on the distribution of the data, appropriate parametric or non-parametric tests will be used. for example, paired t test or wilcoxon test would be applied to assess the differences in baseline variables between both groups and between participants who completed the trial as well as the participants who will be lost to follow up. the primary and secondary outcomes (total and types of hypoglycaemic attacks) will be measured across the randomised groups and compared using the analysis of covariance (ancova), and the appropriate parametric or non-parametric tests dependent on the normality of distribution. subgroup analysis will also be performed using interaction terms in regression models. we will use logistic regression analysis to examine associations between the categorical outcome variable ( frequent vs infrequent hypoglycaemia episodes) and independent variables such as sex, age, educational level, living arrangements, duration of diabetes, number of current medications, experiencing previous hypoglycaemia, the status of self- monitoring of blood glucose, types of anti-diabetic medications, baseline hba c and interactions between the independent variables. the findings will be also presented as odds ratio and % confidence intervals. a p value of less than . will be considered statistically significant. hypoglycaemia rates will be described with kaplan-meier survival curves, considering the time to the first hypoglycaemic attack as the outcome measure. qualitative data analysis the interviews will be audio-recorded then transcribed and translated into the english language. we will use the thematic analysis approach to analyse the collected data for there is a need for protocol amendments through the embedded evaluation process, the changes will be discussed by the supervisory team and will be communicated in writing to the human research ethics committee of the university of lincoln and the institutional review board of kauh. upon completion of the study, we will provide the trial site with an executive summary of the findings in the form of a report. participants would be able to get the results of the study from their health care professionals at the trial site no later than one year after the end of data collection. we are planning to disseminate the study outcomes through peer-reviewed publications and presentations in conferences. we will comply with the authorship eligibility the sugar handshake intervention is designed to be pragmatic and to facilitate transferability of evidence into practice. therefore, pharmacists can easily deliver it to the patients in different working positions including hospitals and community pharmacies. moreover, the delivery of the sugar handshake intervention is cheap and will not cost an extra burden on patients. previous studies concerning the attitudes, religious beliefs, and self-management skills amongst patients with t dm helped in assuring the appropriateness of contextual and cultural delivery as well as the implementation of the study and the sugar handshake intervention. - using blood glucose/hypoglycaemia diaries to objectively report and measure several types of hypoglycaemia is another strength. this will facilitate a more accurate measurement of hypoglycaemic events where the concern that patients may forget to report the experienced episodes is reduced. a plausible limitation that warrants consideration is the short follow up duration ( weeks), which may make it difficult to examine the sustainability of the intervention effect. however, we anticipate that this duration will decrease the dropout rate. moreover, we expect the effect of our intervention to last up to at least six months as concluded by a previous trial. another concern is the enrolment of relatives into different groups upon randomisation, which will introduce contamination bias. therefore, if a patient happens to have a relative who has already participated in the trial, he would be excluded. additionally, participants may not fully adhere to the intervention during the follow-up period. for this reason, they will receive a phone call reminder at week six of enrolment. individualising the intervention according to each patient's lifestyle and potential causes of hypoglycaemia will enhance the adherence to the intervention as well. as this is an open-label study, performance bias and ascertainment bias may result from unblinding participants and the data collectors, respectively. participants in the control group may be less adherent to the trial protocol and more likely to withdraw from the trial. however, efforts will be made to standardise the trial protocol, frequency and time of follow up, and treatment of experienced hypoglycaemia across both groups to minimise performance bias. we also anticipate that the objective measurement of the outcomes would minimise ascertainment bias. the prevalence of diabetes in jordan has been growing rapidly to reach . % in . in light of the lack of awareness regarding diabetes diagnosis, causes, and management we would expect a further increase in the number of jordanians who are diagnosed with diabetes and who would suffer from diabetes-related complications. , while pharmacists are easier to access than physicians, a possible strategy to mitigate the burden of diabetes is to establish and support the pharmacist-led, patient-oriented services. international hypoglycaemia study group ihs. minimizing hypoglycemia in diabetes diabetes care adverse macrovascular events, and inflammation in the edinburgh type diabetes study. diabetes care 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intervention including individualized nutritional recommendations reduces hemoglobin a c level, medication use, and weight in type diabetes. jmir diabetes time trends in diabetes mellitus in jordan between and health care and pharmacy practice in jordan pharmacist-led study in controlling hypoglycemia in older adults with type diabetes mellitus (rose-adam) we would like to acknowledge the university of lincoln and isra university for their support to conduct this study. we would also acknowledge the physicians and nursing staff working at kauh for facilitating recruitment and the study implementation. key: cord- -ymaql hx authors: carr, m. j.; wright, a. k.; leelarathna, l.; thabit, h.; milne, n.; kanumilli, n.; ashcroft, d. m.; rutter, m. k. title: impact of covid- on the diagnoses, hba c monitoring and mortality in people with type diabetes: a uk-wide cohort study involving million people in primary care date: - - journal: nan doi: . / . . . sha: doc_id: cord_uid: ymaql hx background: the covid- pandemic has already disproportionately impacted people with diabetes. timely diagnosis and appropriate monitoring in people with type diabetes (t d) are necessary to reduce the risk of long-term complications. methods: we constructed a cohort of m patients using electronic health records from uk general practices registered with the clinical practice research datalink (cprd), including m patients followed between march and july . we compared trends in diagnoses, monitoring and mortality in t d, before and after the first covid- peak, using regression models and -year historical data. we extrapolated the number of missed or delayed diagnoses using uk office for national statistics data. findings: in england, rates of new t d diagnoses were reduced by % ( % ci %- %) in april , with similar reductions in northern ireland, scotland and wales. between march and july, we estimated that there were more than , missed or delayed t d diagnoses across the uk. in april, rates of hba c testing in t d were greatly reduced in england (reduction: % ( % ci %- %)) with more marked reductions in northern ireland, scotland and wales (reduction: % ( - %)). reduced rates of diagnosing and hba c monitoring were particularly evident in older people, in males, and in those from deprived areas. mortality rates in t d in england were more than -fold higher ( %) in april compared to prior trends, but were only % higher in northern ireland, scotland and wales. interpretation: as engagement with the nhs increases, healthcare services will need to manage the backlog and the expected increase in t d severity due to delayed diagnoses and reduced monitoring. older people, men, and those from deprived backgrounds with t d may be groups to target for early intervention. funding: national institute for health research (nihr) greater manchester patient safety translational research centre the first wave of the covid- pandemic has had major health and economic effects across the world. so far in the uk, there have been more than k covid-related deaths with disproportionate impacts in people with diabetes; nearly a third of all covid-related deaths having occurred in people with diabetes. the impact on the nhs, and in particular on diabetes services, has been enormous, with the suspension of much routine care. as we enter the second wave in the uk, there is an urgent need to minimise the harm done through suspension of routine services and to prioritise care and resources to areas of greatest need. the diagnosis of type diabetes occurs almost exclusively in primary care. timely diagnosis is critically important as delays will increase the risk of long-term complications. there is limited data on the indirect consequences of the covid- pandemic on the incidence and monitoring of diabetes in primary care. likewise, there is limited information on covid- impacts on mortality rates in people with diabetes during and after the first wave of covid- . therefore we used a large primary care longitudinal dataset, broadly representative of the uk population, aiming to compare: i) the uk-wide incidence of type diabetes; ii) the frequency of hba c testing; and iii) mortality rates in people with type diabetes, before and after the nationwide covid- lockdown in march . we compared observed and predicted rates using data covering ten years prior to the pandemic. since older people and more socially disadvantaged groups have been disproportionally affected by covid- infections, and since the same groups may be more adversely impacted by the unintended consequences of government interventions, we aimed to study variation in outcomes by gender, age group, deprivation level and region. we examined primary care electronic health records using the clinical practice research datalink (cprd) aurum and gold databases. , the study population consisted of , , patients from , general practices in england, with a further practices in northern ireland ( , patients), practices in scotland ( , , patients), and in wales ( , , patients). a total of , , patients were included for estimating the expected rates in the pre-covid- period (january -february ). in line with guidance from the cprd's central administration, aurum and gold databases were analysed separately. the cprd contains anonymised consultation records and includes patient demographic information, symptoms, diagnoses, medication prescriptions, and date of death. we also examined practice-level index of multiple deprivation (imd) quintiles, a measure representing an area's relative level of deprivation, ranked within each uk nation. . cc-by . international license it is made available under a perpetuity. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted october , . ; https://doi.org/ . / . . . doi: medrxiv preprint to enable comparisons of rates before the covid- outbreak, during its peak and after the peak, we included patient records from january that established long-term trends and patterns of seasonality. we focussed primarily on reporting observed versus expected rates from / / to / / . first, we estimated incidence rates of type diabetes diagnoses, new prescriptions for metformin (the most commonly prescribed medication in new-onset type diabetes) and insulin, and rates of hba c testing and mortality in people with type diabetes. incident type diabetes was identified from read/snomed/emis codes used in cprd gold and aurum (see https://clinicalcodes.rss.mhs.man.ac.uk). the cprd aurum and gold databases were analysed separately, with data from aurum restricted to english practices and gold providing information on practices in northern ireland, scotland and wales. the use of two discrete data sources also enabled independent replication of our findings. all code lists and medication lists were verified by two senior clinical academics (a diabetologist: mkr, and a senior academic pharmacist: dma). for each patient, we defined a 'period of eligibility' for study inclusion which commenced on the latest of: the study start date ( st january ); the patient's most recent registration with their practice; the date on which data from the practice was deemed 'up-to-standard' by the cprd. a patient's period of eligibility ended on the earliest of: registration termination; the end of data collection from their practice; death. for incident diagnoses and prescriptions, we also applied a 'look-back' period during which a patient was required to have been registered for at least a year prior to the event. flow diagrams illustrating the delineation of the study cohorts using cprd aurum and gold are presented in supplementary figures and respectively. the denominator for the incidence rates was the aggregate person-months at risk for the whole eligible study population. mortality and testing rates in people with type diabetes were calculated using the person-months at risk from all those with type diabetes as the denominator. incidence, mortality and testing rates were stratified by gender, age group (< , - , - , - , - and ≥ years), practice-level deprivation (imd quintiles) and region (in england) or nation (in the rest of the uk). the data were structured in a time-series format with event counts and 'person-months at risk' aggregated (by year and month) with stratification by gender, age group, deprivation quintile and region (or nation in gold). mean-dispersion negative binomial regression models were used to estimate expected monthly event counts from march onward based on antecedent trends since . the natural logarithm of the denominator (person-months at risk) was used as an offset in each regression model. to account for possible seasonality and long-term linear trends, calendar month was fitted as a categorical variable and time as a continuous variable with the number of months since the start of the study serving as the unit of measurement. for each month studied, observed and expected event counts were converted to rates using the observed person-month denominator. the monthly expected rates, and their % confidence intervals, is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted october , . ; https://doi.org/ . / . . . doi: medrxiv preprint were plotted against the observed rates. as they share a common denominator, differences between expected and observed monthly rates are expressed as a percentage 'rate reduction (or increase)'. extrapolated estimates of the number of missed (or delayed) diagnoses of type diabetes were derived using the discrepancy between observed and expected frequencies from march onward, and approximations of the proportional representation of the populations of england and the rest of the uk (in cprd aurum and gold respectively) using data from the office for national statistics. all data processing and statistical analyses were conducted in stata version (statacorp lp, college station, tx). we followed record (reporting of studies conducted using observational routinelycollected health data) guidance (see online supplement). the funders of the study had no role in study design, data collection, data analysis, data interpretation, or writing of the report. our focus was on the impact of the covid- pandemic between march and july . using the inclusion criteria described in the study design, a mixed cohort was utilised consisting of patients whose period of eligibility began before st march and those who became eligible for inclusion between st march and th july . the study cohort was comprised of , , patients (median (iqr) age: ( , ) years, % female) of whom , had type diabetes. of those with type diabetes, the median (iqr) age was ( , ) years, % were female and % lived in an area that was in the most deprived quintile compared to the rest of the uk. in april , the rate of new diagnoses of type diabetes in english primary care was reduced by % ( % ci % to %) compared to the expected rates based on -year historical trends (figure a; supplementary table ). prior to march , rates of type diabetes diagnoses in english practices were higher in older individuals, in men, and in people from deprived areas. these groups experienced the greatest reductions in rates for new type diabetes diagnosis at the time of the first covid- peak (supplementary figure ). the reduced rates of type diabetes diagnosis in april were mirrored by reduced rates of new metformin prescriptions in english practices (reduction: % ( % ci % to %; in april, rates of hba c testing in england were greatly reduced in people with type diabetes (reduction: % ( % ci: % to %)); is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted october , . the reduced rates of diagnosis, new insulin/metformin prescribing and hba c testing increased gradually between may and july though levels remained well below expected rates based on -year historical data ( figure a-d) . overall in english practices, between / / and / / , the rate of diagnosis of type diabetes was reduced by % ( % ci: % to %), metformin prescribing was reduced by % ( % to %), insulin prescribing fell by % ( % to %), and hba c testing in people with type diabetes was reduced by % ( % to %); supplementary table . . cc-by . international license it is made available under a perpetuity. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted october , . ; https://doi.org/ . / . . . doi: medrxiv preprint in april , mortality rates in people with type diabetes in england were more than -fold higher compared to prior trends (mortality rate increase: % ( % ci: % to %); figure a; supplementary table ). peaks in mortality were seen particularly in individuals aged over years (supplementary figure a). mortality rates returned to expected levels in people with type diabetes and sub-groups between may and june (figure a) . overall, between / / and / / , the rate of mortality in people with type diabetes in english practices was increased by % ( % ci: % to %); supplementary table . is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted october , . ; https://doi.org/ . / . . . doi: medrxiv preprint metformin prescribing reduced: % vs. %; supplementary tables and ). over the same ½ month period, the overall reduction in hba c testing in type diabetes was greater in cprd gold practices based in northern ireland, scotland and wales (gold vs. aurum: % vs. %) but the mortality rate increase was lower than in england (gold vs. aurum: % vs. %; figure ; supplementary tables and ). using primary care data from million people in the uk, and -year historical data, we have shown that within the first months of the nationwide 'lockdown' in march , the indirect consequences of the covid- pandemic led to: i) a - % reduction in new diagnoses of type diabetes, with older individuals, males, and people from deprived areas experiencing the greatest reduction in diagnosis rates; ii) a - % reduction in hba c testing; iii) a reduction in metformin and insulin prescribing, particularly in older people with type diabetes, supporting the reduced rates of diagnosis and monitoring; and iv) a short-term % increase in mortality rate in people with type diabetes in england and a % increased mortality rate across the rest of the uk. there is limited prior data on the impact of the covid- pandemic on the diagnosis of type diabetes. a study using primary care data from salford, uk showed fewer diagnoses of type diabetes than expected between march and may , which amounted to a % reduction in activity. here we extend these observations by assessing primary care data across the uk and by providing supplementary data on hba c testing and mortality. we show that the reduced rate of diagnosis applies to all areas of the uk and not just to deprived areas of the uk such as salford. to the best of our knowledge, no study has reported the impact of the covid- pandemic on hba c monitoring in diabetes, and no study has described national variation in mortality rates in people with type diabetes following the first peak of the pandemic. our data have important clinical implications. in early march , gps were advised to minimise the number of face-to-face contacts they had with their patients, including nhs health-checks. our data suggests that this reduction of clinical services has led to major reductions in the diagnosis and monitoring of type diabetes. the concomitant reductions in new prescriptions issued for metformin and insulin further support these findings. type diabetes develops over many years, so it seems unlikely that people's behaviour during the pandemic has reduced the true incidence of these conditions. assuming that the true incidence of type diabetes has remained constant from march , our data suggest that, across the uk, the indirect consequences of the pandemic have led to more than k missed/delayed diagnoses of type diabetes in the ½ months between / / and / / . this figure may be an underestimate if sedentary lifestyles and adverse dietary changes during lockdown have increased obesity rates in the general population. these data are a clinical concern because undiagnosed type diabetes will cause potentially serious long-term complications. the huge reduction in the rate of hba c testing is another important concern for people with type diabetes, because they, and their clinicians, often rely solely on hba c data to make decisions about . cc-by . international license it is made available under a perpetuity. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted october , . ; https://doi.org/ . / . . . doi: medrxiv preprint treatment. the reduction in new prescriptions for insulin was largely observed in older individuals suggesting this reduction was explained by a failure to intensify therapy in people with poorly controlled long-duration type diabetes. there are already concerns in the uk about clinical inertia in diabetes management, with frequent failures to escalate care when glucose control is poor. these hba c data indicate potential further delays in the management of type diabetes that are predicted to cause avoidable diabetes-related long-term complications. a reduced frequency of hba c testing in primary care might also contribute to missing people with non-diabetic hyperglycaemia who might benefit from referral to the nhs diabetes prevention programme. the higher covid-related death rate in people with diabetes has been well-documented, , , and our data support these observations. here, we add to these data by showing national differences in the impact of covid- on mortality rates in people with type diabetes, with higher rates observed in england compared to the rest of the uk. further research is required to understand how population characteristics including ethnicity, population density and deprivation might explain these differences. as engagement with health services increases, and hopefully is maintained during the second covid- peak, our data predict a marked increase in presentations with incident type diabetes. should this occur, then healthcare services will need to manage this backlog, and the expected increase in the severity of diabetes brought about by delayed diagnoses. older individuals, males and people from deprived backgrounds appear to be most adversely affected by reductions in rates of diagnosis and monitoring of type diabetes. as outpatient diabetes services start to open up, these individuals may be a group to target for early intervention, and in particular, for hba c testing and treatment intensification when appropriate. if a second full lockdown occurs, then effective public communications should ensure that patients remain engaged with diabetes services including hba c screening and monitoring, and the use of remote consultations. , our study had several strengths: this is the first uk-wide study reporting the indirect impact of the covid- pandemic on the diagnosis of type diabetes, related prescribing and hba c testing in primary care. our findings in english practices were replicated using data from other parts of the uk. by combining assessments of diabetes coding and prescribing, our data supports the conclusion that reduced rates of diagnoses are genuinely explained by missed diagnoses. our study has some limitations: first, ethnicity coding is not adequately captured in primary care and therefore we had limited ability to explore ethnicityrelated variation in care and outcomes. future studies will incorporate linked secondary care data that has more complete capture of ethnicity data. second, it is possible that some diabetes diagnoses may have been made in a hospital setting following an acute presentation and that the related primary care coding had not been updated at the time of our data extraction. while hospital presentation of incident diabetes may have occurred in some instances, it would not explain the reductions in new prescribing for metformin and this potential explanation does not fit with our local experience. in general, people have avoided hospital attendance during the pandemic. for example, one study documented a % reduction in emergency admissions in the uk. finally, although our results and conclusions are relevant to the uk population, generalisability to other healthcare systems may be limited. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted october , . ; https://doi.org/ . / . . . doi: medrxiv preprint in conclusion, we highlight marked reductions in the diagnosis and monitoring of type diabetes as indirect consequences of the covid- pandemic. over the coming weeks, healthcare services will need to manage this predicted backlog, and the expected increase in the severity of diabetes due to delayed diagnoses. older people, men and those from deprived backgrounds with type diabetes may be specific groups to target for early hba c testing and intervention. should a second full national lockdown occur, then effective public communications should ensure that patients remain engaged with diabetes services including hba c screening and monitoring and the use of remote consultations. the lead authors (mjc and akw: the manuscript's guarantors) affirm that the manuscript is an honest, accurate, and transparent account of the study being reported; that no important aspects of the study have been omitted; and that any discrepancies from the study as planned have been explained. the corresponding author had full access to all of the data and the final responsibility to submit for publication. all clinical codes used in the study are published on clinicalcodes.org. electronic health records are, by definition, considered "sensitive" data in the uk by the data protection act and cannot be shared via public deposition because of information governance restriction in place to protect patient confidentiality. access to data are available only once approval has been obtained through the individual constituent entities . cc-by . international license it is made available under a perpetuity. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted october , . ; https://doi.org/ . / . . . doi: medrxiv preprint supplementary figure : comparison of monthly hba c testing rates in people with type diabetes in primary care by age, gender, deprivation level and by region before and after the first covid- peak in england (cprd aurum) is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted october , . ; https://doi.org/ . / . . . doi: medrxiv preprint supplementary figure : comparison of monthly incidence rates for metformin prescribing in primary care by age, gender, deprivation level and by region before and after the first covid- peak in england (cprd aurum) is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted october , . ; https://doi.org/ . / . . . doi: medrxiv preprint supplementary figure : comparison of monthly incidence rates for insulin prescribing in primary care by age, gender, deprivation level and by region before and after the first covid- peak in england (cprd aurum) is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted october , . ; https://doi.org/ . / . . . doi: medrxiv preprint supplementary figure : comparison of monthly mortality rates in people with type diabetes in primary care by age, gender, deprivation level and by region before and after the first covid- peak in england (cprd aurum) is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted october , . ; https://doi.org/ . / . . . doi: medrxiv preprint supplementary figure : comparison of observed and expected monthly incidence rates for type diabetes in primary care, hba c monitoring and new prescriptions for metformin and insulin before and after the first covid- peak in northern ireland, scotland and wales (cprd gold) is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted october , . ; https://doi.org/ . / . is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted october , . ; https://doi.org/ . / . . . doi: medrxiv preprint uk's record on pandemic deaths risk factors for covid- -related mortality in people with type and type diabetes in england: a population-based cohort study associations of type and type diabetes with covid- -related mortality in england: a whole-population study van den bruel a. opportunities for earlier diagnosis of type diabetes in children: a case-control study using routinely collected primary care records data resource profile: clinical practice research datalink (cprd) diagnosis of physical and mental health conditions in primary care during the covid- pandemic: a retrospective cohort study letter to general practices the silent pandemic: how lockdown is affecting future health legacy benefits of blood glucose, blood pressure and lipid control in individuals with diabetes and cardiovascular disease: time to overcome multifactorial therapeutic inertia? factors associated with covid- -related death using opensafely h(ome)ba c testing and telemedicine: high satisfaction of people with diabetes for diabetes management during covid- lockdown endocrinology in the time of covid- : remodelling diabetes services and emerging innovation video consultations for covid- what is happening to non-covid deaths? reasons: (a) registration ended prior to study period due to death or migration from practice (n = , , ); (b) registration end date defined on or prior to registration start date (n = , ); (c) aged under on study period end ) and/or compare expected and observed rates acceptability defined by cprd as meeting certain quality standards reasons: (a) registration ended prior to study period due to death or migration from practice (n = , , ); (b) registration end date defined on or prior to registration start date (n = , ); (c) aged under on study period end ) and/or compare expected and observed rates supplementary table : comparison of observed and expected monthly incidence rates for type diabetes in primary care, hba c monitoring in type diabetes, new prescriptions for metformin and insulin, and deaths in people with type diabetes between / / and / / and at the covid- peak in april , in england (cprd aurum) key: cord- - bniy b authors: peteranderl, christin; herold, susanne title: the impact of the interferon/tnf-related apoptosis-inducing ligand signaling axis on disease progression in respiratory viral infection and beyond date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: bniy b interferons (ifns) are well described to be rapidly induced upon pathogen-associated pattern recognition. after binding to their respective ifn receptors and activation of the cellular jak/signal transducer and activator of transcription signaling cascade, they stimulate the transcription of a plethora of ifn-stimulated genes (isgs) in infected as well as bystander cells such as the non-infected epithelium and cells of the immune system. isgs may directly act on the invading pathogen or can either positively or negatively regulate the innate and adaptive immune response. however, ifns and isgs do not only play a key role in the limitation of pathogen spread but have also been recently found to provoke an unbalanced, overshooting inflammatory response causing tissue injury and hampering repair processes. a prominent regulator of disease outcome, especially in—but not limited to—respiratory viral infection, is the ifn-dependent mediator trail (tnf-related apoptosis-inducing ligand) produced by several cell types including immune cells such as macrophages or t cells. first described as an apoptosis-inducing agent in transformed cells, it is now also well established to rapidly evoke cellular stress pathways in epithelial cells, finally leading to caspase-dependent or -independent cell death. hereby, pathogen spread is limited; however in some cases, also the surrounding tissue is severely harmed, thus augmenting disease severity. interestingly, the lack of a strictly controlled and well balanced ifn/trail signaling response has not only been implicated in viral infection but might furthermore be an important determinant of disease progression in bacterial superinfections and in chronic respiratory illness. conclusively, the ifn/trail signaling axis is subjected to a complex modulation and might be exploited for the evaluation of new therapeutic concepts aiming at attenuation of tissue injury. interferons (ifns) are well described to be rapidly induced upon pathogen-associated pattern recognition. after binding to their respective ifn receptors and activation of the cellular jak/signal transducer and activator of transcription signaling cascade, they stimulate the transcription of a plethora of ifn-stimulated genes (isgs) in infected as well as bystander cells such as the non-infected epithelium and cells of the immune system. isgs may directly act on the invading pathogen or can either positively or negatively regulate the innate and adaptive immune response. however, ifns and isgs do not only play a key role in the limitation of pathogen spread but have also been recently found to provoke an unbalanced, overshooting inflammatory response causing tissue injury and hampering repair processes. a prominent regulator of disease outcome, especially in-but not limited to-respiratory viral infection, is the ifn-dependent mediator trail (tnf-related apoptosis-inducing ligand) produced by several cell types including immune cells such as macrophages or t cells. first described as an apoptosis-inducing agent in transformed cells, it is now also well established to rapidly evoke cellular stress pathways in epithelial cells, finally leading to caspase-dependent or -independent cell death. hereby, pathogen spread is limited; however in some cases, also the surrounding tissue is severely harmed, thus augmenting disease severity. interestingly, the lack of a strictly controlled and well balanced ifn/trail signaling response has not only been implicated in viral infection but might furthermore be an important determinant of disease progression in bacterial superinfections and in chronic respiratory illness. conclusively, the ifn/ trail signaling axis is subjected to a complex modulation and might be exploited for the evaluation of new therapeutic concepts aiming at attenuation of tissue injury. ( ) cell death induction, e.g., bcl- -associated x protein, caspase- , fas-associated protein with death domain, fas ligand, and tnf-related apoptosis-inducing ligand (trail) dsrna, polyi:c ( , ) iav ( , , , ) sendai virus ( ) trail virus control by apoptosis induction in infected cells iav ( , , ) tissue injury by apoptosis of both infected and non-infected alveolar epithelial cells, lung macrophages iav ( , , ) rsv ( ) necrosis of fibroblasts, dendritic cells, and epithelial cells iav ( , , ) increased cellular infiltration cov ( ) decreased expression of na,k-atpase, impaired epithelial fluid reabsorption iav ( ) introduction in , isaacs and lindenmann ( ) first recognized the potential of a soluble and probably cell-derived factor to combat influenza virus infection and named this factor interferon [(ifn) from latin interferre, to interfere]. since then, three subgroups of ifns have been defined, primarily by their differential receptor usage. while the groups of type i ifn and type iii ifn comprise largely agents directly limiting pathogen spread by improving cellular counter measurements, ifn-γ, the sole type ii ifn, has been mainly implicated in the modulation of innate and also adaptive immune responses ( , ) . accordingly, type i and iii ifns are key signaling molecules in viral control, and lack of both signaling pathways results in increased viral loads and disease severity. still, there is accumulating evidence that not only lack of an antiviral response but that also an unbalanced overshooting activation of ifns contributes to an exaggerated inflammatory reaction, tissue injury, reduced proliferative capacity, and thus enhanced disease severity ( table ) . especially in viral infections, this effect has not only been tracked down to ifn signaling in general but specifically to the exaggerated production of key effector ifn-stimulated genes (isgs) ( ) . a prominent example is the tnf-related apoptosis-inducing ligand (trail) that displays an ambivalent role in viral infection ( - ) ( table ) . whereas first identified as factor produced by immune cells in non-respiratory infection ( , ) , trail is now especially well studied in influenza a virus (iav) infection, where it is released in high amounts from bone marrow-derived macrophages upon pathogenassociated molecular patterns (pamps) recognition and type i ifn production ( ) . macrophage-released soluble trail, but also membrane-bound cell-associated trail, acts via distinct receptors on infected but also on non-infected, neighboring cells. in viral infection, its preliminary role is to drive infected cells into apoptosis to limit virus spread. however, studies performed within the last decade demonstrate that trail's antiviral activity seems to be outweighed by the functional and structural damage it induces not only in infected but also in bystander cells such as uninfected cells of the alveolar epithelium ( , ) . this process is not only relevant in promoting viral disease progression but has further implications in bacterial superinfection and probably also in chronic diseases. the recognition of the ambivalent role of ifn-driven signaling in vivo is a first important step to better understand disease progression and to envision novel treatment options for primary viral respiratory infection targeting distinct host-derived signaling mediators such as trail. it is a commonly accepted concept that-as janeway ( ) already proposed in -immune activation toward invading pathogens is mounted upon recognition of pamps. pamps are evolutionary conserved biomolecules such as proteins, lipids, nitrogen bases, sugars, and complexed biomolecules such as lipoglycans that are essential to the survival of a given pathogen ( ) . pamps are recognized by distinct pattern recognition receptors (prrs) that are germ-line encoded and-similar to pamps-usually show a high evolutionary conservation. the first recognized and probably most intensely studied family of prrs are the toll-like receptors [tlrs; reviewed in mogensen ( ) ; leifer and medvedev ( ) ]. in viral infection, both host cell membrane-localized tlrs (tlr , tlr , detecting viral envelope proteins) and endosomal tlrs (tlr , tlr , tlr , and tlr , nucleic acid sensors) initiate signal transduction cascades leading to ifn production (figure ). tlr activation results in either myeloid differentiation factor (myd ) or tir-domaincontaining adaptor protein-inducing ifn-β (trif) recruitment that both trigger various downstream signaling events, eventually leading to ifn regulatory factor (irf) , irf , and nfκb nuclear translocation as well as map kinase and activator protein (ap- ) activation ( , ) . similar to endosomal tlrs, the cytosolic retinoic acidinducible gene (rig)-i-like receptors (rlrs) are specialized to recognize viral nucleic acid contents and are central prrs relevant to mount an antiviral response, providing resistance to most rna (e.g., orthomyxoviruses) and some dna (e.g., reoviruses) viruses [reviewed in ref. ( , ) ]. both melanoma differentiation-associated gene (mda- ) and rig-i recognize dsrna, ′-triphosphate rna, or the synthetic analog to dsrna, polyi:c ( , ) . both drive the dimerization of the mitochondriaassociated adaptor protein ifn-β promoter stimulation (ips- ) (also named mavs, visa, cardif). a subsequently activated cascade including tradd (tnf receptor type -associated death domain protein), traf (tnf receptor-associated factor ), and tank (traf family member-associated nf-κb activator) induces the phosphorylation of irf and irf , resulting in type i ifn production ( ) . the third rlr, lgp , so far has primarily been implicated to regulate rig-i or mda- as a cofactor; however, a recent study by stone et al. ( ) demonstrated a novel, non-redundant, and independent role of lgp in west nile virus infection. another class of prr, the nucleotide oligomerization domain (nod)-like receptors (nlrs), has mainly been implicated in bacterial recognition ( ) , still several nlrs are activated as well upon virus infection. especially, nlrp is known to recognize rna of different viruses including hepatitis c virus, measles virus, influenza, and vesicular stomatitis virus (vsv) ( ) ( ) ( ) ( ) , resulting in inflammasome formation and caspase- -dependent activation of il- β and il- ( ) ( ) ( ) . in addition, virus infections are sensed also by a structurally diverse group of viral rna and dna sensors residing in the cytoplasm. these include the cyclic gmp-amp synthase that synthesizes the second messenger cgamp. cgamp in turn activates stimulator of ifn genes (sting), tank-binding kinase , and irf , triggering ifn production ( ) ( ) ( ) . moreover, sting itself acts as a prr and has been implicated in dna virus recognition including hsv, adenovirus, vaccinia virus, and papilloma virus and in sensing of retroviral rna-dna hybrids ( ) and rna viruses after being activated by rig-i ( , ) . another cytosolic nucleic acid sensor, pkr, is well known for its phosphorylation of eukaryotic initiation factor α (eif α) in response to viral dsrna. phosphorylation of eif α results in its deactivation, host translational shut-off, and the limitation of viral replication. of note, the pkr-eif α-driven inhibition of protein synthesis can contribute to an ips- -dependent ifn-β induction ( ) . furthermore, pkr has been implicated in efficient type i ifn activation by tlr in response to dsrna ( ) and can mediate-at least partially-activities of irf ( ) . in addition to type i ifns, also type iii ifns exert antiviral activity and are widely expressed after viral recognition, being produced by most cell types including epithelial, endothelial, fibroblast, and polymorphonuclear cells [reviewed in ref. ( , ) ]. like type i ifns, type iii ifns are induced in viral infection by the prr rig-i as well as tlr and tlr and rely on the activation of the same transcriptional activators, including irf , irf , and nfκb. these observations initially led to the conclusion that type i and type iii ifn comprised two completely redundant systems to induce isgs in response to pamp recognition. however, more recent data suggest distinct selection mechanisms for either type i or type iii ifn expression. as such, ips- specifically induces ifn-λ, but not type i ifn, when located at the peroxisomal membrane instead of the mitochondrial membrane in response to rig-i activation by reovirus, sendai virus, or dengue virus challenge ( ) . interestingly, type iii ifn induction is largely independent toward ap- translocation, which facilitates an instantaneous induction of ifn-λ after viral recognition, highlighting it as an important immediate factor driving innate microbial defense mechanisms. release of ifns upon pathogen recognition is a highly conserved mechanism-found from teleost fish to insects and mammals-to prepare the surrounding cells as well as the host defense against the invading threat ( , ). whereas often high-level ifn production relies on specialized sentinel cells such as macrophages or dendritic cells (dcs), mostly all cells of the multicellular organisms are able to respond to at least one type of ifn by expression of respective receptors. receptor binding then induces a signal transduction cascade relying on the janus kinase (jak) and signal transducer and activator of transcription (stat), which results in efficient transcription of a plethora of different isgs in infected as well as bystander cells ( , ) . ifns engage a classical canonical signal transduction cascade employing jak/stat molecules after binding to their respective receptors. herein, type i ifns ligate to their common heterodimeric receptor consisting of the ifn-α receptor (ifnar) and ifnar subunits, whereas type iii ifns act via a interleukin- receptor (il- r )/ifn-λ receptor (ifnlr ) heterodimer that to date has been reported to be restricted in its expression to epithelial cells ( ) . in type i ifn signaling, ifnar engagement leads to the activation of the receptor-associated protein tyrosine kinases jak and tyrosine kinase followed by the recruitment or repositioning of already associated but elsewise latent cytoplasmatic transcription factors stat and stat . consequently, stat /stat are phosphorylated on conserved tyrosine residues, they disassemble, undergo conformational changes enabling their heterodimerization as well as the exposure of a nuclear localization sequence. subsequently, the stat /stat heterodimer translocates into the nucleus where it interacts with the irf to form the trimeric ifn-stimulated factor (isgf ). isgf binds cognate dna sequences, the ifn-stimulated response elements (isre), finally leading to isg induction. also type iii ifn interaction with the il- r /ifnlr receptor complex triggers stat /stat heterodimerization, nuclear translocation, and isgf assembly ( ) . interferon signaling results in the induction of isgs evoking different cellular responses against viral infection, both in infected as well as in non-infected cells, including direct antiviral, immune-modulatory, or cell death-inducing effects to enable an immediate and robust response to a pathogen challenge. many isgs directly interfere with viral replication on an intracellular level. well-studied examples of antiviral isgs comprise ifn-induced transmembrane proteins (ifitms) effective in iav, west nile virus, and dengue virus infection ( , ) , the myxovirus resistance protein a (mxa) that interferes with vsv mrna production and binds the iav nucleocapsid to prevent nuclear translocation of viral genetic material ( ) ( ) ( ) , the - -oligoadenylate synthase (oas), which activates rnase l triggering viral rna degradation, or the prr pkr, which besides activating the ifn response has a major impact on viral protein translation by inhibiting the eif α ( ) . more recently identified isgs include the plasminogen activator inhibitor that blocks iav infection by inhibiting glycoprotein cleavage executed by extracellular airway proteases ( ) or the antiviral isg that limits iav viral replication via its exonuclease activity most likely by interfering with the viral np ( ) . accordingly, ifn pretreatment usually results in the establishment of an antiviral state that limits viral replication and spread from the start of infection and thus favors milder disease outcomes. ifn-α pretreatment has been demonstrated to limit viral spreading of seasonal iav strains and thus decrease morbidity and mortality in mice, guinea pigs as well as ferrets ( ) ( ) ( ) . as shown by a study by tumpey et al. ( ) , this effect can be attributed to the early induction of antiviral isgs including mxa. importantly, type i ifn pretreatment also dampens early replication of highly pathogenic avian influenza in ferrets ( ) . also in respiratory syncytial virus (rsv) infection, treatment with recombinant ifn-α results in significantly decreased lung viral titers, alveolar inflammatory cell accumulation, and clinical disease in rsv-infected mice ( ) . in addition, respiratory infections caused by emerging coronaviruses (cov) can be ameliorated by type i ifn pretreatment strategies. in an in vivo macaque model (macaca fascicularis) of severe acute respiratory syndrome (sars)-cov infection, it could be demonstrated that pretreatment with pegylated ifn-α significantly diminished cov replication and excretion and resulted in reduced pulmonary damage ( ) . macaques also serve as a preclinical model for middle east respiratory syndrome (mers)-cov, and similar to sars-cov, ifn-α in combination therapy with ribavirin reduces viral replication and severe histopathological changes ( ) . in line, genetic alteration leading to an enhanced type i ifn signaling has been demonstrated to limit iav-induced disease outcomes, as a recent study by xing et al. ( ) reported that deletion of trim , a negative regulator of nemo, which leads to nfκb induction and therefore enhanced type i ifn production, is protective in vivo in iav-infected mice. conversely, the genetic depletion of ifn signaling in ifn receptor-deficient mice can result in a lack of viral control, resulting in enhanced viral titers in different viral infections including rsv or iav ( , ) . still, it must be noted that this effect is often mild in ifnar-or ifnlr-deficient animals, which is probably related to a certain redundancy between type i and type iii ifn signaling in limiting viral spreading in epithelial cells ( ) . in contrast, ifnar/ ifnlr-double knockout or stat knockout animals that are deficient in both type i and type iii ifn signal transduction succumb more readily to infection due to excessive viral replication ( ) ( ) ( ) . vice versa, mutations in key isgs such as ifitm are associated with increased iav disease severity in mice and humans ( ) . however, ifn pretreatment and genetic loss-of-function approaches generally are not relevant to human respiratory virus-induced hospitalizations, where patients already present with ongoing respiratory infection and inflammation, and preclinical studies underline that type i ifn signaling in an already inflamed organ is rather detrimental and enhances tissue injury, and lack of type i ifn in vivo may even ameliorate disease outcome. accordingly, in cases where the antiviral defense was not compromised (e.g., in animals with efficient type iii ifn signaling) ifnar-deficient mice infected with sendai virus or iav were reported to be more resistant to infection-induced morbidity and mortality ( , ) . similarly, in sendai virus in vivo infection, wetzel et al. ( ) showed that increased ifn-β levels in the lung homogenate correlates to increased morbidity and mortality, and also for sars-cov, a recent study demonstrates that high type i ifn induction in an already ongoing viral infection contributes to mortality in sars-cov-infected mice ( ) . also for iav infection, type i ifn application after infection has been proven to drive disease severity ( ) . of note, the detrimental effects of type i ifns were especially pronounced in mice lacking central antiviral factors, namely the ifit protein in sendai virus infection and mxa in iav. interestingly, beilharz et al. ( ) demonstrated that application of low doses of ifn-α reduces viral load, which to a certain degree led to attenuated disease progression, whereas high dose application of type i ifn contributed to morbidity ( ) . in line, high expression levels of isgs have been shown to correlate to worse outcomes in ards patients ( ) . this observation corresponds to reports stating that the ifn threshold needed to induce antiviral isgs-showing a beneficial effect in acute respiratory viral infection-is by at least -fold lower than the ifn dose necessary to trigger isgs that show immunomodulatory, death-inducing, or anti-proliferative effects and thus can contribute to disease progression ( ) ( ) ( ) ( ) . altogether, these data demonstrate that ifns may significantly contribute to unbalanced inflammation and tissue injury during respiratory viral infection depending on expression levels and duration of ifn-related signaling events. to date, the underlying mechanisms leading to the ifndependent enhanced disease progression are not fully understood but often result from a dysregulated ifn signaling response. one mode of action of ifn and ifn-stimulated isgs is to stimulate negative feedback loops on ifn signaling. for example, suppression of jak or stat via specific phosphatases, expression of suppressor of cytokine signaling (socs) and socs , or ubiquitination and endocytosis of the ifn receptors ( ) ( ) ( ) ( ) desensitize cells to ifn signaling and allow recovery and the return to homeostasis after microbial challenge. as demonstrated by bhattacharya et al. ( ) , the lack of ifnar downregulation and thus the failure to initiate ifn-desensitization contributes to increased inflammatory signaling, extensive lung injury and, importantly, also impaired tissue regeneration ( ) . moreover, ifns are immunomodulatory and shape the specific responses of cells of the immune system, which has been implied to influence disease progression both positively and negatively. in a recent study, type i ifns have been associated in the regulation of innate lymphoid immune cells (ilc) in iav infection, where they-in concert with ifn-γ and il- -promote an ilc -dependent restriction of immunopathology ( ) . moreover, type i ifns play an important role in stimulating the immune response driven by dcs; they stimulate the expression of mhc molecules as well as the co-stimulatory ligands cd and cd and thus activate t cell responses ( , ) . additionally, ligand-driven activation of ifnar enhances the proliferation of cd positive t cells, especially early in infection. however, late in infection, type i ifns were also implied in decreasing t cell expansion upon sars-cov and arenavirus infection ( , ) , which might potentially be related to the above described desensitization upon prolonged ifn signaling and might be detrimental if initiated too early in infection. in line, pinto et al. ( ) reported an impairment of t cell responses upon type ifn induction in west nile virus infection. in b cells, the lack of ifnar has been demonstrated to result in enhanced release of neutralizing antibodies in iav infection ( ) implying a repressive role for type i ifn in b cell antibody production. however, immunization studies by le bon et al. ( ) reported the necessity of ifnar on b cells for efficient igm and igg production, underlining the need for further studies to understand the detailed effects of ifn-dosage and timing adaptive immunity activity upon respiratory viral infection. type i ifns additionally induce the production of high levels of pro-inflammatory cytokines that have been closely linked to worsened outcomes of acute respiratory viral infection. especially in iav, disease severity and disease progression are linked with an overshooting, ifn-driven inflammatory response, in which further exogenous supplementation with type i ifn in fact correlates with increased morbidity and mortality ( , ) . in non-human primates, iav infection with a highly pathogenic h n isolate evokes a strong induction of type i ifn, resulting in severe lung injury by a necrotizing bronchiolitis and alveolotis ( ) . ifn levels in turn have been demonstrated to cause elevated pro-inflammatory cytokine levels after in vivo iav infection and additionally, in human alveolar macrophages, the release of pro-inflammatory cytokines (e.g., mcp- ) are preceded by a robust type i ifn response ( ) . importantly, also in human infection with h n , levels of pro-inflammatory cytokines are strongly elevated in bronchoalveolar lavage fluid, and cytokine levels have been associated with organ damage and worsened disease outcomes ( , ) . still, it should be noted that due to strain differences in virus-elicited prr activation and, importantly, ifn antagonism by the iav non-structural (ns) protein, ifn levels and disease severity do not always directly correlate; actually, the extent to which ns can suppress the ifn response relates to prolonged viremia and thus can also be a determinant of virus pathogenicity both in human bronchial epithelial cells and in an in vivo model of iav infection ( , ) . alongside iav, also in rsv infection the induction of high levels of pro-inflammatory cytokines has been directly related to type ifn, as rsv-infected but ifnar-deficient mice presented with significantly diminished pro-inflammatory cytokine release, which translated into an attenuated disease course ( ) . also in sars-cov, the late phase type i ifn induction relates to accumulation of inflammatory macrophage populations and elevated lung cytokine levels ( ) . in addition to antiviral, immunomodulatory, and pro-inflammatory isgs, ifn signaling results in the transcription and translation of cell death-inducing isgs. in the context of viral infection, these factors provide a mode to block viral spreading and reinfection by killing those infected cells, in which the internal activation of antiviral isgs is not sufficient to restrict viral replication. thus, the infected cell is sacrificed to prevent the release of infectious progeny virions to limit viral spreading. however, especially in the lung, the disruption of the alveolar epithelial barrier by cell death of infected cells, but importantly also non-infected bystander cells induced by factors such as trail, significantly contributes to worsened disease outcomes. controlled cell death or apoptosis can be induced by intrinsic and extrinsic signals. the intrinsic apoptosis pathway is initiated by diverse intracellular stimuli that influence the expression and activation of b cell lymphoma (bcl)- family proteins that govern the permeabilization status of the outer mitochondrial membrane. once cytochrome c is released from the mitochondria, it binds to the intracellular adaptor protein, apoptotic peptidase activating factor , forming the so-called apoptosome that in turn recruits pro-caspase- ( ). caspases (cysteine-aspartic proteases) exert their action by cleaving other proteins and substrates. herein, initiator caspases such as caspase- and caspase- target other downstream caspases, whereas effector caspases, including caspase- , - , and - , directly cause apoptosis by cleaving and thus inactivating or disassembling a vast array of cellular integral proteins and complexes ( ) . the extrinsic apoptosis pathway relies on an extracellular signal exerted by ligands of the tumor necrosis factor (tnf) receptor (tnfr) superfamily, including trail, tnf-α, and fas ligand (fasl) ( ) . their ligation to their respective cell surface-expressed death receptors (dr) leads via the signal transmission by fas-associated protein with death domain (fadd) to the activation of the initiator caspases- or - , finally stimulating effector caspases including caspase- ( ) . to date, several type i and type iii ifn-induced, proapoptotic factors have been identified ( ) . both caspase- and caspase- have been shown to be upregulated upon type i ifn signaling ( , ); caspase- enhances the fadd-driven extrinsic apoptosis pathway, whereas the less-studied caspase- may promote pro-il- β cleavage and inflammasome-driven cell death (pyroptosis) in macrophages ( , ) . chattopadhyay et al. ( ) demonstrated that sendai virus infection and polyi:c treatment resulted in bcl- -associated x protein (bax) activation and apoptosis induction via one of the key transcription factors of ifn genes, irf . in addition irf was reported to enhance trail-dependent extrinsic apoptosis by nuclear translocation resulting in the translation of to date undefined factors that increase cell death upstream of caspase- activation ( ) . furthermore, both rlrs, rig-i and mda- , trigger the proteins puma and noxa that induce bcl and the ifn/trail signaling axis frontiers in immunology | www.frontiersin.org march | volume | article thus activate the intrinsic mitochondrial apoptotic cascade ( ) . also, pkr influences a cell's susceptibility to apoptotic signals, as it was demonstrated to sensitize to the fadd/caspase- apoptosis pathway upon type i ifn signaling after challenge with iav or dsrna ( ) and the oas-rnasel system has been suggested to contribute to ifn-α-related cell death induction, but the exact mechanisms remain to be elucidated ( ) . finally, also two classical initiators of the extrinsic apoptosis cascade are induced as isgs. both fasl and its receptor fas are upregulated on mrna levels by ifn-α ( ) , and fasl was reported to be induced by type i ifn in iav infection in the murine lung in vivo ( ) . also, the proapoptotic factor trail (or tnfsf , apo l) is induced by ifn-mediated and isgf -executed transcriptional activation, as has been shown by sato et al. ( ) , who revealed the presence of the isre sequence within the trail promoter region ( ) . in iav infection, trail is released in high amounts from infected alveolar macrophages depending on a pkr-and ifn-β-driven autocrine signaling loop. binding of ifn-β to macrophageexpressed ifnar activates a jak/stat-dependent release of trail, which then acts through its receptor dr on the alveolar epithelial cells ( , ) . however, certain prerequisites may decrease the ability of a cell to undergo apoptosis, including a shortage in pro-caspase- availability, expression of cellular fadd-like il- β-converting enzyme-inhibitory proteins (c-flips) that block fadd-driven caspase activation, inactivation, or degradation of fadd itself, or expression of cyld, which acts as a receptor-interacting serine/threonine-protein (rip) kinase de-ubiquitinase and thus stabilizes rip . however, in these cases ifn signaling can still promote a caspase-independent, programmed inflammatory cell death by activating the necroptosis pathway ( , ) . necroptosis is induced by a complex formation by rip and rip kinases that activate both poly-adp-ribose (par) polymerase (parp- ) and/or mixed lineage kinase domain-like (mlkl), leading to atp depletion, calpain activation, par polymer accumulation or cell membrane permeabilization, and release of damage-associated molecular patterns, respectively [reviewed in ref. ( , ) ]. both a type i ifn-dependent jak/stat-driven activation of pkr as well as signaling by the prr dai (dnadependent activator of irfs) initiates necroptosis via rip /rip activation, respectively ( , ) . importantly, the activation of proapoptotic and pro-necroptotic pathways in respiratory infection can result in a structural disruption of the airway and the alveolar epithelial barrier, which is a major hallmark of respiratory disease and its progression to the acute respiratory distress syndrome ( , ) . in virusinduced lung injury, especially expression of trail, which can initiate both apoptosis as well as necroptosis has been correlated with more severe outcomes. as described earlier, trail belongs to the superfamily of tnf ligands and has been reported to be inducible by both type i and type iii ifns. trail has been found to be present in various cells of the immune system, among them natural killer (nk) cells, t cells, nk t cells, dc subsets such as ifn-γ-producing killer dcs and macrophages, and can be displayed in large amounts on the cell surface or be shed upon ifn-and/or pro-inflammatory cytokine signaling ( ) ( ) ( ) . in addition to cells of the immune system, fibroblasts have been shown to produce trail after ifnγ treatment or viral challenge. also, club cells and the alveolar epithelium have been reported to produce trail ( ) ( ) ( ) ( ) . similar to other ligands of the tnf superfamily, trail is a homotrimeric type ii transmembrane protein with a conserved c-terminal extracellular domain that mediates receptor binding and can be cleaved by metalloproteinases to generate a soluble mediator ( ) . however, trail can induce cell death also in its membrane-bound form, that is, similar to trail expression levels and trail shedding, upregulated by type i ifn ( ) . direct cell-to-cell trail-dr interactions have been demonstrated to play a role in macrophage, nk as well as cd + t cell-mediated induction of cellular death ( , ) . in humans, five different binding partners for trail are present: the membrane-bound dr (trail-r ) and dr (trail-r ) that both induce a proapoptotic signaling cascade, the membrane-bound anti-apoptotic decoy receptors (dcr) and dcr , and the soluble interaction partner osteoprotegerin ( ) . in the murine system, only dr has been identified to ligate to trail ( ) . in the human respiratory compartment, both dr and dr have been demonstrated to be present under steady-state conditions ( , ) . however, upon viral infection, cell-sensitivity to trail-induced apoptosis is enhanced, which has been attributed to increased trail receptor expression especially on infected cells, as dr levels are markedly increased in iav-, adenovirus-, and paramyxovirus-infected cells in contrast to non-infected bystander cells ( , , ) . of note, studies on the dependency of dr upregulation upon type i ifn signaling after iav infection have yielded conflicting results in different strains of mice ( , ) , highlighting the complex interplay of ifn-induced cascades in a host-and tissue-specific context, whereas the exact virus-and host-specific mechanisms for dr regulation remain less well defined. moreover, previous assumptions that also dcr expression would correlate with cell-sensitivity to trail-induced cell death could not be experimentally verified ( ) . tumor necrosis factor-related apoptosis-inducing ligand ligation to the proapoptotic receptors dr or dr triggers a trimerization of the receptors. subsequently, depending on additional stimuli, presence or absence of adaptor molecules or inhibitory proteins, different signaling pathways can be activated (figure ) . in the classical trail-dependent extrinsic apoptosis induction, the proteins rip, tradd, and fadd are subsequently recruited to the dr cytoplasmic domain upon trail ligation ( , ) . these factors and the proapoptotic drs all share a cytoplasmic death domain (dd), which is lacking or truncated and thus inactive in the dcr. the dd plays a central role in the concerted formation of the death-inducing signaling complex (disc). disc formation exposes a second functional domain of fadd, the death effector domain that is directly able to recruit pro-caspase- figure | trail/dr -mediated cellular signaling pathways. in presence of rip , tradd, and fadd, trail ligation to dr results in apoptosis induction, which is initiated by recruitment of the pro-caspase- or - to fadd. these in turn activate the effector caspases- and - , which leads to dna fragmentation and apoptosis induction. in addition, tradd can trigger a traf -and jnk-dependent activation of bax and subsequent release of mitochondrial cytochrome c, inducing the pro-caspase- activation. in the presence of cyld, c-flip or absence of sufficient amounts of fadd or pro-caspase- , trail ligation to dr triggers the interaction of rip and rip kinase, which in turn cause cell death via induction of mlkl and/or parp- . in the presence of ciaps, fadd is not recruited to dr upon trail ligation, and tak is activated by tradd/traf interactions. tak induces nemo followed by iκb degradation and nfκb activation, as well as mkk and jnk activation leading to ap- nuclear translocation; both events promote the production of cytoprotective factors such as xiap, ciaps, and c-flip. additionally, tak triggers ampk activation and thus mtorc inhibition, which results in enhanced autophagic activity. abbreviations: ap- , activator protein ; tnf, tumor necrosis factor; trail, tnf-related apoptosis-inducing ligand; dr , death receptor ; rip , receptor-interacting serine/threonine-protein kinase ; tradd, tnf receptor type -associated death protein; fadd, fas-associated protein with death domain; traf, tnf receptor-associated protein; jnk, janus kinase; bax, bcl- -associated x protein; c-flip, cellular fadd-like il- β-converting enzyme-inhibitory proteins; rip, receptor-interacting serine/threonine-protein; mlkl, mixed lineage kinase domain-like; parp- , poly-adp-ribose (par) polymerase ; ciap, cytoprotective factors including inhibition of the autophagic machinery; xiap, x-linked inhibitor of apoptosis protein; ampk, amp-activated protein kinase; mtorc, mammalian target of rapamycin complex; traf , tnf receptor-associated protein ; mkk, mitogen-activated protein kinase. and pro-caspase- . how exactly disc formation induces caspase activation is still under debate. the most probable scenarios include either an autocatalytic cleavage of caspase-pro-domains enabled by the spatial proximity between pro-caspases (generated by their recruitment to disc), by pro-caspase dimerization, or by pro-caspase conformational stabilization ( ) . removal of the pro-domain of caspase- and caspase- results in the activation of the effector caspases- and - , which cleave dna fragmentation factor and lead to apoptosis ( , ) . moreover, trailbinding to dr and dr can induce the jnk either via caspase- or recruitment of tnf receptor-associated protein (traf ) to the disc complex, which results in the activation of the intrinsic apoptotic cascade by bax-dependent mitochondrial cytochrome c release ( ) . in addition, trail signaling is also able to induce necroptosis by both activating the rip /rip kinase downstream effectors parp- and mlkl, contributing to epithelial cell death and tissue injury ( ) ( ) ( ) . it has become apparent in recent years that trail signaling is closely linked to induction of autophagy, a process generally associated with the blockade of apoptosis and necrosis. indeed, autophagy has been reported to improve cellular survival in cell stress by catabolic removal of cytoplasmic long-lived proteins and damaged organelles. it also contributes to viral clearance and the transfer of viral material to endosomal-/lysosomallocated tlr or mhc class ii compartments for the activation of adaptive immunity ( ). several studies outline that trail ligation to dr can result in a traf -dependent activation of tak (map k ) that has been attributed a central role in trail-induced autophagy activation ( ) . tak modulates the ikk-dependent translocation of nfκb, and it also induces jnk activation via mitogen-activated protein kinase. both events lead to expression of autophagy-related factors including inhibition of the autophagic machinery (ciap) , ciap , x-linked inhibitor of apoptosis protein, and c-flip ( , ) . especially, c-flip has been associated with desensitization of cells to trail-induced apoptosis, favoring autophagy-related cascades ( ) . another study revealed that upon trail signaling the amp-activated protein kinase (ampk) is activated. ampk in turn inhibits the mammalian target of rapamycin complex that itself is an inhibitor of autophagy, thus the activation of the autophagic machinery is promoted ( ) . the decision if trail signaling results rather in necroptotic or apoptotic cell death or in activation of autophagy seems to be dependent on the presence of ciaps that promote rip kinase ubiquitination and degradation ( ) , but also on the balance between active caspases and autophagic proteins such as beclin- ( , ) . this suggests a scenario where autophagy is activated as cell protective mechanism until cell stress-as executed by enhanced trail signaling or additional viral infection-increases over a threshold to favor cell death induction. accordingly, as trail signaling is not restricted to infected cells, excessive cell death activation might be limited by autophagy induction in non-infected bystander cells. however, autophagy is not only related to cell survival but can also positively affect apoptosis and induce-even if the exact mechanisms are still under debate-autosis, the autophagy-related cell death, another mode of trail to trigger cell death ( , ) . of note, autophagy activation needs to be placed into its virus-specific context, as some viruses, including dengue virus, poliovirus, and coxsackie b virus ( ) , can exploit autophagic pathways for their own replication and thus promote apoptosis and tissue injury. as discussed above, trail is a potent activator of cell death. however, its signaling outcomes can differ largely depending on its delivered form (e.g., membrane-bound versus soluble), the availability of drs on the target cell membrane, alternate intracellular pathways that might be activated and finally the pathogen itself, as it might exploit trail-induced pathways for its own survival and replication. in acute respiratory infection, trail signaling is often part of an ifn-driven overshooting inflammatory reaction that promotes unspecific tissue injury and thus disease severity by increasing functional and structural changes in infected but also non-infected cells, as will be outlined below. the release and effects of trail have been especially well studied in iav infection in the last decade. earlier studies reported that within days after infection, bronchial, bronchiolar, and alveolar epithelial cells undergo apoptosis ( ) . this early induction of cell death is mainly attributed to direct apoptosis induction by the virus itself, as iav actively promotes apoptosis for efficient viral replication ( ) . herein, the viral ns and pb-f proteins not only play a crucial role ( , ) but also the viral m protein has been implicated in this process as it inhibits autophagy in infected cells ( ) . in addition, our own data revealed that later in iav in vivo infection, the recruitment of bone marrowderived macrophages via the cc chemokine receptor type (ccr )-cc-chemokine ligand (ccl ) axis significantly contributes to alveolar cell apoptosis and structural damage of the alveolar epithelium ( ) . studies by wurzer et al. ( ) had previously demonstrated that iav promotes the production of proapoptotic factors in an auto-and paracrine fashion via nfκb transcriptional activation by iav ( ) . subsequently, brincks et al. ( ) elucidated that human peripheral blood mononuclear cell treated with iav released trail and that increased trail levels correlated with type i as well as ii ifn induction. additionally, trail sensitivity was increased in influenza virusinfected cells. in line, our investigations could elucidate that iav triggers a pkr-dependent translocation of nfκb that results the production of type i ifns. these in turn induce, via ligation to the ifnar receptor complex, expression and shedding of trail by bone marrow-derived macrophages ( ) . in addition, davidson et al. ( ) demonstrated that type i ifn application to iavinfected mice increased morbidity and lung injury, which could be attributed to both dr and trail upregulation inducing epithelial cell apoptosis. importantly, högner et al. also reported that the iav strain used in these studies, a/pr (h n ), which is highly pathogenic for mice, induced an approximately -fold induction in macrophage trail expression, whereas the lower pathogenic virus a/x- (h n ) only stimulated trail by a factor of eight. of note, the relation between trail induction and iav strain-specific pathogenicity also translates to the highly pathogenic avian h n iav, causing severe pneumonia in mice as well as in humans ( , ) . moreover, human infection with both the highly pathogenic h n as well as the pandemic h n iav strains are characterized by a massive influx of mononuclear phagocytes into the alveoli, which is correlated with extensive alveolar epithelial cell apoptosis ( , ) . additionally, macrophages gained from bronchoalveolar lavages of patients presenting with ards caused by the pandemic h n / virus strain showed high surface expression and release of trail ( ) . another recent report demonstrates that in highly pathogenic avian influenza, in addition to macrophages also the alveolar epithelium might be involved in causing elevated levels of trail in the alveolar space ( ) . besides its role in apoptosis, trail signaling upon iav infection has also been implicated in the induction of necroptosis in fibroblasts, dcs, and lung epithelial cells ( , , ) . rodrigue-gervais et al. ( ) demonstrated that lack of cipa promotes rip kinase-mediated necroptosis in response to trail-but also the proapoptotic factor faslreleased from hematopoietic cells. this contributed to severe lung epithelial degeneration and increased mortality, even though viral control was not compromised. nogusa et al. ( ) further elucidated that iav-induced necroptosis depends on rip kinase activation of mlkl, and that rip kinase deficiency, similar to ciap -deficiency, increased iav-susceptibility in vivo. in iav infection, as mentioned earlier, dr expression is elevated on infected alveolar epithelial cells, but not in noninfected cells in vivo, which might impact on trail susceptibility to apoptosis induction ( ) . however, both infected as well as neighboring bystander cells were found to be targeted for apoptosis induction by macrophage-released trail. nonetheless, we could recently show that specifically in noninfected cells within the iav-infected lung, trail severely compromises the function of the ion channel na,k-atpase, which was mediated via induction of the stress kinase ampk ( ) , thereby potentially revealing a cross-link to trailinduced autophagic cell stress pathways in bystander cells both in vitro and in vivo. the trail-induced and ampk-mediated downregulation of the na,k-atpase, a major driver of vertical ion and fluid transport from the alveolar airspace toward the interstitium, resulted in a reduced capacity of iav-infected mice to clear excessive fluid from the alveoli. thus, trail signaling contributes to intensive edema formation, a hallmark of disease in virus-induced ards ( ) . notably, this effect of trail on na,k-atpase expression was induced independently of cell death pathways elicited by caspases, as treatment of cells and mice with a specific caspase- inhibitor diminished apoptosis in alveolar epithelial cells but still allowed for the reduction of the na,k-atpase ( ) . conclusively, treatment of iav-infected mice with neutralizing antibodies directed against trail or the abrogation of recruitment of trail + bone marrow-derived macrophages inhibited apoptosis of both non-infected and bystander cells. thus, lung leakage due to loss of alveolar barrier function was reduced, whereas alveolar fluid clearance capacity was enhanced, resulting in reduced edema, improved survival, and outcome upon iav challenge in vivo. however, trail has also been shown to be upregulated on nk, dc, and on cd + and cd + t cells after iav infection ( ) . studies by brincks et al. demonstrated that especially cd + t involved in cytotoxic t cell responses toward iav and drive iav-infected cells into apoptosis via trail, thus contributing to efficient virus clearance ( , ) . in addition, both fasl and trail are involved in dc-mediated ctl activation and cytotoxicity against iav-infected cells ( , ) . furthermore, studies showed delayed viral clearance upon neutralizing anti-trail antibody administration ( , ) . our data, however, demonstrate that the transfer of trail-deficient bone marrow into irradiated wild-type mice, resulting in loss of trail production by bone marrow-derived macrophages upon iav infection, does not impact on the capacity to fully clear viral particles from the lung at day after infection, suggesting that other compensatory mechanisms are recruited to guarantee viral clearance ( ) . taken together, in iav infection, trail acts both as an important mediator of infected cell killing but particularly as a detrimental factor contributing to tissue injury and impaired inflammation resolution when released in excessive amounts by recruited immune cells. respiratory syncytial virus is an important cause of respiratory tract infections especially in children worldwide. generally, there seem to be virus-elicited anti-apoptotic mechanisms active in the lung epithelium, as rsv-infected primary human airway cells show a minimal cytopathic effect ( ) . however, several cell lines including small airway cells, primary tracheal-bronchial cells, and a and hep- showed increased expression of trail and its ligands dr and dr in an in vitro rsv infection model ( ) . moreover, soluble trail released from leukocytes was elevated in the bronchoalveolar lavage fluid of patients with rsv-associated respiratory failure, suggesting that similar to iav, trail contributes to rsv-induced epithelial injury and disease progression ( ) . also in cov respiratory tract infection, trail levels, but less so fasl, have been reported to be markedly elevated ( , ) . for sars-cov that presents with a severe damage to both the upper and lower respiratory tract ( ) , especially dcs respond with a strong induction of trail production, which was suggested to correlate to increased cellular lung infiltrations present in sars-cov patients ( ) . interestingly, sars-cov infection drives cells into apoptosis by a pkr-driven but eif α-independent pathway ( ) , which might-similarly as seen in iav infection-suggest a pkr-induced and autocrine/paracrine executed activation of apoptosis. also mers-cov, which causes pneumonia and respiratory failure, has been demonstrated to induce profound cell death within h of infection, irrespective of viral titers produced by the infected cells. however, type i ifn expression is strongly reduced in mers-cov in comparison to seasonal human cov in in vitro infection models, including human monocyte-derived macrophages, calu- , and human lung fibroblasts ( , ) , which might also dampen downstream trail induction. therefore, the exact mechanism by which mers-cov promotes cell death remains to be investigated. recurrently, viral infections of the respiratory tract are followed by outgrowth of colonizing gram-positive bacteria that aggravates the course of illness. this is well documented for iav, where "super" infections with streptococcus pneumoniae and staphylococcus aureus are the most frequent and increase viral pneumonia-associated morbidity and mortality ( ) . during the iav pandemic, bacterial pneumonia was evident in most cases ( ) and also during the recent h n pandemic, coinfections were a relevant factor for severe disease in a young patient population without comorbidities ( ) . interestingly, virus-induced elevation of the type i ifn response levels might promote secondary bacterial outgrowth by several mechanisms [reviewed in ref. ( ) ]. in line, it has been repeatedly demonstrated that lack of type i ifn signaling results in better bacterial clearance and increased survival rates in iav-and s. pneumoniae-superinfected mice ( ) ( ) ( ) . herein, ifn-induced apoptosis induction as well as depletion or impaired recruitment of lymphocyte subsets necessary for bacterial control play a critical role ( , ) . bacterial clearance from the lung has been reported to rely on sufficient phagocyte generation, recruitment, and survival. type i ifn has been demonstrated to cause apoptosis in bone marrow-derived granulocytes, affecting the numbers of recruited neutrophils ( ) , but also to impair expression of the cytokines cxcl (or kc) and cxcl (or mip- ), thus inhibiting neutrophil recruitment to the lungs with severe effects on survival of superinfected mice ( ) . a recent report by schliehe et al. ( ) elucidated the mechanistic background for impaired cxcl expression and secretion and demonstrated that type i ifns activate the histone methyltransferase setdb , which in turn represses the cxcl promoter and thus impairs neutrophil recruitment and bacterial clearance. moreover, type i ifn production decreases ccl production, thus inhibiting macrophage recruitment, which as well has been reported to have detrimental effects on bacterial clearance and disease progression in bacterial superinfection after viral insult in vivo ( ) . in addition, type i ifns also impair γδ t cell function and il- release, which was shown to increase susceptibility to s. pneumoniae superinfection after iav challenge ( ) . also in s. aureus pneumonia, a robust type i ifn response is correlated to excessive morbidity and tissue injury ( ) . in a model of polyi:c, s. aureus (methicillinresistant strain, mrsa) superinfection, polyi:c treatment prior to bacterial infection enhanced type i ifn levels and decreased bacterial clearance and survival ( ) . furthermore, shepardson et al. ( ) demonstrated that late type i ifn induction rendered mice more susceptible to secondary bacterial pneumonia in a model of iav-mrsa superinfection. only limited data are available on a direct role of trail in respiratory disease progression due to bacterial superinfections. in a model of iav-haemophilus influenza infection, neither deficiency for cc chemokine receptor type , inhibiting bone marrow-derived macrophage recruitment, nor deficiency of fas or tnfr impacted outcome ( ) . yet, during s. pneumoniae single infection, early cell death of macrophages is thought to limit an exuberant inflammatory reaction and accordingly, a study by steinwede et al. ( ) revealed that neutrophil-derived trail limits tissue injury by inducing cell death in dr -epressing lung macrophages in bacterial mono-infection ( ) . in contrast, in the iav-s. pneumoniae superinfection mouse model, iavinduced trail has a detrimental effect on overall mortality ( ), as trail-induced epithelial injury enhanced bacterial outgrowth of s. pneumoniae-administered at day after iav infection-markedly. importantly, administration of anti-trail neutralizing antibodies enhanced bacterial control by the host organism. thus, the activation of ifn/trail-mediated signaling in viral infection has detrimental implication for outcome of secondary bacterial infection following viral insult, rendering the ifn/trail signaling axis an interesting therapeutic target not only in respiratory viral infections but also in complicating bacterial superinfection. an increasing number of reports connect progression of chronic respiratory disease to acute respiratory virus infection or proapoptotic signaling events. in fact, trail has been reported to be a critical determinant for promoting the development of chronic lung disease in early life ( ) ; targeting trail by genetic deletion or neutralizing antibody application in early-life respiratory infections ameliorated infection-induced histopathology, inflammation, as well as emphysema-like alveolar enlargement and lung function. furthermore, trail was also shown to play a role in the development of allergy and asthma. trail is not only elevated in the sputum of asthmatic patients but has also been reported to be highly expressed in an experimental mouse model of asthma, where it induces ccl secretion by bronchial epithelial cells, thus promoting th cell responses and airway hyperreactivity ( ) . in copd, acute exacerbations driven by viral and bacterial infection are a major factor increasing both mortality and morbidity, and both influenza and s. pneumoniae have been identified among the most common causes of copd exacerbations ( ) . indeed, primary bronchial epithelial cells isolated from subjects with copd show an impaired production of type i ifn ( ) , which has been implied in the enhanced susceptibility of copd patients to respiratory infections; however, even in absence of high ifn induction, both an abnormally elevated loss of alveolar epithelial cells due to apoptosis as well as elevated trail and dr levels were reported ( ) , implying a possible link between viral/bacterial induction of trail and acute exacerbations in copd. trail induction has also been directly linked to cigarette-smoke exposure, a common cause of copd, and trail deficiency resulted in decreased pulmonary inflammation and emphysema-like alveolar enlargement in vivo ( ) . moreover, increased levels of both trail and dr were associated to impaired lung function and increased systemic inflammation in human copd patients ( ) . while alveolar epithelial cell death is closely connected to idiopathic pulmonary fibrosis (ipf), trail and its receptors dr and dr in aec were shown to be upregulated in ipf lungs ( ) . also, in pulmonary arterial hypertension virus infection is considered to be a possible risk factor ( ) , and pulmonary hypertension has been reported to be a side effect of prolonged treatment with type i ifn ( , ) . in line, trail has been closely linked to disease progression in pulmonary hypertension. trail has been found to be increased within pulmonary vascular lesions of patients with pulmonary hypertension ( ) and also in a mouse model of hypoxia-induced pulmonary hypertension, levels of soluble trail correlated with right ventricular systolic pressure, right ventricular hypertrophy, and pathologic alterations ( , ) . importantly, neutralizing antibody-treatment against trail showed positive effects on survival while reducing pulmonary vascular remodeling ( ) . notably, the extent to which infection-induced trail release causes or exacerbates chronic lung disease or in how far trail production in chronic lung diseases affects susceptibility to respiratory viral and complicating bacterial infection remains to be elucidated. respiratory viral infections are major causative agents for lung injury and ards; however, in many cases antivirals are not sufficient to limit disease ( ). besides the fact that most viruses are subject to strong selective pressures that favor quickly evolving, drug-resistant virus variants, recent advances in understanding the processes that contribute to tissue injury and ards highlight a crucial role of immune-related, ifn-driven events. therefore, novel therapeutic strategies often aim to improve the outcome of severe respiratory infection by modulating host cell responses; however, to date, clinical trials trying to improve severe viral infections or ards outcomes by targeting host pathways have not resulted in approval of new drugs ( ) . of note, for establishment of such therapies it has to be considered that the timing and intensity of induction and amplification as well as of dampening and termination of the ifn-driven immune response needs to precisely match the pathogen-and organ-specific requirements of a given infection. a non-controlled regulation of these processes may lead to either an unrestricted pathogen spreading or, on the other extreme, to an overshooting inflammatory response, including the increased production of pro-inflammatory and proapoptotic mediators, elevated levels of recruited immune cells, and/or aberrant repair processes. notably, both too low and too high levels of ifn-induced effects facilitate disease progression with a possible increase of fatal outcomes in ards patients ( ) . accordingly, preclinical in vivo studies of ifn-directed therapies yielded seemingly adverse results, depending on the context, timing, and dosage of ifn modulation. however, in multiple settings of acute respiratory viral infection, studies demonstrate that an exaggerated signaling derived from type i ifn in an already inflamed tissue contributes to worsened outcomes, and importantly, might favor secondary bacterial superinfection [e.g., ref. ( , , ) ]. interestingly, davidson et al. ( ) demonstrated that type iii ifn release upon influenza challenge-in contrast to type i ifn induction-does not trigger an unbalanced inflammatory response that critically contributes to respiratory disease progression in vivo, highlighting it as a possible therapeutic option in iav-induced lung injury. most likely, this effect derives from the lack of the ifn-λr /il- r receptor complex, but presence of ifnar, on immune cells, including bone marrowderived macrophages. nonetheless, other reports identify ifn-λ as a driver of macrophage polarization to an inflammatory m phenotype ( ) that has been attributed to further promote an overshooting inflammatory response, highlighting the need for further studies of type iii ifn biology in pathogen-associated disease progression. as generally ifn-directed therapeutic approaches target various downstream signaling events that might both act beneficially as well as detrimentally on viral replication and pathogenesis, a further approach is to address specific isgs that primarily show detrimental effects on disease progression. as outlined above, trail or its downstream signaling events might comprise a suitable target for adjunct therapies in addition to antivirals. accordingly, our own data in a preclinical mouse model of iav infection demonstrate a clear benefit of the systemic application of neutralizing antibodies against trail at days and postinfection for lung injury, morbidity, and mortality ( , ) . targeting trail as a major determinant of disease severity in respiratory viral infections including iav, but also rsv and cov, may yield therapeutic approaches that are superior to ifn-directed strategies, as they seemingly do not bear the risk of compromising host defense. yet, it should be thoroughly excluded that blocking trail-induced cell death of infected cells will not lead to an overwhelming viral spreading, especially as reports on viral loads upon trail inhibition in preclinical models of iav are controversial ( , , ) . accordingly, additional studies are needed to understand how and to which extent virus-infected cells can be killed or viral spreading can be controlled by other means in the absence of trail. moreover, targeting pathways and signaling hubs downstream of trail/drs, such as ampk ( ), in a well-timed and lung compartment-specific way, may open new therapeutic avenues but requires more detailed preclinical studies on efficacies and side effects. a valid approach might be the use of a combination therapy of such a treatment together with a classical antiviral drug therapy limiting viral replication; however, exact dosage, timing, kinetics, and application routes remain to be defined. cp and sh performed bibliographic research and drafted the manuscript. this work was supported by the german research foundation (sfb-tr b , sfb c , kfo p /p , exc ), by the german center for lung research (dzl), and by the german center for infection research (dzif). virus interference. i. the 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apoptosis-inducing ligand (trail) reverses experimental pulmonary hypertension luks am. ventilatory strategies and supportive care in acute respiratory distress syndrome ifnλ is a potent anti-influenza therapeutic without the inflammatory side effects of ifnα treatment the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- - tapkjb authors: nan title: th escp-nsf international symposium on clinical pharmacy: clinical pharmacy tackling inequalities and access to health care. oslo, norway, – october date: - - journal: int j clin pharm doi: . /s - - - sha: doc_id: cord_uid: tapkjb nan pharmacy, sint maartenskliniek, ubbergen, pharmacy, radboud university medical centre, nijmegen, clinical pharmacy and toxicology, maastricht university medical centre, maastricht, netherlands please specify your abstract type: research abstract background and objective: according to literature adherence to statins ranges from to %. medication adherence is affected by both practical barriers and patient's beliefs about medication. however, physicians also have their beliefs about medication. several studies have shown that these beliefs also impact the decision of patients to agree with a particular treatment or not. as current published interventions on medication adherence (which focus predominantly on patients) are not or just partly effective, physicians' beliefs might be a promising target for interventions to improve adherence. however, there is currently no information available on physician's beliefs about statins and whether these beliefs affect patient's beliefs and adherence. therefore, the objective of this study is to examine whether physicians' beliefs about statins influence the beliefs and adherence of patients using a statin. setting and method: this cross-sectional study was conducted in gp practices and community pharmacies, between september , and march , . physicians' and patients' beliefs about statins were assessed with the beliefs about medicine questionnaire (bmq) specific. patients' adherence on statins was assessed with both the mars- and the morisky- questionnaires. please specify your abstract type: research abstract background and objective: nhs highland and nhs western isles are the most remote and rural health boards in the united kingdom, with high numbers of dispensing medical practices. a pilot is underway in dispensing practices with clinical pharmacists undertaking targeted medication reviews. a previous quantitative service evaluation demonstrated its value, with pharmaceutical care issues identified in almost all patients, the vast majority of which ( . %) were managed by the pharmacist without any need for general practitioner (gp) referral. the objective was to undertake a qualitative exploration of the service. setting and method: all patients and staff involved in the service were invited to participate. a semi-structured interview schedule was developed and piloted. telephone interviews were conducted with all consenting staff and a purposive sample of consenting patients recruited to the point of data saturation. interviews were audiorecorded, transcribed verbatim and analysed thematically. nhs ethics and research and development approvals were obtained. were the most confident with doacs (range from . to . %) please specify your abstract type: research abstract background and objective: patients are at risk of drug-related problems (drps) at transition points during hospitalization. the community pharmacist (cp) is often the first healthcare professional patients visit after discharge. cps lack sufficient information about the patient and so they may be unable to identify problems in medications, which may lead to dispensing the wrong drugs or dosage, and/or giving wrong information. we aim to assess the impact of a complex intervention comprising of medication reconciliation performed at discharge by a hospital pharmacist (hp) with communication between the hp and cp on drps during the days following discharge. setting and method: cluster randomized crossover trial involving medical and surgery care units (each unit corresponding to a cluster) in french hospitals during two consecutive -day periods, randomly assigned as 'experimental'(e) or 'control' c (usual care) periods. during the experimental period, the hp performed a medication reconciliation that was communicated to the patient's cp. main outcome measures: the primary outcome was a composite outcome of any kind of drp (prescription/dispensation, gap or patient) during the days following discharge assessed at day seven post-discharge by phone from patient and cp. the secondary outcomes were /unplanned hospitalizations assessed by phone contact at day after discharge and /the iatrogenic potential exposure scale from to for each patient established by a clinical team. analysis was conducted in intention to treat. results: hospitals corresponding to clusters enrolled patients ( e group v/s c group). no difference was observed on age, sex, autonomy, and number of drugs in home medication at admission and discharge. at day ; ( . %) patients in e group had at least one drp v/s ( . %) in c group (or . ; ic % [ . ; . ] p = . ). intervention was especially efficient for patient discharged from surgery unit (or . ic % [ . ; . ]) and aged less than years (or . ic % [ . ; . ] . although intervention decreased patient exposure to drp with high iatrogenic potential (from . to . % p \ . ), un-planned hospitalizations at day weren't different between groups ( . vs. . % p = . ). conclusion: medication reconciliation associated to communication between hospital and community pharmacists is efficient to decrease patient exposure to drp but not sufficient to decrease un-planed hospitalization. hp-pc : clinical pharmacists bridging health care levels by medication reviews in primary care katherine wendelbo *, , kristine lundereng namsos hospital pharmacy, central norway hospital pharmacy trust, namsos, levanger hospital pharmacy, central norway hospital pharmacy trust, levanger, norway please specify your abstract type: descriptive abstract (for projects) background and objective: nord-trøndelag county is sparsely populated and many inhabitants live far from the hospital. additionally, only half ( of ) of the municipalities have a local pharmacy. traditionally, namsos and levanger hospital pharmacies have performed quality audits of the implementation of drug administration procedures in primary care units. since , a service where clinical pharmacists participate in multidisciplinary medication reviews in municipalities throughout the county has been established. the objective of this poster is to describe the practical approach and design of the service. design: a descriptive report of an implemented clinical pharmacy service in primary care where clinical pharmacists, as part of multidisciplinary teams, perform medication reviews. results: medication reviews are performed on patients admitted to nursing homes and patients in home care, receiving help with handling of their drugs. primary care nurses prioritise patients (by selecting frail elderly with multiple co-morbidities and polypharmacy), usually five patients in each meeting. prior to the review, nurses collect medical information using a checklist including; diagnosis, drug-related symptoms, standard laboratory tests and an updated medication list. the clinical pharmacist receives de-identified medical information by postal mail or e-mail before the meeting. based on this information the pharmacist identifies possible drugrelated problems (drps) and provides recommendations on how to solve them. this is performed in a structured approach according to the integrated medicine management (imm) model. subsequently, the pharmacist visits the municipality and discusses the medication reviews in a multidisciplinary team meeting with nurses and physicians. in addition, the pharmacist gives lectures in a medication related topic (e.g. treatment of insomnia and anxiety, oral anticoagulants and cognitive side effects). following the meeting, the pharmacist reports the drps and suggested interventions to the multidisciplinary team, for further follow-up. during , totally medication reviews were performed in municipalities. in the same period, lectures were given by the clinical pharmacists. conclusion: this clinical pharmacy service enables multidisciplinary medication reviews even in municipalities with limited health professionals and resources. as a part of multidisciplinary teams, the clinical pharmacists contribute with medical competence. camille castel , arnaud de la blanchardière , vincent cattoir , guillaume saint-lorant *, pharmacy, infectious and tropical diseases, microbiology, chu caen, caen, france please specify your abstract type: research abstract background and objective: antimicrobial stewardship have clearly demonstrated their efficiency towards a more adequate use of antibiotics. since , the use of daptomycin, a ''critical last resort antibiotic'' has intensified in our hospital, occasionally outside the scope of its approved indications. this situation has led to the implementation of an antimicrobial stewardship and the drafting of local guidelines. the aim of this study is to analyse the evolution and pertinence of daptomycin prescriptions, after distribution of these guidelines within our institution. setting and method: a monocentric prospective study was conducted between july and november in a -bed university hospital. each daptomycin prescription recorded by pharmacy department was analysed by an infectious diseases specialist in the presence of the prescriber and considering local guidelines and the patient's clinical conditions. main outcome measures: the indicators chosen to determine prescription pertinence were: treatment indication, prescribed dose and other antibiotics associated with the daptomycin prescription. results: daptomycin prescriptions were analysed. observed indications were: sepsis ( %), infective endocarditis ( %), bone and joint infections ( %) and vascular prosthetic infections ( %). identified pathogens were: mrsa ( %), methicillin-resistant coagulase-negative staphylococci ( %), methicillin-sensitive staphylococcus aureus ( %), enterococci ( %) and methicillinsensitive coagulase-negative staphylococci ( %). daptomycin was prescribed as first-line treatment in % of cases. the mean dose was mg/kg/day [ - mg/kg/day] for a mean duration of days [ ; days] . local guidelines were followed in % of cases. daptomycin use was relevant for % of prescriptions. the irrelevant prescriptions triggered the modification or stoppage of antibiotic therapy in % of cases, respectively, generating an % decrease in consumption and an economy of over € for our institution. conclusion: this study shows the efficiency of antimicrobial stewardship in adequately using antibiotics, limitating ecological impacts, improving patient care and decreasing healthcare costs. it also shows that guidelines alone are insufficient to ensure a proper use of antibiotics. without a close prescription follow-up, constant reminders and sustainable evaluations, guidelines only affect a few prescribers. within the context of an ''antimicrobial crisis'', further development of guidelines and antimicrobial stewardship is essential to fight increasing bacterial resistances and requires a close collaboration between all healthcare professionals including pharmacists. interviews were transcribed verbatim and data were analysed using systematic text condensation. results: three major themes were identified: benefits, unrealised potential and criteria and barriers for success. ( ) benefits described by physicians included increased patient safety, increased awareness on drugs, and an ease of workload. drug interaction management was emphasized as one of the clinical pharmacists' most important work tasks, as well as being a resource for collaborating healthcare professions and to the patient himself. ( ) the clinical pharmacists expressed that they had an unrealised potential and could contribute to a greater extent in the multidisciplinary team than they did already. they mentioned education towards physicians and nurses, contribution in treatment decision-making and patient counselling as examples for possible extended work tasks. ( ) as criteria to succeed as a clinical pharmacist, physicians highlighted the importance of oral communication and physical presence on the wards. as barriers for integration in the team, the clinical pharmacists identified the physicians' lack of knowledge about the clinical pharmacists' skills as well as unclear expectations regarding their responsibilities. conclusion: physicians agreed that the clinical pharmacist represent a valuable contribution to the multidisciplinary team, where patient safety and drug interaction management are highlighted as main benefits. clinical pharmacists should to a greater extent educate healthcare professions in drug related topics and provide patient counselling. continuous effort on making the clinical pharmacist a natural part of the multidisciplinary team is crucial for the development of clinical pharmacy. by gathering perceptions from the collaborating professions as well as educating them on what clinical pharmacists can provide, we can develop a multidisciplinary team that enhances patient safety. hp-pc : assessment of dual antiplatelet therapy following acute coronary syndrome using grace and crusade sadeer fhadil * , paul wright, sotiris antoniou please specify your abstract type: descriptive abstract (for projects) background and objective: mortality and morbidity benefits of dual antiplatelet therapy (dapt) following acute coronary syndrome (acs) have been unequivocally demonstrated in a large body of evidence. with the availability of more potent antiplatelet agents, balancing ischemic and bleeding risks to prevent adverse outcomes is an on-going challenge, in particular, recognising that patients with high bleeding risk were excluded from clinical trials. grace and crusade scores stratify risk of mortality and in-hospital major bleeding post acs respectively. these tools should be used to support antiplatelet choice in light of newer more potent agents that equally pose a greater risk of bleeding. design: grace and crusade scores were calculated for patients presenting with acs. clopidogrel was recommended for patients with a high or very high crusade score (greater bleeding risk). ticagrelor was recommended for patients presenting with st-elevation myocardial infarction (stemi) or those with nsteacs with a grace score of intermediate or above (greater ischemic risk) and a crusade score of moderate or less (low bleeding risk). in either case, treatment was at the discretion of the clinician and patients received concomitant aspirin. a registry was collated of risk scores, diagnosis and choice of antiplatelet therapy. results: patients were included in the registry, of which ( %) presented with stemi and ( %) presented with nsteacs. of ( %) patients with a greater ischemic risk received ticagrelor as part of their dapt regime. advanced age, concomitant anticoagulation and those awaiting surgery were the most common reasons for patients with a greater ischemic risk to receive clopidogrel. ( %) had a high or very high crusade score. of these, ( %) received clopidogrel as part of their dapt regime. conclusion: risk stratification was streamlined using the data collection tool and useful to support choice of dapt. european society of cardiology (esc) guidance recommends use of established risk scores for prognosis and bleeding; however evidence to correlate to choice of dapt is lacking. outcome data is currently being reviewed and will provide further evidence to correlate choice of dapt to grace and crusade scores. please specify your abstract type: descriptive abstract (for projects) background and objective: in europe, approximately % of the patients with the human immunodeficiency virus (hiv) infection are co-infected with the hepatitis c virus (hcv). treatment recommendations in hiv/hcv co-infected patients are identical to those in patients with hcv mono-infection. however, potential drug-drug interactions (ddis) between antiretroviral agents and new direct-antiviral agents (daas) imply the need of a careful selection of the hcv treatment regimen. the aim of the present study was to evaluate the need of a change in the antiretroviral therapy (art) due to potential ddis in patients with hiv/hcv co-infection who started treatment for hcv with new daas. we also assessed the effectiveness of hcv treatment weeks after hcv treatment completion. design: we retrospectively registered clinical data about hcv and hiv management: hcv genotype, fibrosis metavir score, initial hcv viral load, hcv treatment and previous art regimen. we recorded the changes in art prior to starting hcv treatment and the reason of this switch (ddi, simplification or duplication of the therapy). results: between february and january , hiv/hcv coinfected patients started hcv treatment with a daas regimen. of them, had advanced liver disease (fibrosis score: f /f ) and were infected with hcv genotype . prior to starting hcv treatment, patients needed a switch in art regimen due to potential ddis with daas. simeprevir and the co-formulation ombitasvir/paritaprevir/ritonavir were the daas most frequently implicated in ddi with protease inhibitors or non-nucleoside reverse transcriptase inhibitors: / and / , respectively. also, we observed some changes of art due to other causes. five switches occurred to adequate the regimen (discontinuation of ritonavir in candidates to take the co-formulation ombitasvir/paritaprevir/ ritonavir or art improvement to decrease pill burden). as for hcv treatment effectiveness, / ( %) patients achieved sustained viral response weeks after therapy completion. conclusion: a large proportion of patients with hiv/hcv co-infection who initiate treatment with daas for hcv need to switch art due to potential interactions that may impact on effectiveness and safety of both treatments. additionally, some changes in art treatment are made to facilitate therapeutic adherence. these results highlight the need of a multidisciplinary approach in which interactions between art and hcv treatments should be carefully assessed. please specify your abstract type: descriptive abstract (for projects) background and objective: the potential impact of polymedication, iatrogenic events and medication error is a serious concern in hospitalized patients. clinical pharmacists can limit these risks by identify high risk. the aim of this study are to identify in six medical units high risk patients by using three predictive scores of rehospitalisation ( ps) , early mortality (charlson) and drug related problems (drp) . design: clinical and therapeutic variables in patients were collected through medical records and prescriptions by clinical pharmacists. scores were calculated during months in six units (internal medicine, n = ; nephrology, n = ; geriatrics, n = ; rheumatology, n = ; cardiology, n = and endocrinology, n = ). the data were analysed by mann and whitney test for the continuous variables and chi square test for the qualitative variables. the coefficient of correlation between the three scores were calculated by a pearson test for normal distribution and by a spearman test for non normal distribution. patients were considered at a high risk for re-hospitalization ( ps [ ) , early mortality (charlson [ ) and iatrogenic events (drp c ) . results: in the general population, the average age was . ± . years old and the sex ratio was . . the average treatment used was . ± . charlson scores were higher in geriatric unit ( . ± . ) follow by medical interne unit ( . ± . ). the ps and drp scores were higher in nephrology unit respectively . ± . and . ± . follow by internal medecine unit . ± . and . ± . . on contrary the rheumatology unit presented the lower level for the three scores. patients were considered at high risk for three scores, % (n = ) in nephrology unit (almost % of unit), % (n = ) in geriatric unit, % (n = ) in internal medicine unit, % (n = ) in cardiology unit, % (n = ) in endocrinology unit and % (n = ) in rheumatology unit. conclusion: knowledge of the variables associated with these predictor scores could help clinical pharmacists to prioritise various medicine units and target those at risk. we identified especially three units at risk: nephrology, geriatric and internal medicine. thanks to these results, clinical pharmacists can rapidly and efficiently target patients who present iatrogenic and/or re-hospitalization risks. design: a retrospective observational analysis was conducted in our hospital, based on medical records of patients presenting atrial fibrillation (af) and treated by doacs from january to may . to identify patients hospitalized due to severe bleeding, we analysed prothrombin complex concentrates (pccs) and activated pccs prescriptions, as well as pharmacovigilance declarations. results: patients were treated with doacs: with rivaroxaban ( . %), with dabigatran ( . %) and with apixaban ( %). fifty-nine ( . %) patients experienced at least one bleeding leading to hospitalization: with rivaroxaban ( . %), with dagibatran ( . %) and with apixaban ( . %). thirty-eight severe bleeding were identified ( . %): occurred with rivaroxaban ( . %), with dabigatran ( . %) and with apixaban ( . %). they included intracranial bleeding ( %) and gastro-intestinal bleeding ( %). seven haemorrhages resulted in hypovolemic shock (dabigatran: , rivaroxaban: , apixaban: ) and of them were fatal (dabigatran: ). rates of bleeding (p = . , v test) and of severe bleeding (p = . , v test) were not statistically different for the three molecules. in case of major haemorrhage, the recommended factor concentrate in our protocols differs between the anticoagulant. with dabigatran, the antidote idarucizumab ( g, intravenously) should be administered, without waiting for plasma concentration results. with rivaroxaban, apixaban or unknown doacs, pcc ( - units/kg) is indicated. in case of pcc failure, activated pcc ( - units/kg) is suggested. pcc, activated pcc or idarucizumab ( ) were used in / patients ( %). in rivaroxaban and apixaban-related haemorrhages, patients received activated pcc: two had a ui/kg dose and one had a ui/kg dose. regarding dabigatran-related bleeding, one patient received pcc instead of idarucizumab. compliance with local recommendations was % ( , p [ . , v test) . pharmacovigilance reports were issued. conclusion: management of doacs-associated severe bleeding in our hospital respects local protocols. it should also be pointed out that patients with life threatening bleeding may benefit from pcc. however, the risk of thrombosis associated with pcc must be weighed against the risk of haemorrhage. since specific antidotes are emerging, like idarucizumab or andexanet alpha, new guidelines for doacs-related haemorrhage are expected. please specify your abstract type: descriptive abstract (for projects) background and objective: this project is part of a prospective quasi experimental proof-of-concept investigation of a clinical pharmacist intervention to reduce drug-related problems among people admitted to a ward in a rural hospital in northern sweden. the aim of this particular study is to explore doctors' and nurses' expectations of having a ward-based pharmacist providing clinical pharmacy services in a rural hospital. design: eighteen face to face semi-structured interviews were conducted with a purposive sample of doctors and nurses working on the ward were the clinical pharmacy service was going to be implemented. semi-structured interviews were digitally recorded, transcribed and analysed using thematic analysis. results: the majority of participants had limited experience or a vague idea of what pharmacists are able to do in a ward. most participants described traditional roles such as inventory, drug distribution and dispensing. most respondents were unaware of the pharmacists' knowledge, skills and competences. for some it was unclear how having a clinical pharmacist in the ward was going to impact on their workload this was particularly important for the nurses. some doctors (mainly experienced) were concerned that having a pharmacist may mean losing or not gaining competence on drugs. for others it was unclear how the pharmacists' will work with patients or what clinical skills they have. however most participants were positive about the implementation of the new service. conclusion: this study provided a rare opportunity to explore the doctors' and nurses expectations of the role of clinical pharmacists before a clinical pharmacy service was implemented. the results showed that the participants' expectations of the clinical pharmacist role were unclear. to successfully implement clinical pharmacy services in a clinical pharmacy ''naïve'' setting; roles, clinical competence and responsibilities should be clearly described. furthermore, it is important to focus on inter professional collaborations between doctors, nurses and pharmacists. practical for the local hospital setting. seven out of experts agreed with pharmacist prescribing for the conditions identified. pharmacists (n = ) were more willing to prescribe antihypertensive and antidiabetic medication ( . %) when compared to oral anticoagulants ( . %). these values are higher than those obtained by vella in . the majority of pharmacists ( . %) recommended that pharmacists should take up further studies to a master or doctorate level degree in a clinical aspect in order to be authorised to prescribe. conclusion: the developed framework for pharmacist prescribing and the guidelines developed for pharmacist prescribing of oral anticoagulants and pharmacotherapy of hypertension and diabetes mellitus were shown to be reliable and were accepted by pharmacists and physicians. please specify your abstract type: research abstract background and objective: valproic acid (vpa) and its derivates and mycophenolate mofetil (mmf) and mycophenolic acid used during pregnancy increase risk of congenital malformation and cognitive impairment. thus, the french national agency for medicines and health products safety (ansm) decided to establish new conditions of prescription and dispensation of drugs containing vpa (may ) and mmf (april ). a signed care agreement and a co-prescription of contraceptives are now mandatory in the drug dispensation for reproductive-age adolescent girls and adult women. this study will describe the impact of these new guidelines on our practice. our objective is to compare the vpa and mmf media coverage and the impact on the prescriptions. setting and method: we compared the mass communication between vpa and mmf on social media, webpages and journal article (public and professional journal) on google and googletrends in the first months around these new rules. we combined different keywords such as ''accord de soins'' and the drug name. in the same time, we collected and analysed vpa and mmf prescribing and dispensing data and compared it to the data for the first months. main outcome measures: results: just before the vpa rule, the vpa was presented in the general press as the new health scandal after benfluorex mediator°w ith google searches in march compared to searches usually per month. simple research combining keywords reveal always more than twice more webpages concerning vpa than mmf. at the same time, a patients association (renaloo for renal failure) wrote to the ansm to contest the new rule with the double contraception and without any consultation of patients association. in our daily practice we also faced some physician reluctant to sign this prescription agreement with patient (too many agreements already asked, decision of ansm without any consultation of learned societies). the care agreements are kept in the patient records, a statement ''care agreement signed'' is reported in the electronic prescription of vpa and mmf. the overall consumption of mmf and vpa increase for respectively the first and months after rule implementation (from + to + %) except for the micropakin mg. the months mmf data will be presented for the final communication. conclusion: the media pressure and the new regulation have an impact on prescription trends. these new prescription and dispensing rules concerned two different contexts: pathology, media coverage, possible drug alternatives. we were faced to some difficulty in implementing the new guidelines, which reveals a certain reluctance of the prescribers or the patients represented by associations. tdmp : vancomycin trough serum concentrations are frequently subtherapeutic in a population of critically ill patients: a prospective observational study please specify your abstract type: descriptive abstract (for projects) background and objective: to design and characterise a framework of international pharmacy standards for pharmaceutical care application on oral anticoagulation for prevention of atrial fibrillation (af) related strokes. design: literature review (including existing international guidelines and quality measures) was conducted to characterise the standards and design an international framework for pharmaceutical practice application on oral anticoagulation for prevention of af-related strokes. expert opinions were sought through a delphi method to reach consensus on the framework domains and standards. results: the framework consisted of twelve overarching standards, which were defined and grouped into four domains as follows; ([personal care package]:-communication with patients, support decision making process, education and counselling, adherence. [medicines optimisation]:-clinical review and therapy optimisation, initiation and control, maintenance, supply and transfer between care settings. [workforce]:-workforce planning, training and development, analysing information; and [governance]:-assurance of service provision) specific to oral anticoagulation in prevention of af-related stroke. each standard was also categorised within dimensions and supporting statements to describe what a quality pharmacy service should deliver. a total of forty-five dimensions and twelve statements were incorporated into the framework. conclusion: a clearly defined framework of international standards was developed as a clinical tool and quality assurance to optimise the delivery of care for oral anticoagulation in prevention of af-related strokes. it will support pharmacists and their teams to develop their professional practice, improve services, and deliver safe and high quality patient care across all pharmacy settings. poster discussion forum i: community pharmacy and public health cp-pc : nurses' and pharmacists' learning experiences from participating in inter professional medication reviews in primary health care: a qualitative study hege t. bell *, , anne gerd granås , ragnhild omli , ingela enmarker , aslak steinsbekk nord university/ntnu, trondheim, hioa, oslo, nord university, namsos, norway, department of nursing, Østersund, sweden, ntnu, trondheim, norway please specify your abstract type: research abstract background and objective: traditionally, drug prescription and follow up have been the sole responsibility of physicians. however, interprofessional medication reviews (imrs) have been developed to prevent drug discrepancies and patient harm. what participating nurses and pharmacists learn from each other during imr is poorly studied. the aim of this study was to investigate nurses' and pharmacists' perceived learning experience after participating in imrs in primary health care for up to years. setting and method: a qualitative study with semi-structured focus group interviews and telephone interviews with nurses and pharmacists with experience from imrs in nursing homes and home based services. the data was analysed thematically by using systematic text condensation. main outcome measures: a qualitative method is useful when looking at objects from the perspective of how they are experienced. results: sixteen nurses and four pharmacists were interviewed. the nurses' perception of the pharmacist changed from being a controller of drug management routines towards being a source of pharmacotherapy knowledge and a discussant partner of appropriate drug therapy in the elderly. the pharmacists became more aware of the nurses' crucial role of providing clinical information about the patient to enable individual advice. increasingly the nurses learned to link the patient's symptoms of effect and side effect to the drugs prescribed. with time both professions jointly spoke of an increased awareness of the benefit of working as a team and the perception of contributing to better and more individual care. conclusion: imrs in primary health care meet some challenges especially concerning how to ensure participation of all three professions and how to get thorough information about the patient. possible solutions might be to use shared communication tools like internet based communication programs and to introduce the patient as a participant at the imrs. please specify your abstract type: research abstract background and objective: international good pharmacy practice guidelines describe how pharmacists should counsel the patients about their medicines, offer additional services where needed, and intervene at drug related problems. daily practice often differs from theory. this study aimed at illustrating the whole process of prescribed medicines dispensing in daily community pharmacy practice. part b of the project focuses on pharmacists' opinions. setting and method: community pharmacies in basel, switzerland, were invited in random order for study participation. one master student in pharmacy performed non-participant observations during day at each included community pharmacy. at dispensing of prescribed medicines, patient data, content of counselling, communication style, and provision of further services (e.g. follow-up offer) were documented on a checklist with predefined themes. interventions were documented systematically. a semi-structured interview on the pharmacists' opinions about the counselling, triggers, facilitators and barriers, and the documentation of interventions was conducted at each community pharmacy. main outcome measures: counselling content at prescription dispensing by numbers and by pharmacists' opinions; barriers, facilitators, and triggers for counselling at prescription dispensing. results: in march and april , of invited community pharmacies participated in the study. out of documented observation periods, encounters were analysed (first prescription: /refill prescription: ). counselling was provided to ( . %) clients with an average of . (± . ) themes per encounter. a total of clients refused counselling. themes most counselled at first and refill prescription dispensing were: drug intake ( / ), dosage ( / ) , and administration ( / ). for the pharmacists (n = ), most important themes to be discussed at first prescription dispensing were indication ( ), administration ( ), and anamnesis ( ); for refill prescription dispensing they were adherence ( ), therapy benefits ( ), and adverse effects ( ). the majority of pharmacists ( ) felt that it was their obligation to ask questions about patients' health during the dispensing of prescription medicines and named trigger (e.g. patient knowledge gap, patient motivation, interactions), but one-third reported difficulties with it. barriers were refusal by patients ( ), communication problems (language, ), lack of medical data ( ) , and lack of time ( ) . conclusion: a discrepancy in counselling content by observation compared to pharmacists' opinions was revealed. this might indicate that pharmacists are aware but hindered by barriers to practice according to good pharmacy practice guidelines. please specify your abstract type: research abstract background and objective: the workforce vision in scotland envisages 'making more and better use of technology …to increase access to services and improve efficiency' across the healthcare interface. services offered by community pharmacy remain limited by lack of shared access to patients' clinical information. in scotland, every patient has a unique identifier, their chi (community health index), which facilitates identification of/searching for patient records. the aim of this research was to explore the experiences of community pharmacists granted clinical portal access to patients' records. setting and method: from april , community pharmacists across nhs tayside (n = ) who had completed technical and information governance training were invited to maintain a portal log of their experiences of using the clinical portal to access patient records. each was asked to record when/why they considered accessing a patient's record and whether their information needs were met. portal logs were subject to independent summative content analysis by two researchers. this study gained ethical approval from robert gordon university. main outcome measures: not applicable. results: clinical portal logs were received from most participating pharmacists (n = / ). two were unavailable (moved to hospital setting; maternity leave). a third had not had occasion to access the clinical portal which he speculated was due to not working at weekends but also raised concerns about gaining patient consent. frequency of seven identified themes provided a partial indication of balance of reasons for usage. firstly (# ), to confirm a patient's prescription (n = ), secondly (# ), for additional information (n = ). less frequently (# ), portal access was to check repeat medications (n = ). other reasons for access were (# ) to check hospital discharge (n = ) followed by (# ) check on multi-compartment appliance aid status (n = ) or (# ) check the emergency care summary (n = ). there were also instances (# ) when portal access was not found to be helpful (n = ) so traditional offline routes were followed. conclusion: preliminary findings indicate mainly positive experiences with no technical issues raised and community pharmacists' information needs largely met. although limited to a small number of pharmacists in only one health board, findings support scottish policy aims, including 'prescription for excellence.' further work is underway around patients' perspectives of community pharmacist access. please specify your abstract type: research abstract background and objective: multicompartment compliance aids (mcas) such as the multidrug punch cards pharmis Ò are used to support patients in the daily management of their medication. solid oral medicines are unpacked from their original packaging and repacked in mcas although controversy exists about the stability of repackaged medicines. different countries published contradictory lists of medicines not recommended for repackaging. we aimed to define and apply criteria able to assess visual alteration of medicines repackaged in mcas. setting and method: eight criteria describing physical alteration of tablets/capsules were retrieved from the who international pharmacopoeia : chipping, swelling, capping, rough surface, cracking, crushing under pressure, mottling, and discoloration. absence of one criteria gives point. a maximum score of points can be obtained. twenty-two critical medicines and three half tablets were repackaged in the multidrug punch cards pharmis Ò and stored at accelerated conditions ( °c/ % rh) for weeks. original blisters of medicines were stored at room temperature as control. each tablet/capsule was visually inspected after , , and days. main outcome measures: score according to eight criteria. results: after weeks, tablets/capsules including of the half tablets showed no visual alteration and were identical to controls. a reduced score ( - points) was given to seven repackaged medicines and one half tablet within weeks: madopar Ò ( ), pravastatin sandoz Ò ( ), carvedilol mepha Ò ( ), plavix Ò ( ), pantoprazol nycomed Ò ( ), adalat Ò cr ( ). swelling and rough surface were the most frequent. chipping and capping were not observed. conclusion: our eight visual criteria are able to detect physical alteration of repackaged solid oral medicines under stress conditions. in absence of reliable data we suggest to apply this simple quality control for repackaged medicines in pharmacy practice. because chemical stability testing is not feasible in practice, pragmatic solutions are sought. further studies are needed for storage at room temperature. cp-pc : drug-related problems and symptom burden in nursing home residents kerstin bitter *, , ulrich jaehde , christina pehe , gabriela heuer , manfred krü ger clinical pharmacy, university of bonn, bonn, aok rheinland/ hamburg, pharmacists' association north rhine, düsseldorf, germany please specify your abstract type: research abstract background and objective: drug-related problems (drp) are common in the elderly due to polymedication. community pharmacies supplying drugs to nursing homes may play an important role in detecting and solving drp in nursing home residents. this project aims to evaluate whether community pharmacists can enhance the medication safety of nursing home residents by solving drp by means of a simple medication review (mr). furthermore, the applicability of a new tool to detect symptoms as potential adverse drug reactions in elderly patients should be tested. setting and method: nursing home residents at the minimum age of years insured by aok rheinland/hamburg and regularly taking at least five drugs per day were invited to participate. pharmacists performed a mr based solely on the patients' medication data, including dosage regimens of the nursing home and self-medication data. the detected and solved drp were counted. additionally, a simple questionnaire (sympel) was distributed to the patients periodically in order to assess their symptom burden. main outcome measures: frequency and type of the patients' drp as well as their symptom burden before and after the mr. results: after testing the feasibility of this intervention in a pilot study, patients were included in the main study so far. in average, the pharmacists identified two drp per patient and reported to the responsible general practitioner (gp) . as in the pilot study, most frequent drp documented by pharmacists were drugdrug-interactions ( %). % of the pharmacists' recommendations were accepted by the gp. % of the patients took at least one drug considered as potentially inadequate in the elderly. out of six different symptoms, patients reported dizziness and bruises most frequently. conclusion: pharmacists can detect many drp in nursing home residents by means of a simple mr. however, the full potential of this service to solve drp can only be exploited if the cooperation with the gps is improved. please specify your abstract type: research abstract background and objective: medicines for rare diseases (rd) are costly and have limited efficacy evidence but they represent around % of all innovative medicines. therefore, countries are facing challenges in providing patient access to them. the purpose of the study was to assess the patient access for slovenia and compare it with other european countries in the last decade. setting and method: the medicines for rd that obtained marketing approval between and via centralised procedure were included in the study based on the orphanet list from january . using the quarterly ims health database, sales data were analysed for slovenia and other european union countries, norway and switzerland. patient access was assessed for each country and comparisons between the countries were made using the three main outcome measures. main outcome measures: the number of medicines for rd available; time to first continuous use after marketing approval; total pharmaceutical expenditure for medicines for rd in euros. results: altogether, medicines for rd were approved between and . complete sales data were available for medicines which were included in the comparison. for germany and the united kingdom the continuous use of ( %) and ( %) was observed, respectively. the following italy, france, denmark and sweden use - % of all the medicines. in slovenia, ( %) medicines for rd were introduced. germany and the united kingdom times to first continuous use were the shortest (median time - months after marketing approval). median times below months were observed for norway, sweden, austria, the netherlands, france and switzerland. other countries were slower in enabling first continuous use (median time from to months). germany, france, switzerland had the largest pharmaceutical expenditure per inhabitant in ( . , . and . euros/inhabitant) while slovenia amounted to . euros/inhabitant. in slovenia more than a half of the medicines for rd approved in europe are used which ranks it in the middle of all the countries in comparison. comparing to the important european pharmaceutical markets like germany, united kingdom, france and italy, slovenia's median time to first continuous use is longer and pharmaceutical expenditure per inhabitant for these medicines is lower. pec : mapping of the dlqi scores to eq- d utility values using ordinal logistic regression and monte carlo simulations: is it plausible? please specify your abstract type: research abstract background and objective: converting dermatology life quality index (dlqi) scores to generic measure data would allow utility calculations and enable cost-effective analysis. this would meet the needs of health technology assessment agencies (htas) such as nice, who preferentially use the general health measure eq- d. the dlqi is a specialty-specific measure unlike the eq- d, a generic measure from which utility values can be derived. often several measures are implemented in studies with increased cost and patient burden. ordinal logistic regression (olr) was used to develop a model to convert dlqi scores to eq- d based utility values for use in economic appraisal of medicines. setting and method: data from patients were randomly divided into estimation and validation sets to fit and test the model. a series of ordinal logistic regressions were fitted in spss v for the five eq- d dimensions based on age, sex and all individual items of the dlqi as predictors. the model produced three estimated probabilities per subject per eq- d domain. using these estimated probabilities, a series of monte carlo (mc) simulations were run for each subject resulting in predicted domain responses. from these, utility values were calculated and compared to actual patient values. main outcome measures: conversion of dlqi scores to eq- d domain data results: there are conceptual overlaps between items of the dlqi and eq- d. the validation data set (which was not included in the creation of the model), demonstrated that the models were highly predictive compared to actual responses, except for minor differences for the pain/discomfort domain. for example, for the eq- d ' mobility' domain, patients answered 'no' (predicted and patients answered 'some or extreme' (predicted ) . we examined the model's latent variables, which also demonstrated high predictability at individual level. for example for the 'usual activities' domain the mean latent variable scores were - . , - . and - . for those responding 'no', 'some' and 'extreme' respectively, showing a clear increase in the scores with response. after excluding subjects with missing variable data there were patients in the estimation set and in the validation set. the model was shown to be highly predictive and repeated simulations demonstrated a stable model. the average predicted utility value for the entire validation set ranged from . to . across the mc simulations compared to the actual average utility value of . . conclusion: using olr, we have developed a method of mapping the disease-specific dlqi onto the eq- d: utility values may then be derived for population data sets, and possibly for individuals. the olr technique could be used to convert data from other disease-specific quality-of-life measures to generic utility data for incorporation into cost-effective analyses, greatly enhancing the potential value of such information. tomi laptoš *, , tanja kersnik levart hospital pharmacy, the division of paediatrics, department of nephrology, university medical centre ljubljana, ljubljana, slovenia please specify your abstract type: descriptive abstract (for projects) background and objective: having to take medication during time in school may present certain discomfort for some children. suboptimal dosing regimens (i.e. dosing more frequent than determined by drug's trough:peak ratio) can be prevented by a clinical pharmacist's overview and therapy optimization. the aim of the study was to review and evaluate dosage regiments in paediatric patients on antihypertensive therapy. dosage regiments are defined as schedule of doses of a therapeutic agent per unit of time and the amount of a medicine to be given at specific time. design: electronic health records (ehr) review, evaluation of actual dosage regiments against regiments recommended in smpcs and clinical database (uptodate Ò ), a consecutive case series study. results: ehrs of patients, admitted to or discharged from the department of nephrology of the division of paediatrics, umcl in with suspected icd- diagnoses from i to i were reviewed. patients were excluded (diagnosis not confirmed or lifestyle-change disease management only). the remaining patients (average age . years (range - )) received daily on average . medications (range - ) in . individual doses (range [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . most frequently used drugs were perindopril (n = ), ramipril (n = ), amlodipine (n = ), bisoprolol (n = ) and doxazosin (n = ). patients received ex tempore oral suspensions. dosage regimen was not optimized in % (n = ) of the patients, among those % (n = ) receiving one medication only and % (n = ) receiving more than one medication where at least one was not optimized. furthermore, % (n = ) patients on stabledosage therapy (no dosage change of either medication in last months with satisfactory clinical outcomes) were eligible for fixeddose combination medication. with optimized dosage regimens patients would receive daily on average . medication (range - ) (- %) in . individual doses (range - ) (- %). the difference was statistically significant ( % ci, p \ . ) in both cases. conclusion: our data show that the majority of the paediatric patients on antihypertensive therapy ( %) received their medication in optimal dosage regimens. however, with an estimated every fifth patient not being on optimal dosage regimen, a multidisciplinary approach is crucial to assure that the individual patient achieves the best clinical, humanistic and economic therapy outcomes. ph : polypharmacy management programmes in the elderly: a case study in greece dimitra gennimata *, , christos kampolis , aggelos vontetsianos , jennifer mcintosh , alpana mair , on behalf of simpathy consortium please specify your abstract type: descriptive abstract (for projects) background and objective: polypharmacy management and medication adherence in the elderly are significant public health issues throughout the european union (eu). simpathy (stimulating innovation management of polypharmacy and adherence in the elderly) is a consortium of organizations representing eight european countries, aiming at stimulating innovation around management of appropriate polypharmacy and adherence, ultimately providing tools for eu policy makers to develop and/or improve, implement and evaluate programs addressing these issues. design: a mixed-methods case study was carried out in greece, to identify policies on the management of polypharmacy and adherence issues in the elderly. a desk review of the polypharmacy and adherence policies at the government, regional and institutional level has been completed. key informant interviews were conducted with policymakers and health professionals responsible for developing and implementing strategies. focus groups consisting of policymakers, clinicians and patients validated the research findings. results: although e-prescription implementation is widespread (& % coverage nationwide) and disease-specific guidelines have been developed, polypharmacy management is only associated with direct economic indicators. no formal policies or programmes are identified. significant contributions are coming from different health professional organizations that have chosen to provide expanded services to their patients, aiming at optimising drug therapy and thus polypharmacy management and medication adherence in the elderly. community pharmacists offer pharmaceutical care to patients, which includes management of prescribed medication, otc remedies, vitamins and supplements and food-drug interactions. hospital pharmacists in some state (public) hospitals review medication for inpatients and out-patients, communicate with prescribers and confirm the ''benefit-no harm'' principle in the prescribed medication (e.g. incompatibilities, side effects, -rights of medication). medical doctors, mostly general practitioners and some specialized ones, usually in primary healthcare settings, keep health records of their patients and have an overview of all administered medication. however, key barriers still remain the lack of coordination of institutions and authorities and overlap of their responsibilities, healthcare workforce and infrastructure shortages and several cultural issues. conclusion: all initiatives to medication management and medicines optimisation are provided without directive from national policies or guidelines. therefore, these activities rely on the goodwill of the health professionals to address pharmacotherapy and therefore polypharmacy management but they are not necessarily representative of what is happening nationwide. a national policy to implement the management of polypharmacy nationwide could mobilise the willingness of health professionals and ensure consistency of care. development and implementation of this policy should build on the grassroots efforts currently underway in this area. ph : clinical profile and treatment discontinuation in a tuberculosis control state programme in brazil: preliminary results from sinan database simone s. bezerra , mara guerreiro *, , , nathany pessoa , maria paula athayde , rodrigo auad , joão josé gomes , josé lamartine soares sobrinho post graduation program in therapeutic innovation, federal university of pernambuco, recife, pernambuco, brazil, centro de investigação interdisciplinar (ciiem), instituto superior de ciências da saúde egas moniz, monte de caparica, please specify your abstract type: research abstract background and objective: challenges remain in tuberculosis (tb) control. discontinuing treatment can leave patients infectious and contributes to the emergence of resistance. this study aimed to describe the clinical profile and cure and discontinuation rates of tb patients enrolled in the pernambuco tuberculosis control programme (pect). setting and method: the study was conducted in three sites in recife, brazil, designated a (one polyclinic plus eight general practice units), b (one hospital for medium-complexity patients) and c (one hospital for high-complexity patients). data were extracted from the notifiable diseases information system (sinan) for all pect outpatients, from / to / (n = ). analysis was performed with the aid of action for excel; there is on-going analysis to further explore differences across sites. ethical approval was granted. main outcome measures: clinical form of the disease, hiv testing, new cases, cure and treatment discontinuation. results: sociodemographic data were available for sites a and b only. most patients were male ( %, n = ), with age raging from to years old ( . %, n = ); most had a low education level ( %, n = ) and low socioeconomic status ( %, n = ). the most common clinical presentation was pulmonary tb. most cases were new ( . %, n = ); recurrence and enrolment after discontinuation were respectively . and . % (n = and n = ). with respect to hiv, . % of patients were seronegative (n = ); about a third ( %; n = ) had not performed hiv test. rates for cure were respectively . % (n = ), . % (n = ) and . % (n = ) in the sites a, b and c. correspondent rates for treatment discontinuation were . %(n = ), . % (n = ), . % (n = ), respectively. tb-related mortality ranged from in site c to . % in site b. conclusion: missed hiv tests may represent undetected tuberculosis/ hiv coinfection. site b presented the highest rates of intrapulmonary plus mixed tb forms, discontinuation of treatment and tb-related mortality; additionally, it had the lowest rate of enrolment after treatment discontinuation. patients co-infected with tb and hiv are firstly referred to this site, which may explain this finding. our findings may help managers allocating resources and assist clinical pharmacists in planning their interventions. findings also suggest the need of more intensive interventions in tb patients, such as pharmaceutical care programmes. please specify your abstract type: research abstract background and objective: pharmacists working in primary health clinics have various roles. pharmaceutical care is one of them. how to provide this service varies across countries and settings. the most optimal way to provide pharmaceutical care is important to define when developing clinical pharmacy services in a new setting such as primary care practices. general practitioners are key stakeholders in this endeavour. the aim of this study was to find the most optimal approach to providing pharmacist-led pharmaceutical care in primary health care clinics in iceland in collaboration with general practitioners. setting and method: action research provided the framework for this research. data was collected from pharmaceutical care interventions with patients, field observations, field notes, and interviews with general practitioners over the period of the study. the study ran from september to june . three separate semi-structured in-depth interviews were conducted with five general practitioners from one primary health care clinic in iceland at different time points throughout the study. pharmacist-led pharmaceutical care was provided to patients (n = ) before and between general practitioners' interviews. the study settings was a primary health care clinic in reykjavik area and the patients' homes. main outcome measures: how to provide pharmaceutical care in collaboration with general practitioners in the icelandic health care environment. results: direct contact between pharmacists and general practitioners over short distances are essential to providing optimal pharmaceutical care services. pharmacist's access to medical records is necessary even though face-to-face communication between pharmacist and patients are most effective in providing pharmaceutical care. pharmacist-led clinical service was deemed most needed in dose dispensing polypharmacy patients. patients require more information about drugs prescribed to them coupled with an accurate drug list with greater detail. conclusion: the most efficient collaboration when pharmacist and general practitioner is obtained when they work side by side at the primary health care clinic. when new services are developed it is vital to identify different requirements of the primary health care clinics to optimize the running of a clinical pharmacist service. ph : accompaniment of patients treated with oral chemotherapy: a survey on patients' experience laure napoly *, , pascal paubel , , sylvie burnel oncorif -regional cancer network, health law and health economics deparment, health law institute, inserm, umr s , paris descartes university, sorbonne paris cité, paris, france please specify your abstract type: descriptive abstract (for projects) background and objective: antineoplastic agents taken orally are more and more used in cancer care. these medicines confer autonomy to patients. although, they may cause adverse effects that can lead to treatment adherence issue, unjustified hospitalizations, or premature treatment interruption. proper accompaniment of patient can prevent these issues. in order to define how to organize this accompaniment, we conducted a survey on patients' experience. the objective is to describe patients care pathway and identify their needs. design: a descriptive, qualitative, prospective, survey was conducted using self-administered questionnaires, in hospitals of ile-de-france region. inclusion criteria were: having being treated with oral neoplastic agents during minimum months, age [ , solid tumor, no concomitant iv chemotherapy. collected data were: patients' sociodemographic and clinical profile, their insight about different steps of care pathway (information, treatment delivery, follow up), their behaviours and interactions with healthcare professionals, and their overall opinion. results: patients were recruited in six hospitals. their sociodemographic and clinical characteristics were variable. % were treated with targeted therapy, % with cytotoxic agent, and % with endocrine therapy. patients showed high satisfaction for given information at the beginning of the treatment. principal source of information identified was the oncologist ( %). while the delivery of treatment, % of patients beneficiated of advices from the pharmacist. accompaniment of patients during treatment seemed unequal, with: a frequency of visits with the oncologist ranging from less than - months; % of patients not knowing any telephone number they can call in case of worries or questions about their condition or treatment; % not remembering any particular accompaniment treatment (visits or calls from nurses or doctors for example). for adverse effects management, three principals actors were identified: oncologist ( %), general practitioner ( %) and pharmacist ( %). regarding the use of oral chemotherapy, patient are satisfied with: it's comfort ( %); being more actively involved in their cancer care ( %); and state not having any anxiety taking chemotherapy home ( %) . asked openly about their concerned and needs three major concepts were identified: high concern about adverse effects, positive feedback about maintaining contact with health professionals between cures and difficulties of communication between health professionals. conclusion: there is a high satisfaction regarding oral chemotherapy and health professionals. the oncologist has a primordial place in patient pathway, whereas implication of pharmacist and general practitioner stays variable. adverse effects are a major concern that needs proactive accompaniment. the variability of our results suggests that accompaniment must be flexible and adapted to the needs of each patients. the key to flexibility could be good coordination and communication between healthcare professionals. ph : general beliefs about medicines among independent elderly adults in sweden: data from an rct lina hellström *, , , victoria throfast the pharmaceutical department, kalmar county council, ehealth institute, linnaeus university, kalmar, sweden please specify your abstract type: research abstract background and objective: there is a need to improve prescription and use of medications by the elderly. the objective of a recent rct was to investigate the effects of e-learning about medicines among elderly adults. a positive impact on the primary outcome measure, knowledge about medicines, is reported elsewhere. a secondary outcome measure, general beliefs about medicines, is reported below. setting and method: the study was a randomized controlled trial in elderly people (aged c years). participants were recruited from patient associations and pensionerś associations. participants were randomized to either an intervention group that participated in the e-learning, i.e. internet modules with video and audio, or a control group that did not take part in the e-learning. post-intervention data was collected using paper-based questionnaires, completed within two weeks after agreeing to participate in the study. the general beliefs about medicines questionnaire (bmqgeneral), comprising the subscales necessity, harm and overuse, was used to elicit beliefs. higher scores indicate stronger endorsement of scale constructs (range [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . main outcome measures: bmq-general subscale scores. results: a total of elderly people were included in the study, in the intervention and in the control group. the mean age in the total population was . years and % were women. eleven percent did not use any prescribed drugs while % used more than five prescribed drugs. the patients' scores were very similar in the two groups for all three bmq subscales. the median ''overuse'' score was in the intervention group versus in the control group, the median ''harm'' score was (iqr - ) in both groups and the median ''necessity'' score was in both groups. in the total population the most commonly expressed negative beliefs referred to overuse of drugs. . % of respondents agreed with the statement ''if doctors had more time they would prescribe fewer medicines'', . % stated ''doctors prescribe too many medicines'', and . % stated ''doctors place too much trust in medicines''. a majority of the respondents agreed with the four items on the ''necessity'' scale. for example ''medicines help people to live a better life ( . % agreed)''. conclusion: the studied e-learning intervention was not shown to have any impact on general beliefs about medicines. beliefs about medicines have been associated with a number of background variables which might explain why increased knowledge about medicines alone cannot change such beliefs. in general, respondents in the study had highly positive beliefs about the necessity of medicines. nevertheless, the results indicate that overuse of medicines is regarded as a problem. ph : maf-plus: pharmacists' contributions to provision of financial assistance for medications ian wee * , charlene ong, niron naganathar changi general hospital, singapore, singapore please specify your abstract type: descriptive abstract (for projects) background and objective: the medication assistance fund plus (maf-plus) is a government scheme introduced in singapore in to provide financial assistance to needy patients who meet pre-set criteria based on means testing. unlike previous schemes, maf-plus provides broader discretion to institutions when providing financial assistance. in our institution, pharmacists reviewed patients' case and medication histories, and filed recommendations to a multidisciplinary committee tasked with approving deserving maf-plus applications. the pharmacists' contributions to the committee, and the outcomes of the applications, are presented in this study. design: all maf-plus applications received between st october and st december were reviewed. pharmacists' comments for each application, where provided, were noted, as well as the range of medicines applied for, review approval rates, and cumulative percentage of available funds utilised. the effect of a recent widening of the scheme's scope to include notable high-cost items was also evaluated. results: between and , maf-plus applications were reviewed, of which ( . %) were approved. of the rejected applications, ( . %) were channelled to alternative financial assistance schemes on the recommendation of the pharmacists. the medicines most commonly applied for were intended for the treatment of cardiac ( . %), respiratory ( . %), and psychiatric ( . %) conditions. pharmacists' recommendations also led to a gradual expansion of our institution's list of pre-approved medicines-from in - to by end- . from onwards, pharmacists previewed increasing numbers of applications for high-cost medicines, particularly those for treatment of retroviral disease, hepatitis c, and rare diseases. cumulative utilisation of maf-plus funds (inclusive of annual replenishment) rose from . % in - to . % by end- , representing an average year-on-year growth of . %. conclusion: using a process of judicious previewing of maf-plus applications, and recommendations to the maf-plus committee, pharmacists contributed to a high percentage of patients receiving financial assistance for medications. despite a steep growth in the number of applications received between and , this approach helped to prevent over-extension in fund utilisation. pharmacists will likely be increasingly relied upon due to an anticipated rise in the number of applications for high-cost medicines. please specify your abstract type: research abstract background and objective: adolescents often treat themselves and take medications without parental supervision. lack of experience and knowledge of medicines in this age group frequently leads to inappropriate use of medicines and adverse drug reactions. data about the use of medicines among slovak adolescents and their knowledge of medicines have not been studied yet. setting and method: for our study we used the questionnaire method. the questionnaire contained multidimensional items with closed-ended and open-ended questions, which focused on the characteristics of the adolescentś health status, use of medicines, also in relation to parents and adolescentś knowledge and perception of medicineś risk. we distributed validated questionnaires for adolescents aged from to at secondary schools in all regions of slovakia. response rate was . %. questionnaires were finally analysed. the differences in the distribution of categorical variables between groups were evaluated using the chi square test. sas . . was used as statistical software. main outcome measures: to determine adolescentś knowledge of medicines in terms of efficacy, self-medication, safety of therapy and analyse which medicines are the most frequently used by adolescents. to compare adolescents with chronic disease and healthy ones from the perception of pharmacotherapy point of view. results: in the analysed group . % (n = ) of adolescents are treated for chronic disease. mostly they suffer from allergy ( . %, n = ) and skin diseases ( . %, n = ). adolescents with chronic disease use regularly prescription medicines ( . %, n = , p \ . ) and over the counter medicines ( . %, n = , p \ . ). this group of adolescents better accept the pharmacotherapy with parental supervision ( . %, n = , p = . vs. healthy adolescents) and they believe in effectiveness of prescription medicines ( . %, n = , p = . vs. healthy adolescents). most frequently prescribed medicines were azithromycin, levocetirizine, ofloxacin and over the counter medicines were ibuprofen, paracetamol, ascorbic acid. we found out in all group of adolescents that . % (n = ) prefer self-medication without check-ups, . % (n = ) used drugs in the last months without a prescription, . % (n = ) take over the counter medications independently without the supervision of parents, . % (n = ) buy medicines themselves in the pharmacy, . % (n = ) do not take medications as recommended, . % (n = ) believe that they have enough knowledge of medicines which they take, . % (n = ) resp. . %, (n = ) believe that prescription medicines resp. over the counter medicines are safe. conclusion: questionnaire analysis pointed out that slovak adolescents have not enough knowledge of medicines. the study provides new information in the field of risk perception and adolescentś knowledge of medicines in the slovak republic and highlights the areas that need to be studied in the future in terms of adolescentś education. federal university of pernambuco, state technical school prof. agamenon magalhães, recife, pernambuco, brazil please specify your abstract type: research abstract background and objective: the effectiveness of tuberculosis (tb) control programmes depends critically on patients completing appropriate treatment. this study aimed to outline the cure and discontinuation rates of patients enrolled in the pernambuco tuberculosis control program (pect), based on dispensing data. setting and method: the study was carried out in three sites in recife public health system, brazil, designated a (one polyclinic plus eight general practice units), b (one hospital for medium-complexity patients) and c (one hospital for high-complexity patients). data were collected between - / , through reports from the stock management software for public pharmacies (horus) for pect outpatients. reports corresponded to a total of patients ( , and in sites a, b and c, respectively). horus defines ''cure'' as medicines collection for three, six or nine consecutive months without interruption, depending on the treatment scheme; discontinuation is defined as non-sequential collection of medicines or treatment interruption for two consecutive months or more. patients were assigned an ''undetermined'' status if treatment was ongoing. data were inputted onto an excel spreadsheet and checked for accuracy. quisquared test, fisher's exact test and bootstrap analysis were performed with r statistical computing. ethical approval was granted. main outcome measures: cure and discontinuation rates for pect outpatients. results: demographic data are not available for the sample. rates for cure were respectively . % ( ), . % ( ) and % ( ) in the sites a, b and c, while rates for treatment discontinuation were . % ( ), . % ( ) and % ( ), respectively. discontinuation rates were significantly different among the sites a, b and c (p \ . ). bootstrap analysis showed that overall the proportion of patients with an ''undetermined'' status in each site did not significantly change these differences. conclusion: only site a had an acceptable discontinuation rate, in light of the world health organization recommendations. this deserves attention as default treatment leaves patients infectious for longer, increases the risk of poor outcomes and fosters resistance to antibiotics. pharmacists could use dispensing data to signal tb patients at-risk of discontinuation, and subsequently tailor interventions addressing its causes. site b had the greater number of patients which discontinued treatment. patients co-infected with tb and hiv are firstly referred to this site, which may explain this finding. our findings suggest the need of more intensive interventions in patients co-infected with tb and hiv, such as pharmaceutical care programmes. please specify your abstract type: research abstract background and objective: many efforts are done to organise good quality and safe pharmaceutical care. in general, the involvement of a hospital pharmacist or hospital pharmacy personnel in the process of medication reconciliation results in a reduction of the number of medication discrepancies. however, in case of emergency admissions this topic is still insufficiently studied. the introduction of good medication reconciliation on the emergency department (er) requires firm logistical and organisational efforts. we investigated the effects of a drug reconciliation intervention by pharmacy personnel during emergency admissions in order to identify discrepancies between medication lists taken by er physicians and by pharmacy personnel. setting and method: this observational, comparative, non-randomised intervention study was performed in . we calculated that a population size of patients was sufficient to perform reliable measurements. inclusion criteria: all patients presented at the er and admitted to a hospital ward \ after presentation with usage of one or more prescription drugs. exclusion criteria: age \ years, residency outside the region delftland, inability to undergo an oral interview, absence of a medication list of the public pharmacy (ozislist), decease of the patient during er-stay and patients undergoing surgical procedures. discrepancies between both medication lists taken by an er physician or pharmacy technician were classified in four categories of increasing severity ( = no discrepancy to = clinical relevant discrepancy) using the index of the national coordinating council for medication error reporting and prevention (www.nccmerp.org). discrepancies were categorised by a panel consisting of a pharmacy technician, a (senior) hospital pharmacist and a th year pharmacy student. statistical analysis was carried out with a statistical software package (spss ) using the mann-whitney u test and chi squares test. main outcome measures: during the intervention measurement we analysed the reconciliated medication by comparing the er's physician's list with the list of the pharmacy technician after a medication verification interview. the number of discrepancies were measured and judged by the panel. discrepancies were given a category , , or as defined. results: during the intervention measurement patients were admitted to the er. sixty-five ( ) patients ( . %) met the in-and exclusion criteria. the number of medication discrepancies decreased significantly after intervention of the pharmacy technician by %, from to discrepancies. the average number of discrepancies per patient after intervention decreased by . %, from average . to . discrepancies per patient. conclusion: medication verification by pharmacy personnel in the er reduces the number of medication discrepancies by half. medication lists generated with a standard interview by pharmacy technicians in combination with an ozis-list on admission of patients at the er is more complete and accurate than the current method. hp-pc : discharged patients: a problem for community pharmacists? information transfer, as well as the role, needs, and objectives of pharmacists when they care for recently discharged patients. setting and method: a focus group was conducted with a sample of six community pharmacists from personal contacts to represent different characteristics. the focus group consisted of different questions and the recording was transcribed, fragmented and categorised. based on these results, a nationwide online-survey was created with the following questions: a) responder's characteristics, b) number and origin of prescriptions, c) role fulfilment of the joint-who/fip-guideline on good pharmacy practice, rated with a -point likertscale, d) information items derived from the focus group discussion grouped into four categories and evaluated for their availability and for their usefulness by likert-scales, e) goals for discharge optimisation, f) additional comments. the questionnaire was piloted and translated forward and backward to french and italian by native speakers. it was sent to all managers of pharmacies belonging to the swiss pharmacist's association in summer (n = ). main outcome measures: conclusions from focus group discussion and responses to questions a-f from the nationwide questionnaire. results: the focus group participants ( . ± . years, % female, % employees) emphasised the importance of an expanded information transfer, especially for medication changes, unclear prescriptions, and information about a patient's medication acquisition. they were concerned about their extensive workload of discharge prescriptions, and mentioned treatment continuity as one of their goals. the questionnaire was answered by pharmacists (response rate . %, . ± . years, . % female). there were . % of responders who reported to fulfil their role (to manage a patient's therapy, function b) not satisfyingly. unavailable but essential information were allergies and the specification of off-label use prescription. unavailable although desired information were the reasons for therapy changes, indications, appointments, contact information, or compounding formulations. concerning design and transfer, information should be written in a structured way but no clear preference for a transfer method was found. goals of community pharmacists were: improved treatment continuity, patient safety, and pharmaceutical care. conclusion: swiss community pharmacists rarely receive sufficient information on discharge prescriptions. appropriate pharmaceutical care is therefore impeded. the knowledge and application of the findings enable directed optimisation of discharge. hp-pc : patients attitude for using antipsychotic medication in the norwegian early intervention in psychosis, tips study rafal yeisen *, , stein opjordsmoen , , , inge joa , , jan olav johannessen , , jone bjørnestad on behalf of centre for clinical research in psychosis, psychiatric division, stavanger university hospital, stavanger, norway background and objective: poor drug adherence in patients with psychosis leads to relapse, re-hospitalization, poor outcome and increased consumption of health services. pharmacoclinical studies have demonstrated that the treatment response decreases with each relapse. it is estimated that % of patients suffering from chronic illness are not taking medication as prescribed after months. the purpose of this study is to investigate which experiential factors that potentially might affect adherence with medication in adults with psychotic disorders. setting and method: in a descriptive qualitative sub-study in the ongoing norwegian early intervention in psychosis, tips study, where twenty-first episode patients ( male, female) participated in semi-structured interviews years after inclusion. they were still using or had used antipsychotics during the last years. data were analysed using interpretative phenomenological analysis. main outcome measures: adherence to antipsychotics. results: the data suggested four main themes, reflecting the patients' subjective experiences and their impact on the desire to adhere to antipsychotics: ( ) admission experience as a psychotic's patient; ( ) information from healthcare staff; ( ) limited involvement in decision-making; ( ) attitude to antipsychotics. conclusion: a number of factors had a positive influence on adherence to antipsychotics. pleasant admission/stay experiences, feeling that antipsychotics had therapeutic effects, mild or no side effects, and believing that antipsychotics are necessary and useful, were typical statements. please specify your abstract type: research abstract background and objective: the hospital-to-home transition is a vulnerable stage in a patient's care. patients can experience problems with medication supply, which possibly lead to therapy interruptions. the objectives of this study were to investigate medication supply after discharge, and patients' and physicians' opinions about the current discharge process and possible optimisations. setting and method: a telephone interview was conducted with discharged patients from the surgical and internal medical wards from the cantonal hospital in baden (switzerland). inclusion criteria were: patients c years old, discharged home with a discharge prescription. patients were called between the nd and th day after discharge and a piloted, structured interview was performed, consisting of questions on experiences and optimisations. afterwards, semi-structured interviews were conducted with five physicians from the study hospital. results from patient interviews and the general discharge process were discussed. main outcome measures: proportion of filled prescription, frequency and type of supply problems including therapy interruptions. opinions of physicians and patients on current discharge process and possible optimisations. results: discharged patients were . ± . years old, % female, % from internal medicine, and % regularly visit the same pharmacy. of the interviewed patients, have not filled their prescriptions yet and had their prescription filled when they were called. of these, % of them visited the pharmacy on the day of discharge, but it took up to the th day until all of them received their medication. supply problems were encountered by patients ( %), mainly because of the medication not being in stock in the community pharmacy. only four patients experienced therapy interruptions, which took up to the rd day post-discharge. patients discharged from internal medical wards had more supply problems compared to surgical wards (relative risk = . , p = . ). patients experiencing supply problems had statistically significant more medicines on a daily basis ( . ± . vs. . ± . , p = . ). physicians were surprised about the late prescription filling and worried about the disease outcomes. however, interruptions were interpreted as unfrequent. when asked if, in future, hospitals should transfer prescription to the community pharmacy prior to discharge, % of patients refused and physicians were undecided, mainly because of a questionable benefit. but both groups indicated that giving some bridging supply would be welcome. conclusion: this study showed that patients discharged from a swiss hospital encounter supply problems, but therapy interruptions are seldom. giving some bridging supply was preferred over an early information transfer by patients and physicians. interventions should consider these opinions and focus on internal medicine patients with high number of medication. please specify your abstract type: research abstract background and objective: adherence to secondary prevention evidence-based medical (ebm) therapies for patients with st-segment elevation myocardial infarction (stemi) is essential to reduce long-term rates of major adverse cardiovascular events. current guidelines recommend the long-term use of low-dose aspirin, highintensity statins, angiotensin-converting enzyme inhibitors (acei)/ angiotensin receptor blockers (arb) and beta-blockers (bb), in addition to p y inhibitors for year. we aimed to assess the adherence to secondary prevention ebm therapies from discharge to one-year follow-up among patients with stemi undergoing primary percutaneous coronary intervention (pci) in contemporary practice. setting and method: observational single-centre study including consecutive patients with stemi undergoing primary pci in a tertiary hospital in switzerland over a one-year period. secondary prevention ebm therapies were assessed at discharge and at one-year follow-up. main outcome measures: prescription of key secondary prevention ebm therapies (aspirin, p y inhibitors, statins, acei/arb and bb) from discharge to one-year follow-up after stemi. bb was recommended only for patients with heart failure or left ventricular ejection fraction (lvef) \ %. results: a total of patients were included. ebm drug prescription at discharge was . % for aspirin (n = ), . % for p y receptor inhibitor (n = ), . % for statin (n = ), . % for acei/arb (n = ) and . % for bb (n = , among patients with lvef \ %). ticagrelor ( . %) was the major p y inhibitor prescribed. overall, ebm drugs were missing at discharge, with of these missing drugs having no justification for no-prescription (contraindications, allergy or intolerance). at one-year follow-up (median . months, n = ), aspirin, statins and acei/ arb prescription rates were . % (n = ), . % (n = ) and . % (n = ) respectively. out of patients ( . %) with lvef \ % received a bb. among patients treated with ticagrelor at discharge, ( . %) were receiving ticagrelor at follow-up, whereas ( . %) were switched to another p y inhibitor. among patients who discontinued ticagrelor (n = , . %), duration of dual antiplatelet therapy was months for % (n = ) and discontinued prematurely (\ year) for % (n = ) patients. reasons for ticagrelor early discontinuation or switch were not specified. conclusion: in a real-world cohort of patients with stemi undergoing primary pci, prescription of recommended secondary prevention medications at discharge is excellent. adherence to ebm therapies at year remains high with more than % of patients receiving all ebm drugs. early discontinuation of dual antiplatelet therapy was observed in % of patients, whereas ticagrelor was switched for another p y inhibitor in . % of patients. these observations highlight key opportunities to improve longitudinal use of secondary prevention therapies after stemi in routine clinical practice. although side effects are less common than traditional chemotherapies, certain ones such as pain, fatigue, nausea and vomiting can still be bothersome. in oncology outpatient clinics, side effects are monitored by oncology nurses; however due to high patient turnover and limited numbers of nurses, the assessment of side effects might not be performed adequately. therefore, aim of this study was to determine side effects of immunotherapy and targeted therapy and to compare the severity assessment of side effects by clinical pharmacist and nurses. setting and method: the study was conducted in the hacettepe university oncology hospital outpatient clinic. the patients who have been taking ipilimumab, nivolumab, pembrolizumab, bevacizumab, panitimumab or cetuximab during october -march were included. the assessment of side effects were undertaken by a clinical pharmacist and nurses separately on each visit using the common terminology criteria for adverse events version- toxicity assessment scale. an independent clinical pharmacist compared the side effects' assessments by pharmacist and nurses for analysis. ethical approval was obtained from hacettepe university ethics committee. main outcome measures: to compare the severity of side effects of targeted drug therapies which were assessed by a clinical pharmacist and nurses. results: during the study period visits of patients were evaluated. a total of side effects assessments were recorded. among those assessments ( . %) was assessed in different ranking by nurses and pharmacist. the differences in the number of assessments were mainly seen in criteria related to pain (n = ; ), sensory loss (n = ; ), fatigue (n = ; ), stress (n = ; ), insomnia (n = ; ) which was performed by nurses and pharmacist respectively. other side effects detected only by clinical pharmacist were oedema, cough, gastrointestinal complaints (heartburn, cramp) and sensitivity of odour which require close monitoring and in-depth counselling by clinical pharmacist. conclusion: this study explores the differences in assessment of side effects by pharmacist and nurses in targeted therapies. routine assessment of side effects between chemotherapy cycles might yield to misinterpretation or inadequate assessment due to workload of outpatient clinic. therefore, inter-professional interactions in outpatient clinics might close the communicational gaps and improve patient care. hp-pc : implementation of clinical pharmacy in the acute psychiatric wards: improving quality of medical treatment across health care sectors amila zekovic *, , signe kristensen , lisbeth lund pedersen clinical pharmaceutical services, capital regional pharmacy, head of clinic, mental health services, copenhagen, denmark please specify your abstract type: descriptive abstract (for projects) background and objective: a study from shows that people with a mental disorder had a two-to threefold mortality compared with the general population in denmark. life style diseases are the major reason for the excess mortality, partly due to undertreatment of physical disease and well known side effects from medicines such as obesity, diabetes, and heart disease. in may , a clinical pharmacy service (cps) was implemented in all acute psychiatric wards (apw) in the capital region as a part of a three-year project funded by the danish health authorities. the objective is to illustrate how the implementation of clinical pharmacy in the apw in copenhagen increases the focus on drug related problems, rational pharmacotherapy and side effects, increasing the quality of medical treatment and patient safety across health care sectors. design: data was collected at the apw in copenhagen which consists of three wards and has a total capacity of beds. inclusion criteria were patients to which two or more of the following apply: • c years of age • c drugs • high risk drugs (clozapine, sertindole and opioids) • combination of antipsychotics and benzodiazepines • diagnosed with liver/kidney disease the secondary inclusion criteria were all patients receiving c drugs as a single criterion. to obtain a valid medication history and secure medication reconciliation, the pharmacist interviewed included patients. the patients were also asked about side effects, compliance, and perceived effects of treatment. a medication review was conducted based on the patient interview, screening for interactions in an interaction database, and consideration of biomedical data in order to evaluate if treatments should be adjusted, initiated or discontinued. the pharmacist's input was discussed with the doctor, as inputs are more likely to be considered if they are communicated orally. finally, all inputs were documented in the patient's journal as a pharmacist note. the model for improvement was used as a tool for implementing the cps and is being used continuously for improving the service. results: between may th and june th , patients were screened at admission to the apw, of which . % met the inclusion criteria ( patients). in this period the pharmacist conducted notes, indicating that . % of the included patients were seen by the pharmacist. in april , patients were in average admitted with . drugs and . inconsistencies between the hospital's medication orders and the medications that the patient had been taking. regarding patients who are discharged to community care shortly after admission, the pharmacist note is sent to their general practitioner for follow up. conclusion: overall, the implementation of a cps in the apw has been successful. medication reconciliation ensures that the patient is provided with correct medicine at admission, transfer or discharge. by performing a thorough medication review based on a consultation with the patient, the service contributes to an increase in quality of medical treatment. please specify your abstract type: descriptive abstract (for projects) background and objective: the importance of the role of a clinical pharmacist resident in the operating room during months, in a private hospital belonging to a group devoted to healthcare for over years. the hospital is recognized as a reference centre of excellence of hospital care in portugal. it has inpatient beds, two surgical blocks with rooms and beds in the intensive care unit. the aim of the clinical pharmacist in the operating room is to ensure compliance with good clinical practice, safety and pharmacotherapeutic effectiveness, as well as optimization of drug costs. design: . logistics restructuring of pharmaceutical services and the need of the physical presence of the pharmacist in the operating room. . furthermore, the workstation of the pharmacist is moved to the operating room and the in-depth study of all medicines used in the operating room. . in compliance with the joint commission, definition, optimization and adjustment of drug stocks to the needs of the service itself. in close collaboration with the nursing staff, consumer kits were created for registration of drugs by type of surgery in order to facilitate registration and ensure billing efficiency. control of the analgesic drug's dispensation circuit in hospitalized surgical patients that stay less than h in the hospital. ensure compliance with the project through which the health regulatory authority evaluates several hospitals in the country, creating a national ranking among hospital specialties. . clinical phase: creation of prescription protocols by type of surgical intervention based on national clinical guidelines. validate prescriptions in the intra-surgical block in compliance with antibiotic prophylaxis, antiemetic and thromboembolic, checking deviations in therapy according to good practice. identify pharmacologic hypersensitivities of patients by consulting the clinical process and anaesthesiology records. provide information on drugs, drug efficacy monitoring and adverse drug reactions in risk management platform. check off label use of drugs. results: of a total of interventions, relate to revenue optimization and relate to clinical interventions. there was an increase of approximately % in billing. on what regards to clinical interventions, the majority of them showed deviations from good clinical practice. the physical presence of a clinical pharmacist in the surgical block is essential as the prescription and administration of drugs is carried out simultaneously, allowing immediate therapy validation, in order to increase the safety and efficacy. the pharmacist has the ability to interact with the multidisciplinary team, as well as monitoring the patient's clinical process, the pharmacotherapeutic profile and drug allergies, allowing the detection of any adverse drug reaction on-time. all these interventions are possible in the pre, intra and postoperative phases. results: counselling (av.(±sd) duration: ± min) was performed in patients ( . % female; av.(±sd) age: . (± ) years; av.(±sd) medicines at discharge: . (± . )). in % of patients mrps were intercepted. the five most common mrps (%) were: need for organisational support ( . , e.g. proper prescriptions' writing), therapy-related discussions ( . ), untreated indications ( . ), errors in documentation ( . ), and medicines without an indication ( . ). patients ( . %) classified for study inclusion, of whom ( . %) consented to be followed-up and ( %) provided data. roughly % of patients report having received information about medicines at discharge, of which three-fourths remember being informed by the pharmacist. more then every second patient ( . %) reported having received valuable new information. changes in chronic-use medicines occurred in . , . , and . % of patients at -, -, and -month, respectively. at -month, in . % of patients chronic-use medicines were newly prescribed, in . % discontinued. medical specialists initiated these changes in . % of patients. one out of five patients couldn't recall the reasons for changes in medication. nearly % of patients showed moderate to little medication adherence at -month. it did not significantly change during the follow-up period. conclusion: clinical pharmacists' counselling prevents mrps at the transition from hospital to home. follow-up data show that changes occur in one out of three patients. medication adherence remains stable, but generally needs to be improved. please specify your abstract type: research abstract background and objective: until , prescription analysis was based in our hospital pharmacy. clinical pharmacy has been deployed in care units since . many clinical pharmacy services were developed: medication reconciliation, patient's therapeutic education and counselling, and prescription analysis unit based. the purpose of this study is to assess the impact of the clinical pharmacist as a direct patient-care team member on prescription analysis. setting and method: we collected pharmaceutical interventions (pis) of the first months of and at the same period of the year in the neurology unit when the pharmacist was unit based. we studied and compared type of pis (medication, drug related problem-drp), rate of pis acceptance and clinical impact. focus was made on high alert risk medications and potentially inappropriate medications. . % in versus none in . when prescription analysis was based in the pharmacy unit, % of drp detected by the pharmacist had a potential clinical impact versus % when the pharmacist performed prescription analysis in the care unit (p \ . ). three drp detected in had serious potential harm. results: ward-based prescription analysis allowed detecting five more times drp with a significant more important clinical impact than pharmacy unit based prescription analysis. the clinical pharmacist as a direct patient-care team member is more efficient in detecting serious potential harm. indeed, the pharmacist has a greater knowledge of the patient's clinical condition. nevertheless the global rate of acceptance of pis was greater when the prescription analysis was based in the pharmacy unit even if the difference is not significant. but prescription analysis is more complex when performed in the care unit, taking account adherence of the patient, and potentially inappropriate medications resulting in much higher risk-taking by the ward-based pharmacist. conclusion: this study showed that unit based prescription analysis is the best way to detect drug related problem. it must be competed by medication reconciliation and medication review to improve medication safety process. hp-pc : qt-prolongation in an acute psychiatric setting: fact or fiction? eva jacxsens *, , hans van den ameele , jü rgen de fruyt , yves vandekerckhove , frank vancoillie , veerle grootaert pharmacy, psychiatry, cardiology, az sint-jan brugge-oostende av, bruges, belgium please specify your abstract type: research abstract background and objective: several psychotropic drugs can induce qt-prolongation, which is a well-known risk factor for developing torsade de pointes (tdp) and sudden death. the clinical relevance of this side effect of psychotropic medication remains unclear, especially in patients hospitalized in an acute hospital. to interpret the clinical importance of psychotropic drug induced qt-prolongation, we investigated the prevalence of these electrocardiographic changes. setting and method: a prospective study was conducted on four psychiatric wards in a general hospital: two acute, short-term psychiatric units (asp and asp ), one addiction service unit (asu) and one geriatric-psychiatric ward (gpw). all adult patients admitted between october st and march th on a psychiatric ward were eligible for inclusion. at admission, an ecg (ecg ) was performed and creatinine and potassium levels were measured. a second ecg (ecg ) was performed at least days after the start of a psychotropic drug associated with a risk of qt-prolongation. qtcprolongation was defined as ms for males and ms for females. clinically relevant qtc-prolongation was defined as c ms. statistical analysis (r software) was done as appropriate. main outcome measures: prevalence of psychotropic drug induced qtc-changes and correlating factors. results: patients (mean age years, %female) were enrolled in the analysis. in patients, an ecg was performed. qtc + were prolonged in . %( / ) of females and . %( / ) of males. no clinical relevant prolongation (c ms) was registered. higher qtc intervals were measured in the geriatric population. . %( / ) of all measured qtc were situated between [ c qtc + b ms] in gpw versus . %( / ) in the other units. significant difference in qtc-changes was associated with sex (p = . ). there was no correlation assessed between qtc-prolongation and age, number of psychotropic drugs or a specific single psychotropic drug (p [ . ). conclusion: in this study qtc-prolongation due to psychotropic drugs is less common than previously described. ecg monitoring may be unnecessary in the follow up of patients without risk factors and could reduce hospital and community costs. however, considering the potential harm associated with tdp, qt-prolongation should be avoided. we recommend recording an ecg before the start of a qt-prolonging psychotropic drug in risk patients: patients with a chronic alcohol or drug addiction, a cardiac history, on concomitant therapy with at least two qt-prolonging psychotropic drugs, or geriatric patients ([ years). hp-pc : implementation of medication reconciliation aase m. raddum *, , anne-lise sagen major sykehusapotekene i midt-norge, sjukehusapoteket i Å lesund, avd. volda sjukehus, volda, sykehusapotekene i midt-norge, sjukehusapoteket i Å lesund, Å lesund, norway please specify your abstract type: descriptive abstract (for projects) background and objective: a correct and accurate medication list should accompany patients at transitions in care from one setting to another, including admission to hospital. complete information on drug use is a prerequisite for all hospital treatment, whereas incomplete information represents a potential patient safety risk. medication reconciliation is defined by the world health organization (who) as ''…the formal process in which health care professionals partner with patients to ensure accurate and complete medication information transfer at interfaces of care.'' the objective of this study was to investigate the quality of the medication history obtained for admitted patients. furthermore, measures to improve the quality of medication histories, i.e. implementation of medication reconciliation, were initiated. design: the study included patients admitted to the internal medicine ward. a comprehensive medication history was determined by performing a standardized patient interview and/or by using relevant sources of information. the primary endpoint was discrepancies between the medication history obtained on admission and the one determined prospectively by a clinical pharmacist. the clinical relevance of the discrepancies was not determined, but sorted according to six major categories, such as: medication not in chart, but patient reports using (omission) and medication in chart, but patient reports not using (commission). further on, in order to minimize the risk of discrepancies, it was focused on implementation of medication reconciliation. a campaign was initiated, where a clinical pharmacist held information meetings regarding the medication reconciliation procedure. for the next weeks, the degree of medication reconciliation was recorded. to spur the degree of medication reconciliation, each ward's weekly numbers were published and the ward with the highest degree of medication reconciliation won a prize. results: among the patients included, a total of discrepancies were revealed. in summary, patients had at least one discrepancy in their medication history, resulting in discrepancies in the medication lists of % of the included patients. at the start of the study, the level of medication reconciliation varied among the wards ( - %), while at the end of the study the levels were increased ( - %). conclusion: all the included wards improved their level of medication reconciliation during the study period. however, these new combinations have potential drug-drug and herb-drug interactions which can affect the safety and effectiveness of the treatment. in our clinical practice, the clinical pharmacist provides patient education about direct acting antiviral drugs (daa) based-regimens, promotes medication adherence and manages potential interactions with hcv treatment. the aim of the present study was to determine the prevalence of use of herbal products in the patients on hcv treatment, and to describe the potential hepatotoxicity of the herbal products and their interactions with hcv treatment. design: we included all adult patients on daa treatment for hcv who were dispensed drugs from / / to / / . we retrospectively recorded demographic data (age and gender), clinical data related to hcv infection (hcv genotype, fibrosis stage, daa regimen and treatment outcomes) and type of herbal products consumed. we then assessed the presence of herb-drug interactions and the potential hepatotoxicity of herbal products. results: we obtained data from patients on daa-based treatment for hcv. the prevalence of consumption of herbal products prior to starting the treatment was . % ( / ). the most consumed herbal products were (prevalence [ % among herbal products users): milk thistle, green tea, chamomile, valerian, pennyroyal, boldo and artichoke. we detected four herbal products with potential hepatotoxic effects according to the literature: milk thistle, green tea, pennyroyal and aloe vera. the prevalence of consumption of these hepatotoxic plants among herbal products consumers were, respectively: . , . , . and . %. we detected herb-drug interactions or potential for hepatotoxicity in out of patients who consumed herbal products. the management of these potential interactions consisted of stopping the herbal product before starting the hcv treatment. conclusion: the consumption of herbal products in our hcv patients was frequent. the management of potential interactions was conservative, recommending to stop herbal products. clinical pharmacists have an important role in the counselling, detection and management of potential herb-drug interactions and herbal products-related hepatotoxicity. poster discussion forum iii: hospital pharmacy and pharmaceutical care please specify your abstract type: research abstract background and objective: anaemia is a common comorbidity of chronic kidney disease. intravenous (iv) iron is used when oral iron formulation became insufficient or to reduce the use of erythropoiesis-stimulating agents (esas) in haemodialysis (hd) patients. the lack of generic group for iv iron sucrose (is) preparations leads to a controversial issue about their clinical effectiveness. in this study, we evaluated the effectiveness of original is compared to is similar (iss) in hd patients. setting and method: a retrospective monocentric observational cohort study was conducted from / / to / / , in a stable hd population to compare is and iss. the follow-up periods lasted weeks and were separated by a one-month wash-out period. original is and iss were administered respectively during the first (p ) and the second (p ) periods. the comparisons were performed using the paired student's t test or the paired wilcoxon test for continuous data and the fisher's exact test for categorical data. main outcome measures: the main endpoint was the difference in haemoglobin (hb) levels between p and p per patient. anaemia parameters (serum iron, serum ferritin, transferrin saturation ratio), the number of transfused patients, the doses of iv is and the doses of erythropoiesis stimulating agents (esas) were compared before and after the switch from is to iss, as secondary endpoints. results: a total of patients were included. there was no significant difference in mean hb value between p and p ( . ± . mmol/l versus . ± . mmol/l p = . ). anaemia parameters were significantly different between p and p (mean serum ferritin, serum transferrin and transferrin saturation ratio) with p \ . , except to the mean serum iron. the mean monthly dose of iv iron per patient and the mean dose of esas were respectively in p and in p : . ± . mg versus . ± . mg (p = . ) and . ± . ui/kg/week versus . ± . ui/kg/ week (p = . ). transfusions occurred less frequently in p than in p (p = . ). conclusion: this study showed that iss was as effective as original is regarding hb levels. however anaemia parameters appeared to be in favour of is; the mean dose of esas seemed to be higher after switching from is to iss. these outcomes should be further explored using prospective comparative clinical studies. please specify your abstract type: research abstract background and objective: the pharmacy residents are sometimes up to deliver chemotherapy when they are on night or week-end duty at the hospital. a dispensation's error (delivery of metoject Ò (methotrexate) for intrathecal (it) injection whereas it doesn't have the indication for this use), led us to test the pharmacy residents' knowledges about the it access in order to underscore the points to be improved. the final aim of this work is to secure the pharmaceutical care of the patient h a day, days a week. setting and method: an online and anonymous survey of questions was sent to the residents of our area. it was composed of three parts: specific general information, questions about the chemotherapy specifically (indication, maximum dose and volumes, molecules used), illustrated questions about real situation for the dispensation on duty. the answers were collected over a two weeks period. main outcome measures: we studied the rate of good answers in global and by respondent. results: twenty-five residents answered the survey, among them % never achieved any internship in a centralized unit of reconstitution of chemotherapy (urc). all the levels of internship are represented: st year (n = ), nd year (n = ), rd year (n = ) and th year (n = ). only % know where a medicine is injected intrathecally on the spinal column, % know on which level of the meninges. three residents think that a nurse can inject intrathecally. they also had to select the molecules which can be injected by this access: % answered vincristine, % vinblastine, % bortezomib; despite these three molecules are mortals if they are injected intrathecally. the majority know the indication of the it chemotherapy: prevention and treatment of cancers' meningeal localizations. sixty percent do not know that several molecules can be injected for the same patient in the same time. the maximum dose of methotrexate is known for half of the respondents, but only % for the cytarabine'one. only residents out of know that ml is the maximum volume allowed to be injected for an adult. six residents would have delivered metoject Ò mg in pre syringe filled if a doctor had asked for an it during a night. lastly, there are only two people who know that aracytine Ò (cytarabine) mg must be reconstituted with sodium chloride for it use and not with the provided solvent containing benzylic alcohol. the score of the residents having already done an internship in an urc is . / compared with . / for those who never did. the respective scores per year of internship are . ( st year), . ( nd year), . ( rd year) and . ( th year). conclusion: results and answers have been presented in a meeting and sent to the residents. we initially note many gaps in knowledge. the residents who already worked in an urc and the elders got better results. all the residents could be on duty at the hospital and all must be formed. a second session will be organized in a month to evaluate the formation's impact. it also has been presented to the assistants during an interactive lesson. this formation is essential to guarantee the dispensation of the adequate product and a secured medical care of the patient. please specify your abstract type: descriptive abstract (for projects) background and objective: patient adherence to prescribed medications is crucial for reaching metabolic control goal. to better understand the impact of polypharmacy on medication adherence, we undertook a detailed survey of medication use among patients with endocrinologic diseases. the aim of this study was to determine medication adherence in a cohort of patients with endocrinologic diseases and to test the hypothesis that adherence decreases with increased number of medicines prescribed. design: we conducted structured interviews to determine self-reported adherence of patient on a scale of (high) to (low observance) (srap- ) and a measurement using morisky medication adherence scales . demographic and medication information were collected from medical record. for statistical analysis, mann-whiney u-test for continuous variables, with chi square for categorical variables and kendall test for correlation were used. results: our cohort included patients, % were women and % were diabetic ( % suffering from type diabetes). the mean age was ± years, the average number of medication was . ± . . ( %) patients were not able to estimate their adherence. patients reported srap- scale with an average of . ± . , this estimation was significantly higher than mmas- with an average of . ± . (p \ . ). the proportion of adherence level were identical between srap- and mmas- with respectively and % of high, and % of medium and and % of low adherence. a significand correlation between srap- and mmas- scales (r = . , p \ . ) was found. however no correlation between adherence scale and number of treatment (r = . for mmas- and . for srap- scale) nor number of daily doses (r = . for mmas- and . for srap- scale). on the medications, % presented difficulties with observance. cardiovascular ( . %), diabetes ( . %) and psychiatric ( . %) treatment are the three most involved drug classes in nonadherence. conclusion: in this cohort, patients reported high medication adherence. we highlighted a correlation between srap- and mmas- scales. surprisingly, we didn't find correlation between adherence scales and number of treatment or dose by day. the next step of this work will be the identification of risk factor of nonadherence using logistic regression analysis. hp-pc : the office of access to healthcare: how to optimize secured access to treatments? claire chatron, adeline flatres, claudine hecquard, guillaume saint-lorant * , alexandra muzard pharmacy, chu caen, caen, france please specify your abstract type: research abstract background and objective: office of access to healthcare (oah) is an organization which offers a medical and social coverage to people who can't access to care and to medication because of the absence of social welfare, living conditions, or financial difficulties. medications are free dispensed thanks to retrocession activity in hospitals pharmacies. the aim of this study is to analyse this activity and to improve communication with patients and access to treatments by an adapted pharmaceutical interview. setting and method: this study includes all dispensations of year . in order to get medications in our hospital, a social worker and the patient come at the hospital's pharmacy. one people of retrocession team (four assistants, two externs, two residents and two pharmacists) dispenses necessary drugs to the patient according to hospital drug formulary and operating protocol. a switch or a special order can be purposed if the drug is not available. then, we give the patient a medication management plan (mmp) to explain him how to take his treatment at home. retrocession team filled a quiz about this activity and ways to improve it. main outcome measures: the main topics included in the quiz and evaluated were: dispensation organization, english talking, feeling during the interview and evaluation of the mmp. results: three hundreds and ten patients were admitted in oah . these patients come mainly from the eastern europe and do not speak french in most of cases. social workers, who can help for communication, are not always present because these patients can come during on-call duty. quiz results showed that weak points occurred during the interview: explanation of the mmp, languages barriers, mention '' if needed '' not understood by the patient. explanation of the order for a particular drug was difficult to operate too. mmp were only drafted in french which was not convenient for foreign people. however, modalities of dispensation were well understood by the retrocession team. following quiz results, mmp was translated into english by the retrocession team. mentions '' if needed '', '' number of maximum tablet a day: … '', ''your medication is in order, thank you for coming to look for this treatment back to the hospital on … '', '' … is the same as …, prescribed by your doctor on your medication list '' have been added and translated. results of the study and new mmp will be presented to the pharmaceutical team and to social workers in staff. an index card for ''communication in english with a patient'' has also been drafted. it contains sentences meadow drafted in english. conclusion: access quality health care service is important to achieve health equity and to increase the quality of everyone's life. these documents improve communication with patients and by the way their understanding about their treatment. the use and the impact of these documents on well understanding will be soon evaluated with social workers and patients. hp-pc : improve the medication in an associated to general hospital nursing home luisa alonso * , marta vidal iglesias, lucia gómez carrasco, guillermo goda, laura garcia, laura marin, ana hernandez, alvaro moreno please specify your abstract type: descriptive abstract (for projects) background and objective: in order to improve the medication reconciliation and to implement training programs for the medical team in an associated to general hospital nursing (asnh) home we measured the discrepancies between pharmacy registered treatments (prt) and medical prescriptions (mp), and we analysed potentially inappropriate prescriptions according to ''american geriatrics society beers criteria'' and ''stopp-start criteria. design: retrospective observational study that included patients admitted in the asnh. the ''consensus document on terminology and classification in medication reconciliation'' was considered for discrepancy classification. data collected: discrepancies between mp and prt. in patients from the original group of , we reviewed potentially severe drug interactions, potentially inappropriate mp and drug classes to avoid in older adults and medications to be used with caution in older adults (according to stopp-start and beers ) . all data were registered, measured and analysed in excel Ò . results: patients and a total of mp were reviewed. discrepancies ( . %) were found between the medical order and the prt, those discrepancies included errors of omission in prt ( . %), absence of discontinuation of medication ( . %), incorrect dosage ( . %). potentially moderate to severe interactions: the most frequent drug groups were proton pump inhibitors (ppis) ( . %), benzodiazepines (bzds) ( . %), oral hypoglycemiants ( . %), other groups with frequency over %, oral antihistaminic, statines, low molecular weight heparine (lmwh), laxatives, calcium salts and iron salts. stopp criteria were identified that affected to mp and the distribution was as follows: laxative combinations ( . %), long term ppis ( . %), cns depressants combinations ( . %), long half life bzds combinations ( . %), aspirin incorrect drug strength ( . %) and other groups with lower frequencies, nsaids and prokinetics. start criteria: being all of them by omission of the drug at the time of admission. beers criteria: prescriptions in the ''avoid prescription in adults'' group of which corresponded largely to concomitantly cns depressants and long term use of ppis in no risk patients. conclusion: the difficult working conditions, the excessive workload and the high staff turnover, where doctors have a patient ratio over / , make difficult to update treatments according to patient daily needs. a clear communication problem between the hospital pharmacy and the asnh prescribers exists due to lack of infrastructures, and it has been demonstrated with the high percentage of discrepancy, that implies an important logistic problem (not a safety problem) since the nurse team works directly with the original medical orders. the analysis of prescriptions showed the need for updating the medical knowledge. the high volume of stop and beers criteria and lack of doctors time made impossible the individual acting upon each patient, so short summaries of continuous training related to most frequent problems have been designed. please specify your abstract type: descriptive abstract (for projects) background and objective: our french university hospital is one of the most active centre for liver transplant ( transplants annually). various professionals are involved in the graft patient care and education. much information and education sessions are exempted before and after the transplant. the objective of this work was to realize a short movie for patients ( ) to get them ready for transplant ( ) to give the key messages to support their transplant ( ) to make family understanding the process and to promote the life behaviour changes. design: three members of the pharmaceutical team with nurses-led care coordination and a surgeon wrote the scenario. we requested two directors for days of shooting. we defined the key points for the patient and places to film, and fixed the duration ( - min). the scenario was validated by the chief of the liver transplant unit and nurses-led care coordination. after the days of film shooting, we selected sequences. results: the movie was a succession of six parts. ( ) the movie has been burned onto cds, put on flash-drives and will be uploaded on the internet. because of the international origin of our patients, the video will be subtitled at least in english. the video will be broadcast to hospitals which do not transplant patients and refer them to our hospital. since the medical team was involved in a collaborative project, the making of the video has permitted to strengthen the cohesion. indeed, this work would not succeed if everybody did not express himself. patients understood the interest to testify about their lived experience with the liver transplant, because they wished to have such information when they were waiting for the graft. conclusion: this movie is very useful for patients and families who are looking for information before and after liver transplant. it is a tool to get them into condition patients. this video presents the advantage of being personalized (local and caregivers that the patient will encounter are filmed). furthermore, it maintains a dynamic involvement of the pharmacy (already well established with clinical pharmacy, patient education and medication reconciliation) in the liver transplant unit. the making of the film has been an opportunity to bind the members of the team together, by valuing the work of everyone. the film could be screened if this abstract is selected for an oral communication. please specify your abstract type: descriptive abstract (for projects) background and objective: numerous procedures on medication management at oslo university hospital aim to minimize the risk of medication-related errors. error reports and observations show great variation in the use of these procedures, primarily due to difficulties in their implementation and maintenance. our aim was to assess the effect of a novel teaching strategy, the impala project, on doctors and nurses compliance with the medication management procedures. design: the project was carried out at general medicine wards at oslo university hospital for a period of weeks at each ward. assessment of medication-related error reports yielded the following areas of focus: (i) correct medication prescription, (ii) specification of doses for medications given on an ''as required''-basis, (iii) double control of medication dosing, (iv) correct and documented generic substitution. weekly presentations by pharmacist(s), lasting for a maximum of min, were given to doctors and nurses as part of daily ward routines. this was repeated over weeks. data on medicationmanagement procedure compliance were recorded before the start of the intervention, during and after each intervention period. the results were presented and made available to both leaders and employees throughout the project period both as an incentive to improvement and as a motivation factor for continued effort. results: there was a marked increase in medication-management procedure compliance among the nurses, especially after the second week of intervention. the most marked increase was shown for double control. increase in medication-management procedure compliance was also present among the doctors, but was less prominent. the data presented gave an extra motivational kick according to the participants. the leaders and the employees stated that the impala strategy was easy to follow and gave results without much organizational effort. conclusion: fifteen minutes presentations given by a pharmacist(s) as part of daily ward routines, combined with presentation of results demonstrated considerable improvement in medication-management procedure compliance. please specify your abstract type: research abstract background and objective: high unexpected serum vancomycin concentrations (svcs) were observed in patients without impaired renal function during the therapeutic drug monitoring (tdm) in our pharmacokinetic service. the aim of this study was to analyse the evolution of the svcs and its relationship with the markers of renal function. setting and method: retrospective study conducted at a university hospital with a follow-up period of months. only adult patients having at least two tdm were selected. trough svcs were measured by cmia (architect i- analyser, abbott Ò ) and fitted to a two-compartment model by using bayesian analysis (pks Ò , abbott). clinical and demographic data and daily dose, as well as timings of vancomycin administration and of blood sample collection were accurately recorded. spss Ò , version . was used to compare data from both tdm by student t-test (parametric data) and wilcoxon (nonparametric data). main outcome measures: concentration-to-dose ratio (cdr: trough concentration * /daily dose); glomerular filtration rates (gfr) estimated by cockroft-gault formula; measured and predicted svcs levels. results: adult patients were included (females: %); median age [ - ] years).the first and the second tdm were carried out after . [ . - . ] and [ . - . ] days from the beginning of the treatment, respectively. in the first tdm, no difference was found between the measured concentrations ( . ( . ) lg/ml) and those predicted ( . ( . ) mg/l. however, predictions were less accurate in the second tdm and predicted concentrations were significantly higher svcs ( . ( . ) mg/l vs. . ( . ) mg/ml, p \ . ). the median cdr in the second tdm was significantly higher than that calculated in the first one ( . [ . - . ] l - vs. . [ . - . ] l- ; p \ . ), indicating a lower clearance and a drug accumulation. however, no statistically significant differences in the glomerular filtration rates were found ( [ - ] ml/min vs. ml/min) in the first and second tdm, respectively. conclusion: although the markers of renal function did not change during the treatment, a decrease in vancomycin clearance was observed. the pharmacokinetic model does not accurately predict evolution of the svcs over the treatment. the introduction of covariates such as the length of treatment or the cumulative dose in the pharmacokinetic model could improve its predictive performance. please specify your abstract type: descriptive abstract (for projects) background and objective: genetic polymorphism or major physiological changes have to be considered in patient therapeutic management. clinical pharmacists have a role to evaluate and optimize the appropriateness and effectiveness of patient's medications. we report here the impact of the clinical pharmacist and his collaboration with the clinical pharmacologist in the therapeutic management of a patient suffered from anorexia nervosa, a psychiatric disorder leading to body composition change that may influence drug pharmacokinetics and efficacy. design: case report. results: the patient was a -year old woman hospitalized for chronic pulmonary aspergillosis previously treated by voriconazole, posaconazole and itraconazole. her medical history included anorexia nervosa since with a body mass index of . kg m - , pulmonary tuberculosis in with relapse in , and chronic pulmonary aspergillosis since . at admission, a treatment by oral voriconazole at mg/ h was introduced. the trough concentration of voriconazole at steady state was . mg/l (therapeutic range - mg/l) despite taking drug on empty stomach. although the voriconazole dosage increased in mg/ h, the trough concentration did not increase significantly ( . mg/l). we hypothesized anorexia led to a significant mucosal atrophy and accordingly, a significant decrease in intestinal absorption surface which is a major determinant of the level of drug absorbed. thus, a switch from oral to intravenous route was performed (voriconazole mg/ h). according to subtherapeutic voriconazole concentrations (trough concentration: . mg/l) despite the use of intravenous route, we decided to perform genotyping to look for mutations of cytochromes p a * , c * and c * , particularly implicated in voriconazole metabolism. the presence of an ultrarapid metabolizer genotype ( * allelic variant of the c isoenzyme) in our patient should lead to increase drug dosage from to %. finally, the patient was treated by intravenous voriconazole at mg/kg/ h (i.e., increase by %). the maximum concentration performed h after iv route initiation was at . mg/l, suggesting a better efficacy. conclusion: this case report highlights the potential complexity of therapeutic management in some patients given anatomical and functional changes or genetic polymorphism, which can affect drug efficacy. clinical pharmacists in collaboration with clinical pharmacologists have to be able to help physicians in this type of situations. please specify your abstract type: research abstract background and objective: posaconazole (pcz) is widely used for invasive fungal infections as prophylactic, pre-emptive or curative therapy in lung transplantation. recently, a new formulation of pcz has been available in enteric-coated tablets. this new formulation improves pcz bioavailability, as compared to the oral suspension, which leads to increase pcz plasma trough concentrations (c min ) in haematological patients. no data related to pcz exposure and its effects on tacrolimus (tac), an immunosuppressant with narrow therapeutic index widely used, exists in lung transplantation. we aimed to assess the consequences of the treatment by pcz entericcoated tablets on pcz and tac exposure in lung transplant patients. setting and method: a single-centre retrospective study was conducted among lung transplant patients receiving tac and either enteric-coated pcz or both galenic forms. main outcome measures: pcz and tac exposure were estimated by the measurement of c min . to overcome the influence of dose (d), c min were adjusted on dose (c min /d) for both pcz and tac. a spearman test (nonparametric distribution) was performed to assess the correlation between pcz c min /d and tac c min /d. results: eighteen lung transplant patients (median age [q ; q ] = . [ . ; . ] years; % female) were included between june and march . eight patients received only pcz entericcoated tablets. pcz enteric-coated tablets were associated to an increase in pcz c min /d as compared to oral suspension ( . ± . l - vs. . ± . l - , p \ . ). overall, pcz therapy initiation led to an increase in tac c min /d ( . ± . l - before initiation vs. . ± . l - after initiation, p = . ). tac c min /d was significantly higher with pcz enteric-coated tablets, as compared to pcz oral suspension ( . ± . l - vs. . ± . l - , p \ . ). a weak correlation was observed between pcz c min /d and tac c min /d, independently to pcz galenic form (r = . , p = . with pcz enteric-coated tablets and r = . , p = . with pcz oral suspension). conclusion: this pilot study in lung transplantation confirms the better bioavailability of pcz enteric-coated tablets as compared to oral suspension. our results show a more important increase in tac exposure with pcz enteric-coated tablets compared to pcz oral suspension, suggesting a concentration-dependent cyp a inhibitor effect of pcz. these findings are of interest in clinical practice to monitor transplant patients treated by the new formulation of pcz. further analyses, including the consideration of confounders, will be conducted. please specify your abstract type: descriptive abstract (for projects) background and objective: within months, two patients receiving apixaban developed agranulocytosis. based on temporal and clinical plausibility as well as published literature, the objective was to determine the causal relationship between agranulocytosis and apixaban. design: description of two agranulocytosis cases reported in our hospital. results: first case is an years old male, admitted to the neurology unit (d ) for ischemic stroke. at admission, blood count showed no abnormalities. four days after admission, treatment administered consisted in: dextrose % infusion iv, sodic heparin iv, acetaminophen, atorvastatin, metoprolol. neutrophils count was normal ( . g/l). heparin was stopped at d and replaced with apixaban according to following dose regimen . mg twice a day. at d , patient presented with hyperleukocytosis (neutrophils count g/l) and high crp ( mg/l). thus, a cytobacteriological urine test was performed. at d , patient presented with hypothermia followed by hyperthermia related to acute sepsis. blood count showed agranulocytosis (neutrophils count . g/l). broad spectrum antibiotherapy was started (ceftriaxone and gentamycin). despite treatment, death of patient occurred at d . the suspected cause of death was septic shock added to severe febrile neutropenia. following haemocultures confirmed sepsis (e. coli) possibly originating from urinary tract infection. second patient is a years old male, admitted to the cardiology unit (d ) for bronchopneumopathy associated with tachycardia and atrial fibrillation. a treatment with heparin was immediately started in association with patient usual treatment (bisoprolol, valsartan, rosuvastatin, hydrochlorothiazide and manidipine). in addition, broad spectrum iv antibiotherapy was started with ceftriaxone and spiramycine followed at d by an oral treatment with cefixime and spiramycine until d . heparin was replaced by apixaban at d ( . mg twice daily). antihypertensive treatment was adapted throughout patient's stay. patient presented neutropenia at d (neutrophils count . g/l), followed by agranulocytosis at d (neutrophils count . g/l) when it was decided to replace treatment with apixaban by fluindione. the following day, neutrophils count was about . g/l and patient received filgrastim. a myelogram showed a possible peripheral neutropenia. in the absence of other confounding factors (hiv, hbv, hcv, cmv), an iatrogenic agranulocytosis related to apixaban was suspected. conclusion: causal association with heparin is unlikely as neutropenia is not an adverse drug reaction known included in the smpc of this drug having a well-established safety profile. since the two patients were taking their usual treatment for a significant period of time, a causal relationship is deemed unlikely. temporal and clinical plausibility seem to indicate a possible relationship between agranulocytosis and apixaban. as this medicine has been recently approved, this might explain why no case has been reported in the literature and the absence of agranulocytosis as an adverse drug reaction of apixaban. please specify your abstract type: research abstract background and objective: taste is tightly connected to children's acceptability of medicines. two ways to overcome lack of acceptability are to administer solid formulations which are easier to taste mask and change to better tasting medicines. dicloxacillin is an antibiotic known for its unpalatability, and taste studies suggest that this might jeopardize its adherence. the aim of this study was to explore if prescription data can be used to estimate acceptability of antibiotics among children on a population level using dicloxacillin as an example drug. the research questions were: when comparing dicloxacillin with other antibiotics commonly used in children, ( ) is there a difference in the age of conversion from liquid to solid formulation and ( ) is there a difference in re-prescription rates on day and after the initial prescription? setting and method: we included all initial prescriptions of oral dicloxacillin, phenoxymethylpenicillin, amoxicillin and erythromycin for children - years registered in the norwegian prescription database (norpd) - due to dicloxacillin mixture being discontinued from the norwegian market in . the age of conversion was defined as the age where half of the children were prescribed liquids and the other half prescribed solid formulations. re-prescription rates were defined as re-prescriptions of a different antibiotic or formulation on day and after the initial prescription, divided by the total number of prescriptions. main outcome measures: age of conversion and re-prescription rates of dicloxacillin compared with other common antibiotics. results: the age of conversion for dicloxacillin was . years, compared to years for other common antibiotics. the average represcription rate for dicloxacillin was . % for children - years and . % % for children - years. the highest re-prescription rate of . % was found in -year olds. corresponding numbers were . , . and . % for common antibiotics. conclusion: the lower age of conversion from liquid to solid formulation and higher re-prescription rate of dicloxacillin mixture compared to common antibiotics indicates that prescription data can be used to identify antibiotics with low acceptability for children - years. further studies are needed to investigate if this also holds true for other antibiotics. please specify your abstract type: research abstract background and objective: attention deficit/hyperactivity disorder (adhd) or hyperkinetic disorders (hkf) is among the most common mental disorders in children, and may persist through adolescence into adulthood. pharmacotherapy used for treating the disorders also has potential for misuse/abuse. the aim was to describe the prevalence and magnitude of use of stimulant drugs and atomoxetine, and compare consumption in the nordic countries. setting and method: a descriptive pharmacoepidemiological study from the * million inhabitants of the five nordic countries in the period - . data were collected from national prescription registers, public drug reports and by correspondence with public health institutions. population data were obtained from official statistical databases or by correspondence with public health institutions. main outcome measures: trend over time, comparison between countries, type of pharmaceutical, gender, age, comparability of data. results: the annual consumption has been increasing from to , both in volume and prevalence of use. denmark had the largest increase in volume, from . to . ddd/ inhabitants/day. sweden had the highest increase in prevalence of use over the period, from . to . users/ inhabitants. iceland had the largest consumption of adhd medications in , . ddd/ inhabitants/day. prevalence data was not available for iceland but sweden was highest in prevalence of use among the other countries in : . users/ inhabitants. males aged - years had the largest volume and prevalence of use in , but females' consumption had been increasing faster both in terms of numbers of users (* . faster) and in volume (* faster) than men's consumption. conclusion: variation in consumption is considerable and cannot be explained by diagnostic and prescription guidelines, as these are similar in the five countries. consumption has been increasing fast in the period in all the countries, and faster for women than for men, although men still consume larger volumes than women, and are more frequent users. please specify your abstract type: research abstract background and objective: in and , regulatory bodies in usa (fda) and europe (ema), issued warnings on use of metoclopramide due to an increased risk for serious adverse drug reactions (adr), especially neurological adrs. ema recommended that metoclopramide only should be prescribed for up to days while fda concluded that treatment longer than weeks should be avoided. metoclopramide is commonly used to treat nausea and vomiting in pregnancy (nvp) and deficient breast milk production ( days course). ema did not make any recommendations concerning use during pregnancy and lactation. the objective of this study is to assess the disproportionality of reporting of adr from metoclopramide, with special emphasis on neurological adrs and women in reproductive age. setting and method: data from whos global adr database vigibase Ò for the time period november to may was used. the measure of disproportionality of reporting calculated was the proportional reporting ratio (prr), and % confidence intervals (ci). analyses were performed according to gender and age. time-toonset of adr was calculated. main outcome measures: proportional reporting ratio (prr) results: vigibase contains over million adr reports. metoclopramide is a suspected/interacting drug in of the reports, most common ( %) are neurological adrs. the majority ( %) of the metoclopramide adrs occurred within the first days of use. a total of % of the reports was received the last years ( ) ( ) ( ) ( ) ( ) . the reporting of neurological adrs was higher for metoclopramide than other medications in vigibase. women in reproductive age ( - years) reported higher proportion of neurological adrs (prr = . , % ci . - . , n = ) than women + years (prr = . , % ci . - . , n = ) but a similar proportion as men - years (prr = . , % ci . - . , n = ). conclusion: there is a . to three fold higher proportion of all reports regarding neurological adrs for metoclopramide than for other drugs. patients initiating treatment with metoclopramide should be informed about risks of adrs and that most adrs occur within days, and instructed to contact health care personnel and stop treatment if adrs occur. please specify your abstract type: descriptive abstract (for projects) background and objective: self-induced drug intoxications (sidi) are one of the most frequent reasons of hospitalization in emergency service ( %) with around - / inhabitants and represent around % of admissions in intensive care unit (icu). it is the most frequently used method of suicide attempts (sa) and the leading cause of hospitalization for young people under . the main objective of our study was to analyse, stratify and pharmaceutically map the different sidi identified in our icu. design: this is a prospective study over months, including all icu patients following sidi from june to january . we have collected psychiatric history and previous sa by sidi, usual treatment, state of consciousness, incriminating drugs, drug classes stratified according to the clinical severity score igsii, evolution, transfer in a specialized centre and average cost of stay. results: ninety-two cases were reported, representing % of icu admissions. the average hospital stay was days for an average cost of . €. this amount is low compared with the average cost of all stays gone through the icu for the period ( , €). ninety percent of patients had a psychiatric history and % a previous sa. the usual treatment was involved in % of sidi. half of the patients arrived conscious with an average of severity score igsii of / , being the highest found for a patient who had swallowed simultaneously pregabalin and nitrazepam. clinical severity of these patients is less than that found on average for all patients in the icu in this period ( / ). eighty-seven percent had a favorable evolution. only one death was observed after ingestion of propranolol. fifty-six and a half percent of patients were then hospitalized in a specialized centre. the great family of psychotropic is the most frequent with benzodiazepines %, neuroleptics %, antidepressants . % and antiepileptic . %. the main drugs involved are oxazepam %, alprazolam %, cyamemazine %, bromazepam % and quetiapine %. antihypertensives then arrive and represent % of sidi. the stratification of severity scores does not appear to show significant differences between drug classes, nor between mono or polydrug ingestions. conclusion: sa by drug ingestion are very common and are often linked to risky behaviours. for these epidemiological and economic findings, it is necessary to continue and develop prevention strategies avoiding the appearance of intoxication (primary), limiting the consequences (secondary), and reducing the risk of recurrence (tertiary). please specify your abstract type: research abstract background and objective: interpretation of quality of life scores to render them meaningful to aid clinical decision-making is an ongoing challenge. interventions often result in statistically significant quality of life (qol) improvement, but may not reach the threshold of clinical importance. the minimal clinically important difference (mcid) is the minimal score change of relevance clinically. the aim of this systematic review was to assess the impact on quality of life of topical, systemic and biologic treatments for psoriasis in randomised controlled trials (rcts). setting and method: prisma guidelines were followed. all available articles describing rcts of therapies for psoriasis that included qol measurements published up to november were identified. six databases were examined with search terms. abstracts of articles were reviewed independently by two assessors: a third adjudicator resolved any opinion differences. risk of bias was assessed using the jadad scale. main outcome measures: reporting of the use of qol endpoints and impact of interventions in psoriasis. results: of screened article abstracts, articles were selected for detailed review: trials met the eligibility criteria, describing research on a total of , patients. reports of psoriasis interventions that fulfilled inclusion criteria have gradually increased over time : - = , - = , and - = ( ) to evaluate the relationship between the use of different therapeutic agents and the severity of osa, and ( ) to determine the effects of commonly used medications on continuous positive airway pressure (cpap). setting and method: patient medical records (n = ) of patients, that underwent sleep studies between the years and were collected over an eight-month period from the sleep laboratory department at mater dei hospital using a random sampling technique. data collected included body mass index, gender, age, epworth sleepiness score (ess), drug history, apnoea hypopnoea index (ahi) and cpap therapy prescription. likelihood ratio chi square test, paired samples t-test and multinomial logistic regression were the statistical tests used for data analysis. main outcome measures: assessment of the drug history in response to osa control using the ess and ahi scores. results: one hundred and seventy ( . %) patients of the patients ( males, females) were diagnosed with osa. forty-five ( . %), ( . %) and ( . %) patients suffered from mild, moderate and severe osa respectively. patients had a mean age of years. angiotensin ii receptor antagonists (arbs) (p-value = . ), sulphonylureas (p-value = . ), insulin therapy (p-value = . ) and non-benzodiazepine sedating agents (p-value = . ) were found to be associated with the presence of osa. a decline in the use of the arbs (p-value = . ), angiotensin converting enzyme inhibitors (p-value = . ) and non-benzodiazepine hypnotics (pvalue = . ) was observed over the study year period. reduction in the cpap therapy benefit was detected with the use of histamine (h ) antagonists (p-value = . ), b-adrenergic blocking agents (pvalue = . ) and antiplatelets (p-value = . ). conclusion: it is confirmed that hypertension and diabetes mellitus type ii are the main co-morbidities associated with the presence of osa. reduction in the use of certain therapeutic agents is observed secondary to cpap therapy use. patients using specific drugs have been identified as being at risk of a reduced cpap therapy benefit. please specify your abstract type: research abstract background and objective: people are using increasingly more common of social networks such as facebook, twitter and youtube for different purposes. many people are using these networks with the aim of getting information and knowledge sharing. there are many groups that pharmacist is a member in social networks at turkey. the largest of these groups has , members. pharmacists are shared common problems, information and experiences in these groups. but the accuracy of the information shared on social networks are not always conclusive. the study aim to evaluate the impact of social network information sharing in the knowledge and attitude of pharmacists. setting and method: clinical pharmacy group has been created to share information on facebook. pharmacist joined this clinical pharmacy group. the group was fed by information which include new drugs, fda alerts, adverse event and case report and also drug related problems during the months. pharmacists were assigned in two major groups, group a active pharmacist who becomes a member of our clinical pharmacy group, share and discuss information through the network and group b who is not a member. a knowledge measurement survey (ams) was given to both of them. main outcome measures: acknowledge measurement survey (ams) was developed and the difference in the score was used to evaluate the difference between the two study groups. results: pharmacists participated in the study, . % of the participants were a member of our facebook group and . % of participants were not. . % of the participants have doctoral degree or student, . % have master degree or student, % have bachelor degree from year-pharmacy faculty, . % have bachelor degree from year-pharmacy faculty. the education level distribution between the two groups was not statistically significant. while . % of the ams questions were answered correctly in the member group only . % were answered correctly in the non-member group. conclusion: the study emphasizes the importance of social network in providing the accurate and fastest information for the daily use of the pharmacists, there is a significant difference in knowledge between the pharmacist who join, share and discuss information on the social network and the one who do not join. cp-ce : impacts of a community pharmacy practice experiences on student professionalism yunn-fang ho , , hung-wei lin *, , fang-ju lin , , sheng-ping chang , yen-ming huang graduate institute of clinical pharmacy, school of pharmacy, college of medicine, national taiwan university, taipei, taiwan, r.o.c please specify your abstract type: research abstract background and objective: professionalism is valued globally and pharmacy schools are expected to nurture competent practitioners to better serve the public with humanity attitudes and behaviours. the study aims: ( ) to understand possible differences in professionalism between pharmacy students and potential community pharmacist preceptors, and ( ) to evaluate student changes in professionalism upon completing the community pharmacy practice experiences (cppe) at the end of the third (p ) year. setting and method: a modified chisholm's pharmacy professionalism instrument ( -item, -point likert scale) was administered to p students, pre-cppe and hopefully post-cppe in september, and community pharmacist practitioners who participated in a two-day preceptor training workshop. participants also provided their significance ratings toward ten traits, namely altruism, accountability, excellence, duty, honor and integrity, respect for others, communication, ethics, humanism, and teamwork. main outcome measures: differences or changes in chisholm professionalism scores. results: thirty-two students and fifty pharmacists participated in the survey. honor and integrity ( . ± . ) and communication ( . ± . ) were recognized by students ( . %) and pharmacists ( . %), respectively, as the most significant trait. humanism was rated the lowest in both groups (students, . ± . ; pharmacists, . ± . ). the -item professionalism scores ranged from . ± . (''i do not expect anything in return when i help someone.'') to . ± . (''i am respectful to individuals who have different backgrounds than mine.'') in the student group; whereas . ± . (''i do not expect anything in return when i help someone.'') to . ± . (''it is wrong to cheat to achieve higher rewards (i.e., grades, money).'') in the pharmacist group. in general, pharmacists' professionalism scores were higher and, in certain items, statistically significant differences were achieved. conclusion: professionalism might grow with professional competency and practice experiences as demonstrated by potential pharmacist preceptors. upon completion of cppe, students could probably exhibit gains in professionalism. more investigations are still underway. please specify your abstract type: descriptive abstract (for projects) background and objective: in france, a significant consumption of benzodiazepines (bzd) is observed in prisons. they are widely used during incarceration to treat or prevent anxiety and insomnia. furthermore, it is known that, an important traffic exists with these drugs because of the releasing properties of bzd in case of misuse. based on these observations, the pharmacist has set up a plan to improve the use of bzd in prison. the purpose of the study was to evaluate the impact of these measures after year of implementation. design: in january , we shared with physicians in a meeting to explain our plan for a better use of bzd and to set up new rules of prescription in prison: • regularly reducing the dose to limit drug tolerance • promoting the use of long half-life molecules which allow reducing addiction and misuse • advising sedatives anti-histaminics to treat insomnia • providing information to patients about addictives risks of bzd on the tv channel please specify your abstract type: descriptive abstract (for projects) background and objective: some drug combinations (described in thesaurus of national agency of drug) are contraindicated because they appear to increase the risk of torsade de pointes. the aim of this work is to standardize our pharmacists' intervention and to propose guidelines for doctors and pharmacists, depending on the situation and drugs, to limit these combinations and to reduce this risk at our hospital centre ( beds). design: a prospective survey was realized over a period of months to identify the drug combinations prescribed in medical prescription software, from the national drug agency thesaurus, that might be inducing torsade de pointes. a multidisciplinary staff was then constituted composed of a cardiologist, a geriatrician, a paediatrician, an anaesthesiologist, a psychiatrist and pharmacists to identify the different situations and to establish guidelines. results: from the survey drug combinations were found to be contraindicated due to increased risk of inducing torsade de pointes on a list interventions realized by pharmacists. the work group identified three drugs with a therapeutic alternative: hydroxyzine, domperidone, escitalopram, the other drugs can't be switched because they are vital or have no alternative. the work group decided to maintain hydroxyzine but only on premedication and child anxiety, to eject domperidone from our therapeutic index and substitute it with metoclopramide or metopimazine, to not initialize escitalopram but to keep it if the patient has no have others risk factors associated or no contraindication. if the patient has a contraindication with a risk factor the doctor could prescribe other ssri. in addition, pharmacists alert doctors about the risk of torsade de pointes on medical prescription software if some contraindications are identified. conclusion: the contraindications identified must not be underestimated. this work allows identification of torsadogenic drugs commonly prescribed and provides guidance for doctors and pharmacists regarding drug combinations. the collective decision will be disseminated to sensitize all the doctors in the establishment. some treatments could not be substituted despite the contraindication; these must be retained but with clinical monitoring. conclusion: a substantial proportion of medication waste in the community pharmacy could have been prevented. unused medicines in the community pharmacy are generally of low economic value, making it unlikely that the costs that pharmacies will make with the redispensing of unused medicines will be covered. therefore, other actions to decrease medication waste in the community pharmacy, such as preventing that too much medicines are dispensed, should be considered. please specify your abstract type: research abstract background and objective: flaws in usage technique for inhalationmedicines is common, as much as half of the users may need some correction measures, to get the active substances down to the lungs and provide the intended effect. inadequate compliance, especially for regular-use preventive medications, is common. good guidance in pharmacies enhances correct use of medicines. the new norwegian pharmaceuticals policy (legemiddelmeldingen) from opened up for paid cognitive services, leading to the first such service being implemented in march . the service can contribute to a more correct use of the medicines and, as a consequence, lead to better control of the symptoms for patients with asthma or copd. our objective was to map the variation in pharmacies' handling of an inquiry regarding lack of effect of an inhalation-medicine. the study was done prior to the implementation of the standardized service ''inhalation-guidance'' in norwegian pharmacies. setting and method: simulated patient (mystery shopper) visits in pharmacies in oslo, akershus and buskerud in november/december . the mystery shopper expressed just having started to use an inhaler because of her asthma, but not experiencing effect. structured data collection sheets were used to register the handling immediately after the visit. main outcome measures: scoring of the quality and contents of the information based on the products' patient information leaflets. results: the issue of inhalation-technique was mentioned in of the pharmacies, whereof asked the ''patient'' to show their inhalationtechnique, in order to correct and advice and used an inhaler or demo-inhaler as an aid in the guidance. going through the instructions or watching a video-demonstration with the simulated patient also occurred, or referring the patient to read the instructions and/or watch the video-demonstration on his own. half of the pharmacies discussed the difference between use for preventive treatment of asthma and inhaler that is being used for treatment of attacks. sixty-five pharmacies gave no information about the importance of regular use of the preventive treatment. conclusion: there was considerable variation in how the pharmacies guided, which indicates a potential for improvement. the new guidance-service, implemented in norwegian pharmacies in march , will contribute to better guidance. please specify your abstract type: research abstract background and objective: in portugal, tobacco addiction was responsible for over , deaths in ( % of the total deaths). the community pharmacist's contribution to control this public health problem is insufficiently documented. the aim of this study is to assess the contribution of the community pharmacist for smoking cessation. setting and method: a retrospective and longitudinal study of a convenience sample of patients integrating quit tobacco consultations, as part of a pharmaceutical care programme implemented by an outsourced pharmacist was performed at several community pharmacies. the smokers, aged or over, were invited to join the programme. patients signed an informed consent and were submitted to a comprehensive approach by face-to-face consultations and telephone contacts. richmond and fagerström tests were used to evaluate motivation and nicotine dependence, respectively. the therapeutic plan (pharmacotherapy and behavioural counselling) was personalised to each smoker. the quit rates were evaluated by patient selfreport and confirmed by carbon monoxide measurements. the continuous variables are expressed as mean ± standard error of the mean. main outcome measures: quit rates at , , and months. results: between january and june , smokers joined the programme, dropouts ( . %). the remaining smokers, ( . %) were male, with mean age of . ± . years. on average, each smoker consumed . ± . cigarettes per day. the mean age of initial tobacco use was . ± . years with . ± . years of consumption. about % reported moderate or high motivation and % medium or high dependence. a total of consultations were held and, on average, each patient received . ± . interventions. all smokers received non-pharmacological interventions (e.g. motivational approach) and ( . %) also accepted pharmacological interventions, usually nicotine replacement products. the quit day was achieved by patients ( . %). a month after quit date, patients were abstinent ( . %). the number reduced to after months ( . %), to after months ( . %) and to after year ( . %). these data upgrade and are consistent with our previously published results ( ). the smoking cessation consultation in the scope of a pharmaceutical care programme in community pharmacy seems to effectively contribute to the reduction of tobacco addiction in portugal. cp-pc : patient counselling at dispensing of oral anticancer drugs in european countries from the pharmacists' perspective andreja eberl * , on behalf of epic working group pharmacy, institute of oncology ljubljana, ljubljana, slovenia please specify your abstract type: research abstract background and objective: the number of oral anticancer drugs (oads) available on the market grows constantly. consequently the number of patients, which have to manage the complex treatment with oads at home is increasing. the pharmacists present an important member of healthcare team, since they are dispensing oads to the patients, which need a high quality information at that crucial moment. therefore, our aim was to evaluate pharmacists perceived confidence and needs for specific continuing education in connection to oads dispensing in european countries. setting and method: we used an electronic mailing approach and a standardized online survey to ask practicing pharmacists in european countries about their experience with dispensing of oads. main outcome measures: frequency of patient counselling and fields of counselling, assessment of knowledge and skills. results: the frequency of patient counselling varied widely in participating countries between ''never'' and ''more than %'' at initial fill of an oad. at following refills the frequency of counselling was generally even lower. counselling mostly encompassed directions of use, the proper use of antiemetics and side effects. however many pharmacists stated, that they do not feel comfortable counselling patients of oads ( %) and even more acknowledged that they were uncomfortable with managing patients' side effects ( %). on the other hand only % of pharmacists believed, that they have received adequate knowledge of oads through undergraduate program, continuing education (ce) events and professional practice. many of pharmacists ( %) have not attended any of ce events related to oncology in last years. pharmacists' responses differed little between the countries. conclusion: the proportion of pharmacists who regularly counsel their patients on oads is insufficient in view of importance of the patients' needs to manage their therapy at home. however the pharmacists seems to be aware of their knowledge deficits and educational needs. the field of oads needs better coverage in under-and postgraduate education. the number of ces has to be increased in order to improve the knowledge and skills in the areas of oads counselling. please specify your abstract type: research abstract background and objective: treatment guidelines for diabetes recommend that patients are well-informed about their disease, treatments and treatment goals, e.g. glycosylated haemoglobin (hba c). the objective was to describe diabetes patients' self-monitoring of blood glucose (smbg) and potential need of guidance. please specify your abstract type: descriptive abstract (for projects) background and objective: in , the international pharmaceutical federation collected data of remuneration models for community and hospital pharmacy and identified large variations between remuneration models and highlighted that the focus is largely on products and not on cognitive services. the aim of the study is to map the remuneration models of different pharmacist-led cognitive services in primary care across europe, with a special interest on medication reviews and to update a prior survey by bulajeva (bulajeva a et al. medication review practices in european countries. res social adm pharm ; : - .). the definition of terms is pivotal for such a european survey to avoid results based on pseudoconceptions. hereafter we present the development of the survey and we will present first results from pilot tests. design: pharmacist-led cognitive services were selected based on a previous study by our group and by searching the literature, official government websites, the pcne wiki and arising links. the definitions of the terms of these services were based on searches in the mesh browser, medline and google scholar. additionally, a search in grey literature and in the internet was conducted to find appropriate foundation for the formulation of the definitions. the questionnaire will consist, of a first part about the remuneration of the pharmacist-led cognitive services. the focus is on country-specific differences in remuneration and the different levels of supply across europe. the second part of the survey is about the different types of medication review services with a focus on e.g. the implementation level, addressed issues, eligibility criteria. this survey will have a cross-sectional study design with an online questionnaire specific for invited participants across europe. to achieve the best quality of answers we will send this survey to at least two researchers with references in pharmacy practice, in each european country (purposive sample). the answers from each country will be checked for discrepancies and these potential discrepancies will be solved by a discussion with the responders. results: by the end of the pre-pilot phase, different pharmacist-led cognitive services were identified and the correlating definitions of the terms were developed. conclusion: at the time of submission the pre-pilot phase has been finished and the pilot will start july . please specify your abstract type: research abstract background and objective: medication adherence is one of the key aspects in assuring optimal health outcomes in majority of chronic diseases. the aim of the study was to evaluate copd patients' medication adherence in slovenia and its association with health outcomes. setting and method: patients were recruited by community pharmacists at the time of dispensing medication for copd. medication adherence was evaluated by using morisky medication adherence scale (mmas- ). patients who scored b points, . - . points and points were regarded to have poor, moderate and good adherence, respectively. quality of life was evaluated by saint george's respiratory questionnaire (sgrq) and the impact of disease by copd assessment test (cat). the study was conducted in september and february . the association between potential predictors and copd impact or quality of life was estimated using multiple linear regression in ibm spss statistics version . main outcome measures: medication adherence rate (mmas- ), quality of life (sgrq total score) and impact of disease (cat score). results: of patients, majority were men ( %) with mean age years. in average, patients were prescribed . medicines for copd and . medicines for other diseases. good, moderate and low adherence to copd medication regimens was found in . , . and . % of patients, respectively. mean cat scores and sgrq scores were . (range - ) and . (range - ), respectively. thirtyeight percent of patients experienced an exacerbation in the past year. linear regression showed no statistically significant association between medication adherence and quality of life or copd impact on patient. factors that statistically significantly predicted patients' quality of life were exacerbation in the past year, education level and number of concomitant medicines for other diseases. the latter was found to be the only factor associated with copd impact. conclusion: the study showed half of the copd patients to be optimally adherent to their treatment and only a small proportion of patients not taking their medicines regularly. due to the nature of the disease medication adherence does not seem to play the most important role in assuring optimal health outcomes in copd patients. please specify your abstract type: descriptive abstract (for projects) background and objective: intermediate care units (imcu) are designed to serve patients in need of more advanced medical care than the ordinary nursing home units can provide. the aim of this study was to see; ( ) how medication information follows patients in and out of icmu and nursing home short-termcare units (stcu) ( ) the type and amount of drug related problems (drp), focusing inappropriate drugs, and ( ) if there are differences between the icmu and stcu in drug use and drps. design: patients c years old admitted and submitted at the imcu or stcu in the study period ( weeks) were included. transfer of medication information were evaluated and given a score. the clinical pharmacist provided medication reconciliation upon admission, medication review and monitoring, and presented identified drps and a suggestion for solving the problem, to the multidisciplinary team. inappropriate drugs, identified by screening tools (stopp/norgep), and systematic medication reviews, were recorded. results: patients from imcu and five from stcu were included. a hospital discharge summary including medical history followed mostly all patients. the score of the medication history was . points out of . by submission from either imcu or stcu, the score was . . systematic drug review identified . drp in the imcu and . in the stcu. imcu patients used . drugs, stcu patients . in the icu, % of the identified drps was inappropriate drugs, none in the stcu. the clinical pharmacist in the multidisciplinary team presented % of the identified drps. the doctors agreed in % of the suggestions for solution, and started immediate changes in %. conclusion: a hospital discharge summary followed the patients, but the medical history part needs improvement. although few patients, the results suggest that imcu patients had more complicated medication and more inappropriate drugs than stcu patients did. clinical pharmacist in a multidisciplinary team provides useful contribution to identify, solve and prevent clinical relevant drps, including inappropriate drugs. please specify your abstract type: research abstract background and objective: lack of clinical effects of medication review on health-related quality of life of older people may be due to insufficient focus on health-related complaints. goal attainment scales (gas) are an instrument to formulate specific health-related goals. the objective of this early process-evaluation of the dreamer-study (drug use reconsidered in the elderly using goal attainment scales during medication review) is to investigate if pharmacists are able to formulate gas during a medication review of older people with polypharmacy. setting and method: older patients aged years or older using or more medicines are included in this study. half of the patients were randomized into the intervention group, where they received a medication review. during the patient interview, the pharmacist formulated gas in concordance with the patient. recommendations were made to reach these goals in collaboration with the gp. main outcome measures: number of performed medication reviews, total number of formulated gas and the three most frequent types of gas. results: until now patients have been included in the drea-mer study ( % of the target). half of them ( ) were randomized into the intervention group. by now ( %) of these patients have received a patient interview. goal attainment scales were formulated yet. the number of gas ranged from to per patient. the four most frequent gas were: polypharmacy-reducing the number of medicines ( ), reducing pain ( ), increasing mobility ( ), reducing fatigue ( ). conclusion: gas seem to be a feasible approach during medication review that increased focus on patient's needs and health-related complaints. cp-pc : oral transmucosal fentanyl citrate: a regional survey of dispensing practices in community pharmacy please specify your abstract type: descriptive abstract (for projects) background and objective: oral transmucosal fentanyl citrate (otfc) is an opioid analgesic indicated for management of breakthrough cancer pain in patients with malignancies who are already receiving and who are tolerant to opioid therapy for their underlying persistent cancer pain. otfc are usually use off-label prescription, especially in noncancer patients or patients without opioid maintenance treatment. this practice can expose to iatrogenic risks, lack of efficacy, abuse and addiction. the observatory of drugs, medical devices and therapeutic innovation of upper normandy, conducted a study to assess the knowledge of pharmacists on these medications and assess dispensing practices (pharmaceutical analysis and advice to patients). design: between june and september , two quizzes were sent to the pharmacists and pharmacies in upper normandy: one included questions of knowledge and general practice, the other assess dispensing practices of otfc prescriptions received at the counter, regarding indication, dosage and associated opioid medication. results: of the pharmacists who participate in the survey, % know the all of the oftc specialties, % of them confuse transdermal and transmucosal fentanyl specialties. indication, dosage, titration methods and the main interest of oftc are known by , , and % of them. only % have dispensed oftc more than times over the past months, % never have. they already have dispensed oftc in noncancer patients ( %) or without opioid maintenance treatment ( %). they consider not know enough about these drugs to be able to provide the necessary advice to patient ( %) and would like specific training on oftc ( %). of the analyzed prescriptions, only % are consistent with the marketing authorization: otfc medicines are prescribing in noncancer patient ( %) and/or dosage is higher than four units per day ( %) and/or there is no prescribed opioid maintenance treatment ( %). only two prescriptions have been discussed with the prescriber, and all were approved and dispensed. conclusion: otfc specialties are occasionally dispensed and often misunderstanding by pharmacists. a good knowledge of otfc is necessary to achieve the pharmaceutical analysis and provided appropriate advice to patients, in order to guarantee the good use of these medicines. support tools for dispensation, recalling indication, . the most frequent interventions were drug substitution (n = ), dose adjustment (n = ), and clarification of information (n = ). common services were reconstitution of suspension (n = ), provision in advance for continuing supply (n = ), and follow up offers (n = ). conclusion: the observation of the dispensing process in community pharmacies revealed a broad range of tasks performed by the pharmacy and identified several variables likely to influence the counselling. in addition, pharmacy activities could be pictured by the documentation of pharmaceutical interventions. please specify your abstract type: descriptive abstract (for projects) background and objective: medication reconciliation (mr) is a multidisciplinary process to correct medication errors resulting from miscommunicated information at transitions of care. development of this activity is essential but it is hindered by the time required for its implementation. we must carefully choose which services can develop this activity. as it was recently introduced in cardiac surgery unit, this study aims to evaluate impact of this process to hospital admission (severity of potential harm of medication error intercepted) and to determine the relevance of this activity in this unit. design: prospective study conducted from january to april . the data is recorded in an excel table, filled after each mr. there are five items: patient's age, best possible medication histories (bpmh), implementation period of the mr, inadvertent discrepancies (ids) and clinical impact. to assess the severity of ids, a scoring method was used (doerper et al. ) with the cooperation of surgeon and pharmacist. results: eighty-two patients (mean age ± years old) were included in the study, which represents % of the patients hospitalized in this service. the mean number of drugs per patient was ± . the bpmh were obtained within h to h of admission to hospital. a total of ids were detected, with a mean of . ids per patient. the most frequent type of ids was omission ( %, n = ), error of dose ( %, n = ). the three most common classes involved in ids were hypolipaemic drug (n = ), antidiabetic drugs (n = ) and the drugs for acid related disorders (n = ). the mean of ids per patient ( . ) as well as the percentage of patients affected by a ids ( %) are less important in cardiac surgery than those observed in other services of the institution and in the literature. about clinical impact, % of patients presented with ids considered as minor, % significant and % major. among the major ids, none was evaluated as critical or catastrophic. in our study, this process remains retroactive. conclusion: one of challenge experienced when implementing mr process in hospitals is demonstrating its clinical impact. in order to address this concern, we found that the little ids with a serious clinical impact in this unit. mr is an interesting process to detect drug errors. to optimize our study we will improve our organization in order to be closer to the patient and to strengthen the doctor-pharmacist collaboration. please specify your abstract type: research abstract background and objective: special packaging like multidose drug dispensing (mdd) may optimize medication use in patients with a decreased ability to manage their own medication. however, it remains unclear how a 'decreased ability to manage medication' is defined. the objective of this study is to assess potential medication problems that contribute to a decreased ability to manage medication in patients starting with mdd compared to patients who use manually-dispensed drugs. setting and method: patients starting with mdd (cases) and patients using manually-dispensed drugs (controls) were interviewed in community pharmacies. questions to assess potential medication problems covered three domains; medication adherence ( ), practical management issues ( ) and medication knowledge ( ) . every potential medication problem was scored with one point. cognition was assessed with the mini-cog and frailty with the groningen frailty index (gfi). main outcome measures: mean scores of potential medication problems on the domains medication adherence, practical management issues and medication knowledge. results: patients starting with mdd and patients using manually-dispensed drugs were interviewed. patients starting with mdd scored more potential medication problems on all domains: adherence . versus . , practical management issues . versus . , medication knowledge . versus . . on the three domains together, patients starting with mdd scored . [ . - . ] potential medication problems compared to . [ . - . ] for patients with manuallydispensed drugs. forty-two percent of the patients starting with mdd might be cognitive impaired and % was classified as frail compared to and % respectively of the patients using manually-dispensed drugs. conclusion: patients starting with mdd reported significantly more potential problems on three domains that may contribute to a decreased ability to manage their medication. cp-pc : fifteen key questions to assess patient knowledge on new oral anticoagulants corina metaxas * , valerie wentzky, sonja luginbü hl, kurt e. hersberger, isabelle arnet please specify your abstract type: research abstract background and objective: knowledge on new oral anticoagulants (noacs) is crucial for their safe and effective use. validated tools that assess patient knowledge exist for vitamin k antagonists, but not for noacs. we aimed to identify which questions are relevant for patient knowledge on noacs. setting and method: based on a systematic literature search, questions were compiled for the assessment of noacs knowledge. key questions were selected through three rounds of ranking by an expert panel (four physicians, four pharmacists, four nurses). round (online survey; importance): the questions grouped into the nine educational topics of wofford,adapted for noac (disease, mode of action, risk-benefit, adherence, accessing healthcare professionals, diet/life-style, lab-monitoring, medication interactions, self-care) were to be rated as important/not important and educational topics were to be ranked according to decreasing importance. round (online survey; relevance): the questions were to be ranked according to decreasing relevance. round (focus group): number of questions was reduced by voting. main outcome measures: ranking of educational topics and questions ( = most important/relevant) in march/april . results: experts ranked adherence ( . ± . ) as the most important topic, followed by risk-benefit ( . ± . ), disease ( . ± . ), accessing healthcare professionals ( . ± . ), self-care ( . ± . ), lab-monitoring ( . ± . ), medication interactions ( . ± . ), diet/life-style ( . ± . ) and mode of action ( . ± . ). one question was judged as unimportant by all experts. out of the remaining questions, ( . %) were selected as relevant for basic knowledge, ( . %) were combined into four questions and one new question was generated. a total of key questions remained after the focus group discussion. conclusion: a multiprofessional expert panel was able to select key questions retrieved from literature and ensured content validity. the selected questions will be compiled into a tool to assess patient knowledge on noacs. background and objective: medicines use review (mur) was defined by the slovene chamber of pharmacies in december and an education program was set to assure pharmacists competencies. in june the first pharmacists were certified and implemented the service in the community pharmacies. additionally, an online database was established to collect mur reports and provide feedback on pharmacists' performance. the aim of the study was to evaluate identified drug related problems (drp) as well as pharmacists' interventions from mur documentation. setting and method: a preliminary retrospective analysis of documentation for mur services provided in the first year after implementation was performed. drps were classified using a slovenian drp classification system, which is based on the pcne classification v . [ ] . data were analysed with descriptive statistics measures. main outcome measures: number and type of identified drp and pharmacists' intervention. results: a preliminary analysis was performed on mur cases, performed by certified pharmacists. in total drps were identified: ( . %) manifested and ( . %) potential. patient had on average two drps, however patients had none. main risk factor for potential drps was inappropriate use of medicines. adverse drug events (ades) presented . % of manifested drps; the main risk factor was again inappropriate use. in two cases ades happened due to an allergic reaction. different medicines were the cause of ades; mainly statins resulting in muscles pain and sleeplessness. another frequently manifested drp was insufficient effectiveness of treatment. drug interactions were risk factors in cases of manifested drps, mainly in connection with antidepressants: serotonin syndrome due to escitalopram, bleedings in concurrent use of escitalopram and ginkgo, sleepiness, etc. pharmacist intervened independently in . % of cases; times recommendations were given to physicians. however, in . % of cases the outcome of intervention is unknown. the preliminary results of the first mur cases points to a high number of identified manifested drps. however, the knowledge of intervention outcomes is lacking and therefore more attention has to be put on establishing adequate follow up on this issue. official definition represented harmonisation of several similar activities that have already been performed in slovenian pharmacies and also provided an educational program to assure pharmacists competencies. in may the first pharmacists were certified and implemented the service in the community pharmacies. therefore, the aim of the research was to get an insight into the implementation of mur in slovenia from the perspective of the first community pharmacists that provide the service in practice. setting and method: a focus group with seven community pharmacists, that provide mur in practice, was run in february . guided discussion included three main themes: the development and assurance of competencies, experience with the provision of service in practice and the future of the service. the discussion was voice recorded and analysed with the nvivo . written consent from included participants was obtained. main outcome measures: views, challenges and opportunities for the medicines use review service in slovenia. results: in total themes were identified and organized in three main categories: competencies for quality provision of mur, mur's recognisability and organizational aspects of mur provision. participants emphasized broad knowledge in pharmacotherapy is pharmacists' key competence and advantage in performing mur when compared with other healthcare professions. recognisability of mur among other health care professions as well as participants' work environments is low. hence a comprehensive approach in marketing of the service is needed. positive patient's feedbacks were reported, however persuading patients to attend mur presented a challenge. another barrier was the time to perform mur, which could be overcome by suitable work organization and special time intended for mur. conclusion: participants of the focus panel had positive experience with the development of competencies and implementation of the service in the practice. several challenges were presented connected with the recognition of the service by patients, physicians and health care payer. they strongly believe that continuing professional development forms the base for quality of the service in the future. cp-pc : evaluation of rational antibiotic dispensing in the community pharmacy setting: a simulated patient study betul okuyan * , mehmet ali savan, fikret vehbi izzettin, mesut sancar please specify your abstract type: research abstract background and objective: in the present study, it is aimed to evaluate rationale antibiotic dispensing without prescription in the community pharmacy setting; this will be done by using a simulated patient methods. setting and method: this study was conducted in malatya, located in the east part of turkey. the simulated patient visited the community pharmacies to meet the pharmacist, posing as the husband of a patient with acute uncomplicated rhinosinusitis. the simulated patient was trained regarding the standard information to be provided by the researchers and informed about the privacy of all information that would be gathered during the present study. the sample size was sixty-seven pharmacies, with a confidence interval of % and error of margin of %. the study was conducted over a total of pharmacies. all the pharmacies were listed alphabetically and were randomly selected and allocated random numbers by a computerbased program. main outcome measures: after each community pharmacy was visited, the simulated patient filled the check list which had been drawn up for the purpose of the present study. due to ethical concerns, no audio or video records were used during the study. any suggested medications were not purchased from the community pharmacy. results: of the total community pharmacies that were visited . % of them had female pharmacists and . % were run by male pharmacists. the mean number of questions asked by pharmacists to the simulated patient was . ± . . only eleven pharmacists did not suggest any medication for the simulated patient. however, thirty-two ( . %) pharmacists recommended various medication regimens, including antibiotics. of them, . % referred the simulated patient to a physician. conclusion: in conclusion, it was observed that dispensing antibiotics without prescription was still high, pharmacists did not take comprehensive medical or medication history from patients, and pharmacists provided insufficient medication information to the patient regarding suggested medications at community pharmacy setting. to avoid irrational antibiotic dispensing, it is essential to educate both health care providers and the general population. although dispensing antibiotics without prescription is illegal in some countries, it is necessary to actualize new regulations to avoid antibiotic dispensing without prescription. please specify your abstract type: research abstract background and objective: the medication adherence is an important part of active (as well as passive) attitude of a patient to the disease treatment. it represents the level of keeping the treating procedure as well as the recommendations of doctors, pharmacists and other healthcare professionals. this study deals with the adherence in patients with hypertension. the hypertensive patients are a substantial part of patients, daily visiting the community pharmacy to pick their prescriptions. these patients represent group of patients with typical asymptomatic disease. this means that they do not take the medicines or use them according to their own will. the result of their non-adherence could lead to later complications. the aim of the study was to evaluate the level of adherence and its relation to the clinical outcome-the blood pressure in hypertensive patients. setting and method: the methodology was based on a single anonymous questionnaire survey combined with the blood pressure measuring in a community pharmacy in slovakia. the modified morisky -item medication adherence tool was used in this study. main outcome measures: the results of medication adherence were evaluated as follows: - points = full adherence, points = partial adherence and - = non-adherence. each participant should use at least one antihypertensive agent and fulfil the anonymous questionnaire in the community pharmacy. the pharmacist measured the blood pressure in each participant twice, within the interval of min and used the average value in data sheet. results: the research included hypertensive patients ( . % females and . % males). the results showed that almost % of the respondents were non-adherent to the prescribed pharmacotherapy ( . % of those were males and . % were females). the group of partially adherent patients consisted of . % of the respondents ( . % of those were female). only . % respondents were fully adherent according to modified morisky score ( . % of those were women). fully adherent patients reached an average blood pressure . / . mmhg; partially adherent hypertensive patients recorded an average blood pressure . / . mmhg; and in the non-adherent patients has been observed the average blood pressure . / . mmhg. the results showed an alarming situation, and confirm the published data. non-adherent patients could not goal the good clinical outcomes. this leads to adding of another medications, raising the risk of interactions and adverse drug reactions, complications of undertreated disease, and finally, to pharmacotherapy costs increasing. please specify your abstract type: research abstract background and objective: in psychology, depression is a mental state characterized by feelings of sadness, dejection, inner tension and indecision. in psychiatry, the depression is defined as a severe mental affective disorder which paralyzes clarity of thought, psychomotorics, sleep cycle and raises pessimistic and depressing emotions often lead to pathological changes of personality. during treatment of depression is often needed psychotherapy and pharmacotherapy as well. using of antidepressants requires the sufficient level of medication adherence in patients. non-adherence to antidepressant medication significantly contributes to the undertreatment of depression in primary care populations. the aim of this study was to evaluate the level of medication adherence to antidepressants to better understand the socio-behavioural factors associated with non-adherence. setting and method: the anonymous, face-to-face questionnaire survey was set in the community pharmacy in slovakia. questionnaire obtained questions on socio-behavioural factors and adherence tool-modified -item morisky score (mmmas- ). main outcome measures: respondents were patients ( males, females) using at least one antidepressant. the results were evaluated as follows: points = full adherence, - points = partial adherence and - = non-adherence. results were evaluated in relation to socio-behavioural factors. results: average level of the medication adherence in our group was , which means the line between partial and full adherence. the results showed non-significant higher medication adherence level in males ( ) compared to females ( ). the highest level of medication adherence ( ) has been shown in patients - years old, the lowest average adherence level (non-adherence) was observed in patients up to years old ( ). patient living in the city were more adherent to their medication ( ) compared to patients living in countryside ( ). the highest level of the partial medication adherence has been shown in secondary educated patients ( ). partial adherence level was higher in patients with monthly income over € ( ) compared to non-adherent patients with monthly income up to € ( ). in patients using no other medications, only antidepressant, we have observed the highest partial adherence ( ). conclusion: our survey showed the partial antidepressant medication adherence levels in our study group. poor adherence results in low stabilization of clinical state in patient, in using more types of therapy and in increasing costs. there might be very important role of the community pharmacists and other health care professionals to improve the medication adherence and persistence through counselling and education patients on importance and need of antidepressant medication. ( ) and medication regimen complexity was assessed by using the medication regimen complexity index (mrci) ( ) . five and more medication usage has been defined as polypharmacy. results: a hundred and two elderly subjects ( . ± . ; male) were included in this study. of them, . % had two and more chronic diseases. the most common chronic diseases determined in study population were cardiovascular diseases (especially hypertension), diabetes and hyperlipidaemia. the polypharmacy has been defined in . % of them. the mean of mrci per elderly patient was . ± . . one or more pims use was observed in seventy-four elderly subjects ( . %). of all elderly subjects, . % were dispensed one and more medicines with a potential for drug-disease/ syndrome interaction. pims use was more frequently determined in patients with polypharmacy ( . vs. . %, p \ . ). the total score of mrci was significantly increased with elevated number of pims (r = . , p \ . ). conclusion: this study highlights a significant association between utilization of pims and both polypharmacy and higher total score of mrci in elderly patients. pharmacists could be evaluated utilization of pims in especially elderly patients with used five or more medications and/or higher total score of mrci. please specify your abstract type: research abstract background and objective: nursing home patients with multimorbidity often use multiple drugs simultaneously, which makes these patients more susceptible to adverse drug events. several studies have pointed to a need to increase the quality of prescribing to this population. to achieve this there is a need for reliable information about patients' diagnosis, and what is recorded as the drug's indication in different electronical and handwritten health records. the aim of this study was to examine the registered diagnoses, and indications for drug use in nursing home patients. we also wanted to study the extent to which diagnoses are untreated with drugs, as well as the extent to which drugs have a registered indication for use and a suitable recorded diagnosis. setting and method: data was collected for long-term patients, on average years old, and % females from four nursing homes in tromsø municipality, norway. we retrieved information about patients' diagnoses and indication for drug use from the electronic health record and written drug charts. two pharmacists conducted the linkage between the reported diagnoses and drug use. main outcome measures: percentage of untreated diagnoses and the percentage of drugs with a registered indication for use. results: as considered by the pharmacists, % of the registered diagnoses was untreated with drugs. dementia, gout and osteoporosis were the most commonly untreated diagnoses with, , and %, respectively. in comparison, the indication for use listed on the patients' drug charts was reported for % of the drugs. the drugs with the highest percentage of recorded indications were acetylcysteine (n = ), oxycodone (n = ) and zopiclone (n = ), where , and % had a listed indication, respectively. conclusion: a high percentage of nursing home patients' diagnoses seem to be untreated. however, most drugs that patients received were listed with indication for use in the drug charts. to increase quality of drug prescribing, one should put emphasis on improving the recorded information in electronical health records. cp-pc : personal changes in drug regimen: dangerous for health system? inga urtane, raivis pastars, dace bandere please specify your abstract type: research abstract background and objective: patient compliance is a key factor for a successful treatment and lack of it is the main reason for predicting treatment failure. in multiple researches patient adherence is determined to be as low as %. therefore it is important to identify the reasons of patients not following their drug regimen. objective. to analyse the patient comprehension of their drug regimen depending on the duration of hypertension and received treatment. setting and method: during the period from december to march a quantitative survey was conducted to include respondents who have been diagnosed with arterial hypertension and whose regimen includes at least one fixed dose combination drug. main outcome measures: in an anonymous survey data was collected about their demographic information, co-morbidity, other prescribed medication, intake regime, the average blood pressure during treatment, and patient's assessment of the prescribed therapy. collected data was analysed with spss. results: the study included participants, most of whom ( . %) were women. participants average age was . ± . years and the median arterial hypertension duration was ( ; ) years. the study participants, who sometimes consciously adjusted dosing regimen, observed arterial hypertension for a longer period of time compared to the group, which follows the prescribed regimen according to their doctor's recommendation, respectively, ( ; ) vs. ( ; ); p = . . group of respondents (n = ) receiving c prescription drugs, more often deliberately adjusted treatment regimen compared to respondents (n = ) treated with b prescription drugs, respectively . versus . %; p = . . respondents who deliberately adjusted drug were more often not satisfied with the number of longterm daily use of tablets (n = ) compared to the group (n = ), which had to intake fewer tablets every day, respectively, . versus . %; p = . . conclusion: arterial hypertension duration was associated with more frequent conscious adjustment of therapy without consulting a doctor. more individual prescriptions (c ) and an increase in the number of tablets per day at the same time also increases the risk of patients deliberately changing their dosing regimen. long-term drug users should receive additional attention during pharmaceutical care process to their respective treatment schedule in order to promote proper use of medication. please specify your abstract type: research abstract background and objective: diabetes is a health issue and real burden for in belgians. better adherence to the treatment could potentially reduce complications, decrease morbidity and mortality, and have a beneficial economic impact due to fewer consultations and hospitalizations. setting and method: a one-year program was started in belgian pharmacies to accompany diabetes patients taking dpp- inhibitors and encourage them to be compliant with their treatment. this study concerns of these pharmacies, all part of the same cooperative group. all pharmacists received prior training in motivational techniques and reviewed the bases of diabetes therapy with an e-learning program. materials developed for the patients included brochures on diabetes and its treatment, nutritional advice, physical exercise, foot care and tips and tricks for diabetics. main outcome measures: the impact on pharmacological adherence was measured using mmpr and pdc. two control groups were included: a historical control group and a group of patients that were not included in the project. non-pharmacological adherence was assessed using questionnaires. results: in the subgroup of pharmacies, patients were included in the program. by the end of april , only of them had completed the program; patients came only once to the pharmacy. they either stopped their treatment after one prescription, or were occasional clients. adherence rates were found to be high in all groups ( . - . % of patients with mmpr b %). only for the pdc, a statistically significant difference was measured between the intervention and control group ( . vs. . %; p = . ). no other statistically significant impact was measured (neither pharmacological, nor non-pharmacological). conclusion: adherence was very high in all groups. the underlying reasons still need to be investigated (choice of adherence measure, healthy user effect, etc.). however, both patients and pharmacists were very pleased with this type of program. this new role of the pharmacist will definitely be more developed in the future. please specify your abstract type: research abstract background and objective: oral anticoagulants (oac) have a beneficial effect on the long term survival of patients with atrial fibrillation and venous thromboembolism. however oacs have also side effects such as bleeding, especially when used inappropriately. pharmaceutical care interventions aim to optimize medicines use and improve patient health outcomes. the literature lacks a review on the impact of pharmaceutical care interventions in patients using oac. therefore, we systematically assessed the impact of pharmaceutical care interventions on the effective and safe use of oac compared to usual care. setting and method: a systematic review was performed in pubmed and embase with synonyms/detailed specifications of the terms oral int j clin pharm ( ) . it was motivate for the need to sort the instruments for urm, including professional participation, and on the basis of the clinical management unit, and reduce variability in decisions. the p&t or ''multidisciplinary commission rational use of medicines'' is constituted by people: one hospital medical director (president), head of pharmacy (secretary), and three directors of healthcare centre, three directors of department of specialities, one epidemiology, one hospital pharmacy, one primary care pharmacy and one paediatric. because some of these members are far between them, and normally dose not have too much time, we create an online platform to work, discuss and download all the necessary documents. setting and method: we used the facilities of the andalusian agency for healthcare quality (www.acsa.junta-andalucia.es), and as a base the law of the administrative decision. we have organized a session to discuss methodology with the participation of all members. main outcome measures: number of meeting and number of internal discussion emails. drug or protocol decisions. design of the platform. results: the design platform consists of five tabs: ( ) has the member information, position, telephone and address, ( ) email forum, following a subject line, ( ) a place for meeting requests and then hang up the meeting minutes. ( ) a tool allows you to upload documents to the portal ( ) a search engine. two sessions are schedules and total of mails. we have of members who have never participated online. at this moment we have adopted two decisions. conclusion: it is an online experience of one andalusian p&t committees, the low turnout makes go slower than expected, therefore physical meetings are necessary in this moment. we are working how to get more participation and involve in the project the committee members. please specify your abstract type: research abstract background and objective: liver cirrhosis can have a major impact on drug metabolism, requiring evaluation of drug safety and dosage in individual patients. currently, there are no guidelines on safe prescribing for medications in patients with liver cirrhosis, and these patients have many questions about safety and side effects of medication. the objective of this study is to explore the patient's needs on information about medication. setting and method: qualitative, semi-structured interviews were performed in patients with a (history of) liver cirrhosis. the patients were approached through an item in the newsletter of de dutch association of liver patients. topics in the interview guide were preferences about information about medication, side effects, safety, drug dosage, and how patients preferred to receive this information. interviews were audiotaped and transcribed verbatim. interviews were analysed using thematic content analysis. main outcome measures: the experiences and needs of patients with liver cirrhosis concerning information about medication. results: patients indicated they had received sufficient information about the indication, possible drug-drug interactions and the duration of treatment. they preferred (more) information about how medications work, what adverse drug reactions could be expected and practical aspects concerning intake of medication. informational needs were related to questions 'how to act': patients with more informational needs took a more active role in responsibility for their own medication management. patients needed information to know what to do, e.g. in case of adverse drug reactions or when a dosage was forgotten. the doctor and internet were the preferred sources of information: doctors because of the personal contact and internet because of the accessibility. facilitating factors were 'taking time' in healthcare provider-patient contact and 'everyday language' for texts on the internet and in package leaflets. a combination of verbal information by the healthcare professional and written information was preferred. conclusion: patients with liver cirrhosis need information about medication to take an active role in their drug management. comission for medicines and medical devices, chu de toulouse, toulouse, france please specify your abstract type: descriptive abstract (for projects) background and objective: due to its common use, insulin is often considered as a harmless medication by lots of health professionals while an overdose can lead to dramatic consequences and death. between january and june , in our university hospital, % ( out of ) of the declared adverse drug events have involved insulin: were caused by prescription errors and by administration errors. all were discovered after the medicines have been administered but thankfully none had serious consequences. the british national health service (nhs) and the french medication safety national agency (ansm) made a list of ''never events'': avoidable events which should never happen and misadministration of insulin is among them. the objective was to increase patient safety in the hospital by setting different actions to promote and improve the appropriate use of insulin and warn health professionals about the real dangers of this medicine. design: different actors participated in the implementation of these actions: the commission for medicines and medical devices (which is composed by doctors and pharmacists) directed a group made by physicians and clinical pharmacists from the department of cardiological and metabolic diseases'. results: in addition of the usual analysis of any adverse event linked to medication declared in the hospital, several actions were set up: • a didactic document summarizing all the ''sensitive'' steps during the prescription, stocking, dispensation and administration of insulin has made the front page of the hospital's intranet and was also diffused throughout the establishment. • a chart resuming all the different insulins commercialized in france has also been diffused. it contains their types, durations and onsets of action, conditions of storage and pictures of their packaging. • the computerized protocols involving insulin are going to be reviewed in order to lower their numbers and harmonize their content. • a revision of the list of insulins available at the hospital is in progress to reduce their number and avoid any confusion between the different products. • an evaluation of insulin's computerized prescription practices will be made via a data request. : this topic about insulin shows a greater willingness to secure the medication circuit in the hospital. other action plans such as this one will be set up involving other medications among the never-events list. meanwhile, the commission for medicines and medical devices pursues its actions of promoting the appropriate use of medication. please specify your abstract type: descriptive abstract (for projects) background and objective: one of the hospital pharmacist tasks is to suggest substitutions to ensure conformity of medical prescription with the hospital formulary. indeed, when an eye drop isn't available at the hospital, there is a specific supply circuit which has to remain exceptional: it's ordered directly to the pharmaceutical wholesaler. in this context, ophthalmologists and clinical pharmacists created a table proposing therapeutic equivalencies with eye drops available at the hospital. after approval by the commission for medicines and medical devices, this tool has been diffused within the establishment via the intranet website since the beginning of to the medical and paramedical staff. the purpose of the study is to evaluate professional practices concerning the use of the eye drops' equivalence chart. design: in the study, we compared eye drops' orders made to the pharmaceutical wholesaler before and after the table's diffusion, thus between january and december . for each order, we used the table available at this time to determine if equivalencies could have been proposed or not. if so, we identified the hospital ward and the pharmaceutical specialty. market changes have also been considered. results: we noticed a decreased frequency of eye-drops ordered despite available equivalencies: % in (before the table's diffusion), % in (after its diffusion), % in and % in . prisons units are responsible of % of these orders: they have the lowest rates of substitutions. their most ordered pharmaceutical specialties are ophthalmic glaucoma agents: % ganfort Ò (bimatoprost . mg/ml/timolol . %), % xalacom Ò (latanoprost + timolol . %), % azopt Ò (brinzolamide) for which the authorized substitutions are for the first two specialties: monoprost Ò (latanoprost) + ophtim Ò (timolol . %) and for the third: dorzolamide Ò . conclusion: equivalence table diffusion throughout the hospital has facilitated and improved the prescription and substitution of eyedrops. orders of pharmaceutical specialties despite authorized equivalencies available have declined by half. probably for practical reasons regarding long-term treatments, prison units make less substitutions but an awareness campaign will be carried out to reduce these rates. please specify your abstract type: research abstract background and objective: the patient's education and information is a mean to reduce medicine misuse and it can be performed with support of a leaflet or informative material about medicines. in brazil, there is a lack in regulation about this type of informative material to compounded medicines. the aim of this study was to evaluate the quality and effectiveness of leaflets developed to compounded medicines' users through knowledge's level and medicine treatment adherence. setting and method: analytical and quantitative study; month prospective study through interviews, at time zero (t ) and after days (t ) in a university pharmacy in goiás, brasil; fisher's exact test to measure effectiveness; ethics committee number / . main outcome measures: categorization into high adherence and low adherence by morisky test; categorization into sufficient, regular or insufficient knowledge about medicine prescription; perceptions and suggestions about delivered leaflet in medicine dispensing process. results: of patients ( . % female, mean = . years), . % considered as relevant the leaflet's content, as well . % of them kept it and . % of them read it. suggestions of . % included a desire in increase font size, more emphasis on drug interactions and images. there was a predominance of regular knowledge in both analysed times ( . % e . %), however there was a decrease in high adherence to medicine treatment ( . - . %). among patients who read the leaflet, no statistically significant association was found on these two variables at t and t (p = . and p = . , respectively). knowledge about ''administration schedules'' showed a significant improvement after intervention (p = . ). . % of patients considered that there was no need to obtain more information. conclusion: this study demonstrates the evaluated leaflets had relevance to patients and demonstrate clinical relevance. however was not observed statistically significance. this highlights the need of using different ways to measure the effectiveness of an informative material to promote rational use of medicines and depth studies and stimulation of greater attention from the health professionals to the topic. di : chlormethine gel: effectiveness and tolerance to treat mycosis fungoides françois dugre *, , anne lefebure , sonia martelli , marion pin , eve maubec , philippe arnaud pharmacy, dermatology, bichat-claude bernard hospital, paris, france please specify your abstract type: descriptive abstract (for projects) background and objective: to determine the effectiveness and tolerance of chlormethine gel in treating mycosis fungoides. design: mycosis fungoides is the most common form of cutaneous t-cell lymphoma (mf-ctcl). early stages (ia and ib) can be controlled by skin-directed therapies such as chlormethine and carmustine. these drugs which are solutions for injection are usually used for skin application. chlormethine or mechlorethamine gel is an alkylating agent representing an alternative for previously treated patients diagnosed with mf-ctcl, in case of therapeutic failure and intolerance, or in case of chlormethine and carmustine solutions supply disruption. a retrospective observational analysis was conducted based on medical records of all patients treated by chlormethine gel in our hospital from the first of july to the first of september . the following data were collected with an excel table: body surface area or bsa affected by disease, location of the lesions, therapeutic management, effectiveness and treatment tolerance. results: fourteen patients ( women, men, mean age [min ; max ]) were treated with chlormethine gel in our hospital. twelve ( %) were treated three times per week, ( %) once a day. before treatment by chlormethine gel, ( %) patients were treated by dermocorticoids, ( %) by dermocorticoids and phototherapy, and ( %) by bexarotene, all of them stopped their treatment on account of inefficacity. one ( %) patient was treated by carmustine and dermocorticoid, and ( %) by only carmustin, all of them stopped it because of supply disruption. one ( %) patient received it in first line therapy. ten ( %) patients showed a response (partial or complete), one ( %) experienced a stabilization of his disease. before treatment with topical chlormethine, seven patients ( %) had an involved bsa [ % and four of them ( . %) experienced adverse effects. seven patients ( %) had an involved bsa \ % and three of them had ( . %) side effects. a total of seven patients ( %) presented at least one adverse effect. five patients ( %) stopped the treatment on account of adverse effects; two of them ( %) interrupted it temporarily. reported side effects were: irritant dermatitis and erosive toxicity ( ), rash ( ) and telangiectasia ( ) . conclusion: our results indicate that chlormethine gel can be effective to treat mycosis fungoides. however, it involves side effects that seems to be more frequent than those observed with chlormethine solution (used for skin application). indeed, the french national authority for health reports % of adverse effects for chlormethine solution versus % in our study for chlormethine gel. moreover, telangiectasia was never documented with chlormethine. this significant number of side effects of chlormethine gel can be explained by the gel formulation which induces patients to apply more product, especially in patients with plaques affecting more than % of the bsa. it is important to explain to patients to apply a thin film of chlormethine gel to involved skin areas and allow the skin to dry completely. sophie dumas *, , capucine devaux , nathalie le guyader diaconesses croix saint-simon hospital, diaconesses croix saint-simon hospital, paris, france please specify your abstract type: descriptive abstract (for projects) background and objective: aprepitant, a neurokinin- receptor antagonist, prevents nausea and vomiting due to high and moderate emetogenic chemotherapy in combination with other antiemetic agents. it induces cytochrome p (cyp) c and moderately inhibits cyp a . drug-drug interaction could occur with intravenous anticancer or antiemetic drugs metabolised by these isoenzymes. it may lead to adverse effects or loss in efficacy. regarding recent international antiemetic guidelines, emergence of new intravenous chemotherapy and lack of bibliographic data, a report on aprepitant interactions is performed in oncology. the aim of this study is to review pharmacokinetic interactions with aprepitant in order to prevent potential toxic effects of intravenous anticancer or antiemetic agents and provide the best patient care. design: anticancer and antiemetic agents metabolised by cyp a and c were identified. pharmacokinetic literature review was performed using medline Ò database and laboratory data. clinical assessment and non-aprepitant pharmacokinetic studies were excluded. a table was established to summarize data. results: ten intravenous anticancer agents used in oncology are identified as cyp a substrates. pharmacokinetic assessments are achieved for docetaxel, cyclophosphamide, vinorelbine, irinotecan and trabectedin. studies dealing with the five other drugs are strictly clinical assessments. among the different pharmacokinetic studies, only trabectedin showed relevant interaction with aprepitant. in this association, aprepitant dose needs to be adjusted. cyp c catalyses the cyclophosphamide activation pathway with minor contribution. however, it would have few repercussions on cyclophosphamide pharmacokinetic. corticosteroids and hydroxytryptamine type ( ht- ) receptor antagonists are also metabolised by cyp a . aprepitant significantly increases corticosteroid plasma concentrations. in this case, corticosteroid dose adjustment should be applied. furthermore, no interaction has been found with ht- receptor antagonist. conclusion: regardless of the emetogenic level of anticancer agents, all drugs have been studied because of theirs potential combinations. two relevant pharmacokinetic interactions have been demonstrated leading to dose adjustment recommendation. corticosteroids doses, in association with aprepitant, should be reduced one-fourth for intravenous form and one half for oral form. aprepitant first dose should be decreased to mg when it is co-administrated with trabectedin. these two results lead us to re-evaluate our prescription practices. please specify your abstract type: research abstract background and objective: nsaids are associated with serious adverse reactions which in turn are responsible for significant risks of morbidity and mortality. the aims of this project is to identify risks involved in nsaid administration including over-usage and significant drug interactions, and to analyse occurrence of side-effects. the trends of nsaid prescribing by physicians and pharmacists are also determined. setting and method: a pharmacy from each electoral district was chosen by stratified sampling. a sample population (n = ) was obtained from pharmacies in malta. data was collected through the completion of questionnaires carried out by the patients. the trends of nsaid prescribing were determined by another questionnaire directed to pharmacists and physicians that was available online. main outcome measures: use of nsaids by patients and prescribing trends. results: back pain (n = ), muscular pain (n = ), headache (n = ) and arthritic pain (n = ) accounted for the most frequent use of nsaids. diclofenac accounted for the most commonly administered nsaid, taken by of the patients, of which use the mg dose. chronic disorders of symptoms experienced by the patients included hypertension (n = ), heartburn (n = ), dyspepsia (n = ), asthma (n = ) and a history of helicobacter pylori infection (n = ). other disorders suffered by single individuals include epilepsy, crohn's disease and renal dysfunction. more than half of the respondents (n = ) admitted to self-prescribing regardless the fact that the majority of nsaids are prescription-only medications. epigastric pain ( . %), stomach ulcers or gi bleeding ( . %) and elevated blood pressure ( . %) were the most common sideeffects that pharmacists and physicians come across. nsaids were frequently found to be co-administered with antihypertensives ( . %) and ssris ( %) regardless of their significant risks of interacting with nsaids. . % of the pharmacists and doctors believe that nsaids are being over-used and . % state that closer monitoring of nsaid adverse effects is necessary. conclusion: the risk involved with nsaid administration due to over-usage and drug interactions is identified, and healthcare professionals are aware of this risk. pierre leduc, antoine lanneluc, christophe gellis * , sylvie poux, dominique plats, regine larnaudie corrèze, ch brive la gaillarde, brive la gaillarde, france please specify your abstract type: research abstract background and objective: proton pump inhibitors (ppi) are widely prescribed in hospital while their long-term use may be responsible of many potentially serious long-term side effects (hypomagnesemia, neutropenia, gastric cancer) and drug interactions (ppi are inhibitors of cyp c ). the objective of this study was to assess the appropriateness of ppi prescriptions in a geriatric department in order to optimize their conditions of prescriptions. setting and method: this prospective study involved patients hospitalized between january and april in a geriatric department. the accordance of the prescriptions with the marketing authorization indications and the french guidelines was analysed. data collection was done using a table excel. main outcome measures: collected information were related to patients (age, sex) and ppi prescriptions (active substance, administration route, dosage, duration of therapy, therapy indication and reassessment of ppi therapy). results: ninety-one patients were included: sr: . , mean age: . years [ ; ]. ppi therapy prevalence over the period was %. the ppi were prescribed in the geriatric department in patients (mostly esomeprazole) whereas patients had ppi therapy (mostly esomeprazole) at the admission, for more than years in patients. oral route was the most frequent one (n = ). ppi were administered once a day and only three ppi were administered in the morning. % of ppi prescriptions were considered unjustified; the indications were prevention of haemorrhage with antiplatelet therapy (n = ), prevention of haemorrhage with corticoid (n = ), prevention of haemorrhage with anti-vitamin k (n = ), dyspeptic disorders (n = ), gastralgy (n = ) and others reasons (n = ). % of ppi prescriptions were considered relevant. the reassessment of ipp therapy (n = ) lead to prescribe another dosage (n = ), to stop therapy (n = ) or no change (n = ). conclusion: the study showed that the majority of ipp prescriptions were not in accordance with french guidelines. limiting the prescription to the indications, reassessing the therapy or respecting the therapy duration should reduce the risk of long term side effects and the economic burden of ppi in a long term use. please specify your abstract type: descriptive abstract (for projects) background and objective: to evaluate the effectiveness and safety of the use of high dose of tigecycline ( mg followed by mg every h) a tertiary care hospital. design: retrospective observational study. period: january to december . inclusion criteria: episodes use of tigecycline ( mg followed by mg every h. exclusion criteria: time less than days treatment. data source: corporate program stories electronic health. results: we identified episodes in patients ( men, mean age: years ( - )). treatment was directed to multidrug-resistant organism infection in cases (seven klebsiella pneumoniae oxa- , two enterobacter cloacae, two enterococcus faecium and one methicillin resistant staphylococcus aureus. in one episode they coincided e. cloacae and e. faecium). in cases had severe sepsis or septic shock (seven abdominal focus, six respiratory focus and one unknown focus). the median number of days of treatment was ( - ). tigecycline was administered as monotherapy in three cases, bitherapy and triple combination therapy in . the antibiotics were associated were: beta-lactam ( ), aminoglycosides ( ), quinolones ( ) colistin (three, two inhaled cases), cotrimoxazole ( ) and vancomycin ( ) . in episodes produced clinical and/or microbiological resolution and antibiotics are rotated by progression picture or lack of improvement, death occurred in three cases and was suspended on suspicion of hepatotoxicity. among the seven episodes of klebsiella pneumoniae oxa- infection there were four pneumonias, three with favourable evolution and one patient died, two bacteraemia, both with resolution clinical and microbiological, and one urinary tract infection resolved. among the episodes in severe/ septic shock were five cures, six cases of antibiotic rotation progression or lack of improvement and three deaths while patients receiving therapy tigecycline. patients showed an adverse effect possibly related to therapy tigecycline: diarrhoea after days of treatment and case of liver toxicity after days of tigecycline and piperacillin-tazobactam which led to their withdrawal. int j clin pharm ( ) : - conclusion: tigecycline has been used in double dose defined in data sheet especially in situations of severe sepsis or septic shock and infection multiresistant microorganisms. the effectiveness is conditioned by the clinical situation patient, being worse in severe/septic shock sepsis. tigecycline high dose was well tolerated and there was only a case of stopping the medication for suspected damage hepatic. di : wikipedia and medicines: who edits medicine articles on the english wikipedia? kristian husvik skancke , kristian svendsen *, department of history, uit -the arctic university of norway, hospital pharmacy of tromsø, tromsø, norway please specify your abstract type: research abstract background and objective: the medical profession and pharmacists are divided on the usability of wikipedia for looking up health information. nevertheless wikipedia is widely used, more than half of us physicians and percent of all medical students use wikipedia as a source of health-related information. there is a potential for incorrect and biased information being added by the pharmaceutical industry. the aim of this project was to examine who edits wikipedia articles on medicines and to investigate whether the pharmaceutical industry edits these articles. setting and method: two different groups of articles has been examined; the top ten bestselling medicines (substances) in the world in and the ten most recently approved medicines on the european market (until december ). the top ten medicines were selected from a consultancy report by evaluatepharma/ep vantage. the ten most recently approved medicines (new substances) were found on the european medicines agency webpage. we queried the english wikipedia on january and information from the edit history and the editors' user information were extracted. unregistered editors were checked using a whois service. for the new medicines all editors were checked, while for the bestselling medicines large edits and initial edits was checked. main outcome measures: edits suspected of being made by the pharmaceutical industry. results: ten bestselling medicines: there are many users editing these articles and/or watching them, limiting the risk of misinformation from the industry. there was no indication that the pharmaceutical industry had edited any of the articles. ten most recent medicines: no article existed for dasabuvir. for the nine other substances there were relatively few editors and watchers. in four out of the nine articles we found evidence of edits from the pharmaceutical industry. these edits, were done by registered editors with very few edits except for the medicine in question and they had made large additions to the articles sometimes even before the medicine was marketed. conclusion: the pharmaceutical industry seems to edit articles about medicines on english wikipedia however we found no evidence of harmful edits and bestselling medicines have many editors monitoring the quality of articles. please specify your abstract type: research abstract background and objective: the pharmaceutical professional service of the monitored dosage systems (mds) tries to improve the adherence of the patients to the treatment. the aim was to analyse the relevance of the repackaging of the most sold medicines in our country being used by patients included in the mds professional service and to determine the information discrepancies according to the source used by the pharmacist. setting and method: cross-sectional descriptive study. community pharmacy and healthcare institutions. all the patients included in the pharmaceutical professional service of mds on june , . data source: patients' records in the professional service, database of medicines ranked by sales in units in our country to december ( medicines), information sources on medicines: ( ) vademecum of medicines and ( ) the centre of drug information of our agency of medicines. main outcome measures: number of institutionalized and ambulatory patients included in the professional service of the mds and demographic characteristics, sum of different repackaged medicines belonging to the studied patients, analysis of the repackaged medicines of major use, number of discrepancies on the repackaging of the medicines according to the information source. results: patients were included in the professional service of the mds. of them were institutionalized (average age: . years, . % men, . % polymedicated defined as using c prescribed chronic medicines) and the remaining were ambulatory (average age: . years, . % women, . % polymedicated). different medicines prescribed in the institutionalized patients were taken into account, of them included in the sales ranking in our country. according to the first source, of medicines were eligible for repackaging, medicines could be repackaged according to the laboratory manufacturer and the remaining ones could not be repackaged. according to the second source, of medicines could be repackaged, and the remaining ones could not. different medicines prescribed in the ambulatory patients were taken into account, of them included in the sales ranking in our country. according to the first source, of medicines could be repackaged, medicines would depend on the laboratory manufacturer and the remaining ones could not be repackaged. according to the second source, of medicines could be repackaged, and the remaining ones must remain in the original package. discrepancies were observed in the information for ( . %) and ( . %) medicines in institutionalized and ambulatory patients, respectively, based on the sources used. conclusion: a considerable number of discrepancies in the information on the relevance of the repackaging of medications in the mds were found between two analysed sources. these findings have already improved the quality of this professional service. it would be necessary to alert the pharmacist of the existence of the above mentioned discrepancies to be able to prevent errors from occurring at the time of repackaging the medicines in the mds and, thus, increasing patient safety. please specify your abstract type: research abstract background and objective: despite the global advances of pharmacy practice and subsequently pharmacy education, students experience insufficient opportunities to practice the activities, tasks and processes essential to deliver pharmaceutical care. objective: to describe the development, implementation, and assessment of a clinical pharmacy practice (cpp) experience course in internal medicine, cardiovascular, respiratory clinics and drug information centre that is newly integrated into pharmacy curriculum at a university in north cyprus. setting and method: a weeks structured pharmacy practice experience was designed for fifth year students. student competence was assessed using formative osces and summative written exams before and after the course, and mapped in eight main cpp competences. the course utilized a wide variety of learning and practical activities including rounds participation, morning case reports, interdisciplinary activities, carrying interventions, role-play, direct patient care, formal case presentations, journal clubs and answering drug queries. competencies tested and strengthened include: taking medication history, response to the symptoms, pharmacotherapy knowledge application, comprehensive patient assessment, data interpretation using evidence-based approach, public health counselling, drug related problems management, patient counselling and communication skills. student perceptions and experience was assessed using semi-structured group interview and a questionnaire. main outcome measures: student scores in osce; student's perceptions. results: student reported that the course met pre-set objectives with substantial learning in different areas of cpp. students scored best in communication skills ( . ± . %), public health promotion ( . ± . %) and patient counselling ( . ± . %) than in resolution of drps ( . ± . %) and pharmacotherapy application ( . ± . %), while they significantly enhanced in di manipulation ( . ± . %) compared to baseline assessment ( . ± . %)(p = . ). conclusion: the course provided a rich experiential learning environment rather than just theoretical knowledge of clinical pharmacy. students well perceived the course structure assessment and knowledge attained. this could be implemented in other faculties of pharmacy through turkey. please specify your abstract type: descriptive abstract (for projects) background and objective: clinical pharmacy and clinical pharmacology have many similar aspects. both areas present professionals who have groundings in drug therapy principles and who aim to optimize the efficacy and safety of therapies for patient's benefits. however, there are clear distinctions. clinical pharmacologists are in general doctors with an additional education in clinical pharmacology. many of these are prescribers of drugs in practice but are in usual connected to academic parts responsible for education and research. they belong to a well-recognized but small sub-specialty of medicine. in contrast, clinical pharmacists are part of a much greater group of professionals working in most hospitals in developed countries. while the former one is restricted and subordinate to distributing the drugs requested by the medical prescribers, the role of the pharmacist has increasingly developed to encircle monitoring outcomes of medicine treatment and report management, patient safety and budgetary responsibilities. pharmacists are currently capable to take on prescribing responsibilities in developed countries and have been actively involved in collaboration in practice of prescribing with doctors. they also take on a great part in education related to rational prescribing that was once thought the area of the clinical pharmacologist. given the difference in size of the two areas there is understandably increasing confusion in the minds of managers in health services as to the continuing role and identity of clinical pharmacology. this may illuminate, in part, the diminishing in numbers and visibility of clinical pharmacologists in certain countries. in fact, some might see the continuous development of clinical pharmacy as a direct danger to the viability and future existence of the specialty of clinical pharmacology. however, clinical pharmacy and clinical pharmacology working synergistically would serve for the well-being of the public. design: . results: . conclusion: . maxime apparuit *, , lea boissinot , ngauv melodie , stephanie charles weber , isabelle lopez , françois chast pharmacy, hopital cochin, pharmacy, hopital hotel-dieu, paris, france please specify your abstract type: descriptive abstract (for projects) background and objective: hereditary angioedema (hae) is a rare disease characterized by episodic attacks of swelling which can be life-threatening. treatment for hae involves prophylaxis and management of acute attacks. the objective of this study was to evaluate patients' knowledge of their disease and their treatment. design: a questionnaire about the disease and drug treatment has been implemented. it was distributed to patients through either a pharmacist during patients stay at the hospital, or the french association des malades souffrant d'angioedèmes (amsao). answers were collected by electronic or conventional mail. results: patients completed the questionnaire. the average patients age is . ± . years. all of those interviewed could name their disease. for % of patients, the crisis happened unpredictably but in most cases a triggering factor was described, such as stress ( %), fatigue ( %) or an emotional shock ( %). oedema were located mainly in extremities ( %), abdomen ( %), ent sphere ( %) or face ( %). patients ( %) reported having more than crisis each year (eligible to prophylaxis), among them, patients ( %) said they had no preventive treatment. all patients knew the difference between prophylactic and curative treatment of crisis. among the patients receiving treatment for crisis, were able to define which treatment to be used depending on the intensity and location of the crisis. the majority of patients used icatibant during a crisis, but the most frequently cited prophylaxis treatments were tranexamic acid ( %) and danazol ( %). for injectable drugs to treat acute episodes, icatibant (subcutaneous) and c esterase inhibitor (intravenous) were self-administrated respectively in and % of patients. conclusion: this study showed that patients generally knew their disease and its treatment. however, they are insufficiently informed on drugs to be used according to the clinical situation and especially intravenous self-administration. therefore, it seems necessary to increase pharmacist involvement in patient's information about therapeutic strategy and drugs routes of administration. this for a major objective: an optimal self-care in a skilled patient. please specify your abstract type: descriptive abstract (for projects) background and objective: hospital pharmaceutical educations (hpe) on patients with oral anticoagulant (oa) can improve their overall management by providing skills on proper use. an ambulatory monitoring is necessary to ensure good compliance and understanding of the treatment. our study aimed at the establishment of hpe for patients with oa, the establishment of a hospital-city link in burgundy, and an evaluation of the expectations of ambulatory health professionals (ahp). design: the development of hpe has been performed in our centre for patients with oa and assessed between may and september . in order to ensure continuity in their support, patients then received a binding document to the attending physician, pharmacist and nursing home stating the treatment and acquired skills. a satisfaction survey, with anonymous electronic questionnaire circulated by the representative boards evaluating the expectations of ahp, took place in order to improve and make the programme more attractive. results: two hundred and ninety-one patients could benefit from hpe and came out with an oa. one hundred and forty-three answers were collected: officinal pharmacists and nurses. ninety-seven percent of ahp have judged relevant the following stated security goals: the name of the drug, its use, its risks and to be able to inform all ahp. ''associated pathologies and treatments,'' ''the last coagulation test'' and ''potential factors for non-adherence'' seem necessary for the binding document. more than % of participants found that this action will facilitate the establishment of pharmaceutical anticoagulant educations in cities, the dialogue around the oa with the doctor, patient's compliance and will secure the treatment. conclusion: hpe certainly help patients. its implementation for patients with oa in our hospital has generated a real interest. the addition of an ambulatory link allows continuing at best their support. the questionnaire has also allowed us to know the opinions of ahp involved and some improvements to the binding document may have been done. participants were asked to associate the task to the profession by determining whether each profession had the main responsibility for undertaking the task, a supportive responsibility, or whether they should not be involved at all. data was analysed using spss Ò, version . the chi squared test was used to assess any significant association between categorical variables. main outcome measures: perception of the oncology pharmacist's role by healthcare professionals. results: from a total of completed questionnaires, it was found that for tasks listed as ''patient education and counselling'', % were considered as the pharmacists' main responsibility, whereas % were believed to be supportive roles. main tasks included educating the patient regarding which medication to avoid during their treatment. for tasks listed as ''drug related problems'', and % of tasks were found to include pharmacists as having main and supportive roles respectively. supportive tasks included dose calculation of anti-tumour therapy required per patient. in the ''authorisation of medication'' category pharmacists' main roles carried a total of % and supportive that of % of the total number of tasks. this included ordering anti-tumour medication. further analysis of data revealed that years of experience did not have a significant association with results obtained (p-value = . ); however physicians, pharmacists and allied healthcare professionals were found to involve the pharmacist most extensively (pvalue = . ). conclusion: tasks associated with the pharmacist were representative of the current role they possess within the oncology setting; however this association was limited to professionals having a close working relationship with pharmacists. this may be due to the lack of an established multidisciplinary team approach within this scenario thus limiting the perception of the oncology pharmacist's contribution. an implemented multidisciplinary team may improve communication between the professionals involved and optimises patient care. the aim of the study is to analyse from a qualitative and quantitative point of view the pharmacy resident's activity in pneumology service. setting and method: the study included all the daily prescriptions of three units of pneumology from january to april . pi and data were extracted from the software pharma Ò and collected in a summary excel Ò table: nature of potential errors, nature of the proposals offered by residents, way of transmitting pi, and rate of pis' acceptance. main outcome measures: potential errors are collected by following the validated and standardized criterions of french society of clinical pharmacy. results: over months, lines of prescriptions from patients aged years old (median [ - ]) were evaluated. sex ratio (m/f) was . . one hundred and two medication problems have been found: overdose ( . %), contraindication (ic) ( . %), under dosage ( . %), wrong rhythm of administration ( . %), forgotten treatment ( . %), dose unit error ( . %), antibiotic indication missing ( . %), drug not listed in the hospital formulary ( . %), potassemie unchecked ( . %), dose unadapted to renal function ( . %) or to inr ( . %), treatment not indicated ( . %), wrong administration route ( . %), antibiotic unreevaluated ( . %), redundancy ( . %). the proposals made to the doctors were: stopping treatment ( . %), posology adaptation ( . %), substitution ( . %), dose unit modification ( . %), adding information about the indication ( . %), treatment renewal ( . %), administration modalities changing ( . %), biological monitoring ( . %), therapeutical monitoring ( . %), antibiotic treatment reevaluation ( . %). all pi were made by informatical way. all medicinal classes were found in this study. hydroxyzine, cyamemazine and escitalopram were often found in contraindication errors. they are involved in cardiac disorders with qt extension. pis' acceptance rate was %. conclusion: this study shows the importance of pharmaceutical analysis on the quality of access to healthcare. the statement of pi allows us to identify the most frequent errors, warn and prevent doctors from these potentials errors by proposing solutions. the rate of acceptance is high which means that doctors agree with our proposals. pharmacists' implication in clinical pharmacy activities and their participation to medical rounds will improve this activity and by the way optimization of the management of the patient. please specify your abstract type: descriptive abstract (for projects) background and objective: ppis consumption is largely practiced in europe, because of their excellent tolerance in short time, and their misuse with regard to indications, dosage and treatment duration (in , france was the nd,ppi consumer in eu). the result is drug iatrogenic disease and unjustified expenses in health insurance. objectives: assess the ppis consumption and appreciate conformity according to the latest recommendations for relevant prescriptions of ppis. design: prospective study via an audit (model created internally), every hospitalized patients with a ppis prescription, in two hospitals, on a given day. data collected through the patient's medical record. prescriptions conformity defined, by taking account of indication level ( : approved by the ma (marketing authorization), : non-valid but certified by international publications or learned society, : nonvalid without scientific proofs, : non-indicated), dosage and treatment duration. analysed situations with no conformity (inappropriate dosage despite conform indication, treatment duration unjustified and ppis prescribed in wrong indications ( and ) . results: patients have ppis prescriptions ( male, £ years] among patients ( %). % ppis prescriptions began during the hospitalization. ( %) of the ppis prescriptions are in accordance with the experiment (indication + dosage +treatment duration), as well in community than in hospitals. details: indication level ( . %), indication level -gi bleedings-( . %). of the ppis prescription aren't in accordance. details: treatment duration ( %), dosage ( %), indication level -prevention of iatrogenic bleeding risk without nsaids prescription-( %), indication level ( %). regarding level indications, ppis are always taken with anticoagulant and/or platelet aggregation inhibitors and/or corticoid. conclusion: the part of ppis prescriptions in this study is high. the majority of non-conformity is caused by ppis prescribed with an indication level . the improvement program will involve feeding back ppis' good use, to educate physicians (junior and senior) about the relevant ppis prescription and give advice in complex situations (indication and ). in collaboration with prescribers, shutdown protocol of ppis, prescribed in long term, could be implemented in order to avoid the acid rebound effect after brutal treatment discontinuation. hp-ce : impact of a self-management program on inflammatory bowel disease patient in a university hospital caroline egon * , xavier pourrat please specify your abstract type: descriptive abstract (for projects) background and objective: inflammatory bowel disease (ibd) is a group of chronic inflammatory diseases that affects the colon and the small intestine. crohn's disease and ulcerative colitis are the principal types of ibd and involve severe diarrhoea, pain, fatigue and weight loss. ibd affects young adult with an increasing annual incidence ( . million concerned people in europe). patients with ibd are affected by somatic or psychosocial problems and patient education may contribute to their well-being. since september , individual educational sessions have been set up and since september , collective educational sessions. these sessions have been developed to improve patient's understanding of treatment options and medical adherence. the aim of this study was to demonstrate that a therapeutic education program (tep) could have a significant effect on ibd patient's skills with regards to their disease. design: after individual education sessions with a nurse, a group education session was introduced for outpatients with ibd. the collective session include approximately six to ten patients and is organized in a half day workshops (about disease and treatment) conducted by a multidisciplinary team. the workshops were performed by an education nurse, two hospital gastroenterologists, two hospital pharmacists and a community pharmacist. these sessions were wrapped up by a short satisfaction and knowledge questionnaires. results: in total, ibd outpatients participated to the educational program, patients with crohn's disease and patients with ulcerative colitis ( . % male; median age: ). for the individual educational sessions, two competence questionnaires were performed about anti-tumour necrosis factor alpha (tnfa) therapy: one about general knowledge, another one about self-administration subcutaneous injection. patients completed these questionnaires. for the collective educational session, the competence questionnaire developed consisting of six questions covering few items: disease, symptoms, treatment and complications. patients completed this questionnaire. after the questionnaire, each participant received a summary document about drugs, side effects, therapeutic and medical advice. conclusion: the patient education program contributes to the improvement of self-management skills when it comes to ibd. pharmacists joining medical specialists and nurses provided pharmaceutical care with a positive impact on compliance, which is a determining factor for the success of the treatment and the quality of life in patients living with an ibd. this program will be continued and a new program for teenagers is to be established as well. hp-ce : desensitization study of paclitaxel and carboplatin drug in the ovary tumor protocol in cuf descobertas hospital miguel  . freitas * , daniela brites, ana bota pharmacy, hospital cuf descobertas, lisbon, portugal please specify your abstract type: descriptive abstract (for projects) background and objective: the hospital pharmacy should be an integral part of the multidisciplinary team and implement strategies that meet the patient's needs. pharmacy, in oncological area, is in constant renewal. josé de mello saúde uses paclitaxel/carboplatin protocol as first line in ovarian tumor. although the antineoplastic agents are essential for the treatment of cancer, they can also cause hypersensitivity reactions, which may carry serious consequences. both immunoallergologist and oncologist create a desensitization protocol, which allows the reintroduction of the drug with greater security. the desensitization protocols involves the gradual administration of small quantities of the drug, resulting in a refractory period of the white blood cells (mastocytes) and a lower production of cytokines until the dose has been totally administrated. objective:to evaluate the efficacy of methods used to prevent and treat hypersensitivity reactions of carboplatin and paclitaxel, in order to carry on the treatment. design: a retrospective review of the patient files was performed in the day hospital between and . we included only patients with moderate to severe immediate hypersensitivity reactions (b h) receiving carboplatin and paclitaxel. the desensitization protocol brigham and women's hospital was applied using three solutions with increasing concentrations (dilution : , : and : ) in twelve successive steps for about h. results: in the period - were desensitized five patients with platinum group drugs, carboplatin (n = ) and paclitaxel (n = ) and the total elapsed six desensitization. almost all patients reached the scheduled daily dose, except a patient, which suspended the desensitization program for disease progression. conclusion: the desensitization protocol allowed the successful reintroduction of antineoplastic drugs in patients with a history of hypersensitivity reactions, in order to treat the disease. please specify your abstract type: research abstract background and objective: in the context of harmonization of clinical pharmacy activities within our region, a common medication reconciliation project was developed between two general hospitals. the objectives of this study were to initiate, a common medication reconciliation activity in the two hospitals, to analyse the results, and to communicate to all professionals in the area. setting and method: a working group composed of pharmacists of each hospital was formed to develop analytical documents. a -month prospective study was conducted in two general hospitals: in the first one, in an emergency department, and the other one, in a medicine department. patients included in the study were either elderly and/or had polypharmacy and/or were hospitalized for iatrogenic reason. at int j clin pharm ( ) : - the point of admission and discharge for each patient, the pharmacist has completed a conciliation record, and has detected potential discrepancy. unintentional discrepancies were reviewed and corrected by doctors. at the discharge, medication changes were sent to general practitioner and community pharmacies. a satisfaction survey about this process was sent to healthcare professionals (gp, pharmacist and nurses). a medication reconciliation's workshop was organized for a hundred healthcare professionals in the area. main outcome measures: at the point of admission, the conciliation record included the list of patient's home medication, admission medical orders, and the types of discrepancies. at the discharge, drugs prescribed were compared to admission medical orders. the satisfaction survey included seven questions to assess the process. results: during the study period, patients were included corresponding to prescription lines. reconciliation process required about min per patient. we identified at admission unintentional discrepancies. the most common unintentional discrepancy was the omission of medication ( %). % concerned alimentary tract and metabolism group. at the discharge, no discrepancies were found; the process required min per patient. % of healthcare professionals answered to our satisfaction survey to date. % are satisfied and believe that the process of medication reconciliation secures the patient medicinal treatment. healthcare professionals were present at the medication reconciliation's workshop, indicating an interest in the process. conclusion: in this experience of medication reconciliation, due to unintentional discrepancies observed, we had better implement this activity in the two general hospitals. a pharmacist devoted to this activity will be hire in each hospital. this relevant practice is well accepted by clinician. thus, we will improve communication with gp and community pharmacies. please specify your abstract type: descriptive abstract (for projects) background and objective: the sickle cell disease (scd) is a genetic, chronic disease, paroxystic in its unpredictable and polymorphic acute events. this most frequent genetic illness in the world is a major public health concern in french overseas territories. haute autorité de santé (has) recommendations for the care of scd advocate the development of therapeutic patient education (tpe). in martinique (french west indies), we consider the population of patients with scd in among which are followed in the adults sickle cell centre (ascc). one of the actions carried out by the ascc of our hospital is the tpe. the objective is to set up an original (because specific in the scd) tpe method, which enables the patient to live better with his disease on a daily basis, by teaching him and his family to recognize prematurely certain complications. design: we analysed needs from the outcomes of a national french survey has which one participated martinique and retained the following themes: the red blood cell, the genetic transmission, the main symptoms, the role of the water, the medicinal treatments and the questions of everyday life. we chose the innovative educational tools called the ''malles des savoirs Ò **'', a set of unusual experiments, accessories and models, which, by using a method of active pedagogy ''omca*: observer, manipuler, comprendre, agir ***'', value the learner by offering to him to manipulate and to experiment by himself. results: in , healthcare professionals (doctors, pharmacists, nurses) and a president of patients with scd association followed one week of formation in the omca* method for the animation of six workshops for - teenagers and adults. every ''malle des savoirs Ò **'' contains the necessary material for the animation and a guide of the organizer, including, for every tackled issue, a generic introduction, a presentation of the themes, the index cards of educational animations proposing the activities and one time of synthesis grouping the approaches concepts. the interactive manipulation allows the appropriation of the discoveries become then long-lasting experiences. a final evaluation allows to spot the problems met by learners to understand, to analyse the difficulties and to proceed to the useful adaptations during the next activity. conclusion: this tool, playful and perfectly adapted to the scd, engages, accompanies and helps patients in the construction of their own knowledges to return them actors of their disease. in , we shall estimate the impact of the development of this specific tpe programme of the patient with scd. please specify your abstract type: research abstract background and objective: to investigate the frequencies and clinical relevance of unintentional medication discrepancies, between preadmission medication lists and discharge medication lists, at discharge from hospital. a discrepancy is considered unintentional if there is no documentation explaining the intent of the medication change or if it is unintentional according to the prescribing physician. setting and method: systematic literature review. main outcome measures: frequency of unintentional medication discrepancies per patient and per medication; frequency of clinically relevant medication discrepancies. results: of the patients included - % experienced at least one unintentional medication discrepancy. of the medications used by the patients, - % were involved in unintentional medication discrepancies. of unintentional medication discrepancies found in five studies, - % were clinically relevant. conclusion: the review documented a high frequency of medication discrepancies, of which many were clinically relevant. ensuring sufficient communication of correct and complete medication information in transitions of care is a process which should be better implemented, to enhance patient safety. please specify your abstract type: research abstract background and objective: to investigate the frequency of medication changes not documented in the discharge letter, at discharge from hospital, for both regular, as needed and over-the-counter medications, supplements and herbal remedies (otc). secondary, differences between variables and patients with undocumented medication changes were investigated. setting and method: the patients included were all part of the intervention groups from an intervention study, conducted by one of the authors (tg), from april to december . the best possible discharge medication list was compared against the medication list in the discharge letter and any discrepancy between the two lists was noted, taking into account the text in the discharge summary. main outcome measures: the proportion of patients affected by at least one undocumented medication change at discharge and proportion of medications with undocumented changes. the proportion of patients was compared using a test according to gender, age, number of preadmission/discharge medications and length of hospital stay. results: two hundred patients were included in the study. the proportion of patients experiencing at least one undocumented medication change for the three subgroups: regular medications; as needed; otc, were , and % respectively. the proportion of medications involved in undocumented changes for the three subgroups were , and % respectively. the proportion of patients experiencing undocumented medication changes was significantly higher in patients with more than five regular medications at admission, (p \ . ) and at discharge (p \ . ). in both regular and as needed medications, the proportion of patients experiencing undocumented medication changes was higher in patients hospitalized longer than days (p \ . and p: \ . respectively). for otc, the rate of patients experiencing undocumented medication changes, was higher in females (p: \ . ). conclusion: a high proportion of patients are affected by at least one undocumented medication change and many medications are involved in undocumented changes. correct and complete medication information at admission and discharge may resolve many of these errors, ensuring patent safety at transitions of care. hp-ce : participation in courses at learning and mastery centre and the impact on patients' beliefs about medicines merethe nilsen *, , erik oie , kirsten k viktil diakonhjemmet hospital pharmacy, department of internal medicine, diakonhjemmet hospital, oslo, norway please specify your abstract type: descriptive abstract (for projects) background and objective: patients with chronic diseases are referred to learning and mastery centre (lmc) where the main objective is to support patients to cope with chronic diseases. education about the disease(s) (by a physician) and the medication treatment (by a clinical pharmacist) are important elements of these courses. little is known about how the participation at lmc influences the patients' beliefs about medicines. design: patients c years participating at a days course at lmc regarding acute coronary disease or atrial fibrillation were included in the period september -december . the patients filled out 'beliefs about medicines questionnaire'(bmq) before and immediately after the course, and also months after the course to evaluate their concern (bmq-concern) and necessity (bmq-necessity) of their cardiovascular medications. the bmq scores were dichotomized at scale midpoint (scale - ) to evaluate high and low concern and necessity, and these scores were combined to calculate the 'ambivalence'and 'acceptance', 'sceptical', and 'indifferent'rate to medications, and also the mean scores of the bmq were calculated. results: fifty patients were included, mean age years, % were women, using a mean of . cardiovascular drugs taken regularly. fifty-eight percent of the patients had high concern prior to the course, whereas and % had high concern immediately after and months after the course, respectively. ninety-nine percent of the patients assessed their medication as highly necessary before the course, % immediately after, and % months after the course. the mean score for bmq-necessity was . (sd . ) prior to course and . ( . ) and . ( . ) immediately after and months after the course, respectively. the corresponding scores for bmq-concern were . ( . ), . ( . ), and . ( . ), respectively. the proportions of patients classified to be 'accepting'were , , and % at the three time points, respectively, and the corresponding numbers for patients classified as 'ambivalent'were , , and %, respectively. conclusion: the lmc course had an immediate positive influence on the patients' concern about their medicines and on 'acceptance'. however, the effect seems not to persist over time. a closer follow-up could be discussed. please specify your abstract type: research abstract background and objective: the narrative-based medicine was intended primarily for health care professionals, and the use of narratives can be applied in any settings to better understand the meaning of own profession, to rediscover/strengthen the motivation to work as a team. the italian society of hospital pharmacist (sifo) promotes a qualitative study aimed at getting the real picture of pharmacist's role within the national health system (nhs), the interaction with other health professionals and patients through the narratives of under specialization pharmacists (ui) and pharmacists already working in the nhs (hp). these data can be further investigated to increase the perceived value/role of the pharmacist. setting and method: sifo hps and uis joining the national pharmacy school specialization network were invited to participate. all pharmacists participating to the study were given a semi-structure interview. the methodology was developed within the conceptual framework of the grounded theory (gt) a research methodology that arises in the context of qualitative research. gt is a systematic methodology involving the construction of theory grounded in data systematically gathered and analysed. main outcome measures: analysis of narratives. narratives were analysed according to the classifications of kleinman, frank and launer and robinson together with transitional analysis (ta). results: a total number of narratives were collected ( ups and hps). narratives from both group of participants show the need of strengthening the professional identity already in the early years of the pharmacy curriculum and more effectively during the years of specialization as well as the need of being educated to deliver patientcentred care as members of an interdisciplinary team. conclusion: this is the first step of a study that also includes patient's contribution to the definition of pharmacist's professional identity. hp-ce : impact of pharmaceutical counselling on cancer patients' information desire and treatment satisfaction stephanie wuyts *, , jacques de grève , veerle foulon , hilde collier , pieter-jan cortoos pharmacy, medical oncology, university hospital brussels, brussels, faculty of pharmaceutical sciences, catholic university of leuven, leuven, belgium please specify your abstract type: research abstract background and objective: appropriately educating onco-/haematological patients is a prerequisite to improve patient empowerment, satisfaction and outcomes. objective: to quantify patients' information need and satisfaction on cancer drug therapy and how this can be improved by clinical pharmacist's counselling. additionally, the pharmacist's impact on therapy quality and costs is assessed. setting and method: setting: prospective, randomised study in the ambulatory ( beds) and in-hospital onco-/haematology unit ( beds) in a tertiary hospital. inclusion criteria: adult patients on intravenous or oral cancer therapy, with informed consent. methods: all patients were asked to complete standardised surveys (extent of information desired, eid; patient satisfaction with cancer treatment education, ps-cate and cancer satisfaction of treatment questionnaire, ctsq) on three occasions (at the start of a new therapy, during the second cycle and after months). patients in the intervention group received additional counselling by a clinical pharmacist including medication reconciliation and review. control patients received standard of care (information on drug therapy was provided by the onco-/haematologist, followed by limited administration instructions by nursing staff). main outcome measures: patient information desire and satisfaction on cancer treatment results: patients were included over a period of months (control (n = ); intervention (n = )). no significant differences were found between contact moments or patient groups for eid, ps-cate and ctsq-scores. however, scores for ps-cate on medication side effects were positively correlated with contact moment (r s = . ; p = . ). multiple linear regression analysis showed a similar trend (b = . ; p = . ). patients receiving first-line therapy (b = . ; p \ . ) and ambulatory patients (b = . ; p = . ) were more satisfied on treatment education. the clinical pharmacist documented more drugs than were recorded in the patient file ( vs. . drugs/patient; p \ . ). on average, each patient required two pharmacist's interventions per occasion. intervention acceptance rate on drug related problems was high ( %). during the study, interventions shifted from therapy adjustments towards advice on supportive measures ( st contact: %; rd contact: %). improved medication stock control on the ward led to a savings of € , . conclusion: the clinical pharmacist can play an important role on the onco-/haematological ward, leading to improved drug reconciliation, patient counselling and cost savings. hospitalised patients and patients receiving salvage therapy appear to have higher educational needs, making them possibly overlooked target groups. finally, pharmaceutical counselling should be repeated and primarily focused on side-effect management to have a meaningful impact on patient satisfaction. please specify your abstract type: research abstract background and objective: europe is ahead of the usa and canada on approval, regulatory and marketing aspects of biosimilars. however, there is still uncertainty about interchangeability and substitution of biosimilars. the aim of the study is to assess pharmacists' perceptions about biosimilar interchangeability. setting and method: a cross-sectional study was carried out in june-july . hospital pharmacists from quebec and france were invited to respond to an online survey of nine questions (surveymonkey Ò , palo alto, ca, usa). the survey focuses on pharmacist's exposition to biosimilars (general knowledge, dispensing) and their perceptions about biosimilar interchangeability. a -item likert scale was used to answer to statements based on key issues about biosimilar interchangeability. main outcome measures: levels of agreement on biosimilar interchangeability key issues. results: a total of pharmacists responded ( % in quebec vs. % in france). the global response rate is: % ( % quebec vs. % france) (n = / ). % attended at least to one conference on biosimilars ( vs. %). % had already dispensed biosimilars ( vs. %). more than % of the pharmacists knew that: biosimilars can cause immunogenicity, clinical studies are requested for their approval, automatic substitution is not permitted. % considered that post-marketing surveillance for biosimilars should be reinforced. pharmacists considered that biosimilars are cheaper than the reference product ( vs. %). there was no difference between the level of agreement of french and quebec pharmacists for the statements. pharmacists agree that a list of biosimilar and interchangeable biologic products is necessary ( vs. %), using the international nonproprietary name to prescribe a biological product can create confusion between the reference product and its biosimilar ( vs. %), pharmacists should check if patients already experienced an immunogenic reaction before dispensing a biological product ( vs. %). pharmacists disagree that a biosimilar can be used for all the indications of the reference product ( vs. %). conclusion: perceptions of quebec and french hospital pharmacists about biosimilar interchangeability issues are very similar. this study highlights the need to deal with the lack of clarity of national guidances. clinical studies on biosimilar interchangeability must be conducted in the future to help pharmacists and physicians to take clear-headed decisions. please specify your abstract type: research abstract background and objective: analgesics are essential drugs in hospitals and especially in emergency units. medical and nurse staffs are used to the narcotic status of opioids. for some drugs, a regulatory change to narcotic status can discourage their use. for others, it could limit their access particularly in developing countries; that's why who did not recommend ketamine to be placed under international control (http://www.who.int/medicines/access/controlled-substances/ recommends_against_ick/en/). yet, the french drug agency has recently considered to register drugs containing ketamine as narcotics. the aim of this study was to assess the impact of this possible regulatory change on the pharmaceutical and medical practices in some paediatric french hospitals. setting and method: the survey was conducted in january-february in four parisian paediatric hospitals: four pharmacies, paediatric neurology and anaesthesia departments, intensive care units and pain management services. main outcome measures: pharmacists, clinicians, health managers and nurses were interviewed, using a standardized questionnaire with closed and opened questions, on the drug circuit including ordering, storage, distribution, prescription, administration and destruction. results: all the health professionals (five pharmacists, ten clinicians, five nurses) indicate that the change to narcotic status would not preclude the use of an analgesic drug. they consider that the pharmaceutical aspects (dispensation, storage and transport, etc.) are not limiting, provided that clinical usefulness is demonstrated: short action onset allowing rapid efficacy, short duration of action allowing the replacement by another drugs if needed, and moderate clinical monitoring. change to narcotic status was rather seen as advantageous since allowing better traceability, use and prescription. half of the pharmacies (n = / ) had a computerized register of narcotics and % of care units (n = / ) had a drug staffing in addition to nominative prescriptions, which was used in all care services. the drugs were kept into secured rooms. none of the emergency units (n = / ) had a computerized secured cabinet. conclusion: according to this survey, narcotic status is not a limiting factor for a drug use in paediatric hospitals, when its clinical usefulness is clearly demonstrated. to promote its use, it is important to inform medical and nurse staffs and include it into care protocols. beyond the nominative prescription, implementation staffing is a key step. please specify your abstract type: research abstract background and objective: port-a-cath is an implanted venous access device most commonly used for frequent or continuous chemotherapy administration. however, the procedure and its subsequent maintenance are not free of complications and requires additional intervention by the clinical pharmacist who can provide further patient care to make a positive impact on. to assess the effective provision of appropriate patient counselling offered by a clinical pharmacist on reducing port-a-cath relatedcomplications in cancer patients. setting and method: a controlled prospective observational study carried out on patients newly diagnosed with cancer eligible for chemotherapy administration at the oncology unit. assessment of port-a-cath related-complications were assessed at regular schedule of chemotherapeutic protocols administration. main outcome measures: to assess, reduce and solve port-a-cath related-complications. results: the most significant port-a-cath related complications were skin rash . % (p \ . ) with occurrence in males (n = ) and females (n = ), skin erythema . % with equal occurrence in both genders, followed by skin discharge . % with also equal occurrence in both genders. a high occurrence of skin rash . % occurred among diabetic cancer patients. a significant improvement in port-a-cath related complications after the provision of patient counselling by the clinical pharmacist was observed as skin rash ( . %), skin discharge ( . %), and skin erythema ( . %). conclusion: results of this study pointed out the essential role of clinical pharmacist in argumenting patient care and improving port-a-cath related-complications in cancer patients. please specify your abstract type: research abstract background and objective: polytherapy, frequently used in the elderly, is associated to an increased risk of potential drug-drug interactions (pddis) and adverse drug reactions (adrs). literature demonstrated that medication reconciliation and medication review performed by hospital pharmacists are correlated to drug related problems (drps). aim: to define a structured and feasible model where hospital pharmacists support clinicians identifying drps and promote the safe use of medicines. setting and method: prospective, feasibility study conducted in four internal medicine wards of a hospital in northern italy. inpatients (c years old, treated with c drugs) were consecutively included; the recognition/reconciliation process was performed by pharmacists in order to identify changes between prescription profile at home and during the admission (active principles, dose, administration route). these changes were classified as intentional documented discrepancies (id), not documented (ind), not intentional (ni). prescriptions during the first -hours of hospitalisation were analysed to retrieve drps (ddis, inappropriate medications for elderly, off-label, over/ under dosage, duplications, adrs) then discussed with clinicians. based on literature, referring almost drp in % of patients, a sample size of patients should allow an estimate of drp rate over % (need of intervention) with a % power and a confidence interval of % (software stata version . ). main outcome measures: rate and type of: discrepancies, drps at admission and discharge, pharmacists consultations accepted by clinicians. results: ad interim results are presented. between october/ -february/ , inpatients ( male, . mean age) were included. overall, patients were admitted with drugs used at home and prescribed during the first -hours; pharmacists retrieved discrepancies ( %id, %ind, %ni) and drps, of which % ddis, % off-label, % overdoses, % duplications, % inappropriate drugs, % not notified adrs. the % of drps was known to clinicians and % considered clinically relevant for the patients. please specify your abstract type: research abstract background and objective: hypertension is a major risk factor for cardiovascular morbidity and mortality worldwide, for which management is based on two principal, complementary approacheslifestyle modification and lifelong treatment with antihypertensive medication. adherence to hypertension therapy is a major public health challenge, despite the availability of multiple classes of antihypertensive agents. factors contributing to non-adherence are multifactorial and include intolerances to drugs at standard doses that result in therapy discontinuation. medication intolerance (mi-htn) refers to patients who experience adverse drug reactions (adrs) to at least one antihypertensive medication, without a known immunological mechanism and the need to discontinue them. we sought to determine factors associated with mi-htn and to identify patients' beliefs and concerns about their antihypertensive treatment and medication in general. setting and method: a cross sectional survey consisting of selfreported questionnaires including beliefs about medicines questionnaire (bmq), perceived sensitivity to medication (psm) and quality of life was undertaken in an unselected patients attending a hypertension centre of excellence out-patient clinic based in london. main outcome measures: to determine factors associated with mi-htn and the impact of health beliefs and self-reported perceived sensitivity to medications on mi-htn and bp control. chi squared tests for comparisons between cases/controls and multiple logistic regression analysis were used for statistical analysis. results: participants were included, of which ( %) participants had mi-htn. two-thirds were female (p = . ) with a mean age of ± years (p . ), of whom . % had uncontrolled hypertension (p = . ). calcium channel blockers were the most commonly reported intolerance by drug class followed by diuretics. being female and age [ were statistically associated with a greater likelihood of reporting medicines intolerance (p \ . ). patients who believed that medicines are harmful were [ -times more likely to report mi-htn (p = . ) and -times more likely to have uncontrolled bp ([ / mmhg) (p = . ). patients with high self-perceived sensitivity to medication was -times more prone to mi-htn (p = . ). conclusion: our findings suggests the need for greater focus on behavioural change interventions to both improve patients' perception of the necessity to persist with lifelong antihypertensive medication and allay concerns regarding harmful effects of drugs may help with long term control of hypertension. please specify your abstract type: research abstract background and objective: today, the number of medical problems in heart transplant recipients has increased due to aging and complications common to immunosuppressive drugs. the co-existence or emergence of other disease states such as renal dysfunction, infection, diabetes, obesity, hypertension, hyperlipidaemia, malignancies, and osteoporosis necessitates the use of other medications. the use of these medications in combination with immunosuppressive agents increases the risk of drug-drug interactions. the aim of this study is to identify the frequency and significance of drug-drug interactions for the patients who received cardiac transplantation. setting and method: this retrospective study was conducted at a cardiovascular specialty hospital. all patients who received cardiac transplantation from the same surgery team between and ( years) were included in the study. all data were collected from the medical records of the patients. only the most recent prescription before discharge was analysed for the presence and significance of drug-drug interactions. drug-drug interactions were checked using micromedex(r) interaction checker. main outcome measures: main outcome measures were the frequency and significance of drug-drug interactions. results: a total of patients met the inclusion criteria and prescriptions were analysed. each prescription contained an average of drugs. a total of drug-drug interactions were identified: . % was classified as moderate; . % as major and . % as contraindicated. almost half of all interactions (n = ) included immunosuppressive agents ( . % was classified as moderate; . % as major and . % as contraindicated). conclusion: cardiac transplant recipients were found to have a high number of drug-drug interactions. in order to advise on these interactions which increase with poly-pharmacy, drugs with narrow therapeutic index or drugs that require intensive monitoring, it is recommended to include a transplantation pharmacist in the transplantation team. please specify your abstract type: research abstract background and objective: the aim of our study was to assess the impact of patient education provided by the pharmacist on gylcemic control, medication knowledge level and medication adherence of patients with type diabetes. patients who were diagnosed with type diabetes for at least one-year time and were receiving at least one antidiabetic medication, attending to the outpatient diabetes clinic for the control visit were informed about the study and invited to participate in the study. patients who gave their informed consent were included in the study. setting and method: the setting is a diabetes outpatient clinic of a state hospital. the medication knowledge levels, medication adherence scores, fasting blood glucose levels, hba c levels and blood pressure of the patients were measured before pharmacist's education. after provision of standard information and individualized patient education all these parameters were measured again after monthstime and the impact of the education was assessed. main outcome measures: main outcome measures are change in the clinical parameters (hba c; fasting blood glucose; blood pressure), as well as improvements in medication knowledge and adherence levels. results: the study was conducted on patients who met the inclusion criteria; none of the patients were lost to follow-up. majority ( %) of the patients was female and the mean age was . years. pharmacist intervention resulted in positive outcomes at all clinical parameters. systolic blood pressure decreased by mmhg, while diastolic blood pressure decreased by . mmhg (p \ . ). hba c level decreased by . % (from . to . %; p \ . ) and fasting blood glucose level by . mg/dl (p [ . ). on the other hand, the number of patients reaching the blood pressure goal increased from to ; and those reaching to hba c goal increased from to (p \ . for all). similarly, the medication knowledge level [usual range - ] increased from . to . (p \ . ); and the medication adherence score [usual range - ] increased from . to . (p \ . ). conclusion: it can be concluded that pharmacist's contribution results in positive outcomes in glycaemic control and management of co-morbid conditions of type diabetic patients by improving medication knowledge and adherence levels of the patients. pharmacists should take active role in management of chronic diseases. hp-pc : impact of a pharmaceutical care program on glycemic control, medication knowledge and medication adherence levels of type diabetic patients residing at a nursing home nimet saglam *, , sule apikoglu-rabus , betul okuyan , fikret v. izzettin , nuran yildirim clinical pharmacy department, marmara university faculty of pharmacy, darulaceze nursing home, istanbul, turkey please specify your abstract type: research abstract background and objective: the aim of our study was to assess the impact of pharmaceutical care provided by the pharmacist on glycaemic control, medication knowledge level and medication adherence of patients with type diabetes residing at a nursing home. setting and method: this prospective cohort study was conducted in a state nursing home (darülacaze nursing home) in istanbul, turkey on patients who completed the whole study. all the patients received pharmaceutical care provided by the pharmacist. this pharmaceutical care program was held for months. it consisted of an initial visit, followed by ''care and control'' visits and a final control visit; each visit was held at two-week time intervals. at the initial visit, demographic and general clinical data were collected and medication knowledge and medication adherence levels of the patients were also assessed. pharmaceutical care needs were identified for each patient and recommendations addressing these issues were structured. education regarding the medications of the patients was provided in both verbal and written forms using the standard patient education leaflets prepared by the pharmacist. at each visit pharmaceutical care needs are assessed and pharmaceutical care is tailored accordingly. main outcome measures: main outcome measures are change in the clinical parameters (hba c; fasting blood glucose), as well as improvements in medication knowledge and adherence levels. results: majority ( %) of the patients was male and the mean age was . years. pharmacist intervention resulted in positive outcomes regarding hba c levels. hba c level decreased by . % (from . to . %; p \ . ) and fasting blood glucose level by mg/dl (p [ . ). similarly, the medication knowledge level [usual range - ] increased from . to . (p \ . ); and the medication adherence score [usual range - ] increased from . to . (p \ . ). conclusion: it can be concluded that pharmacist's contribution results in positive outcomes in glycaemic control of type diabetic patients by improving medication knowledge and adherence levels of the patients. pharmacists should take active role in management of type diabetes at the nursing home setting. please specify your abstract type: research abstract background and objective: haemoglobin variability is related to mortality and morbidity in haemodialysis, renal transplantation and pre-dialysis patients. some demographic, haematological and pharmacological variables may affect hb variability. but there are some controversies about the influences of different erythropoiesis stimulating agents (esa).the objective of this study is to determine the influence of different esa on haemoglobin variability in pre-dialysis patients. setting and method: we conducted a prospective observational study with chronic kidney disease patients recruited from outpatients of nephrology department of a tertiary university hospital (from january to june ). exclusion criteria were: stage i and ii, not treated with esa, haemodialysis, peritoneal dialysis, renal transplantation, thalassemia, and deficit of glucose- -phosphate dehydrogenase . main outcome measures: patients included were treated with esa in maintenance phase (stable months prior).hb variability was calculated by standard deviation (sd) and residual standard deviation (residual sd) of hb levels. statistical analysis was performed with spss . (spss inc, chicago). observation period was months and data were recorded from the clinical records. ( ) . %, sofosbuvir/daclatasvir/ribavirin ( ) . %, sofosbuvir/simeprevir ( ) . %, sofosbuvir/ledipasvir ( ) . %, sofosbuvir/ledipasvir/ribavirin ( ) . %, dasabuvir/ombitasvir/paritaprevir/ritonavir ( ) . %, dasabuvir/ombitasvir/pari taprevir/ritonavir/ribavirin ( ) % ombitasvir/paritaprevir/ritonavir/ ribavirin ( ) . %, sofosbuvir/ribavirin ( ) . %. viral load at week was \ iu/ml in patients and at the end of treatment . conclusion: the results of rapid viral response at end of treatment were similar to those obtained in studies published to date. due to its recent access to these treatments it is necessary to continue monitoring these patients to assess virologic sustained response at weeks after end of treatment. please specify your abstract type: research abstract background and objective: fragile patients are considered those vulnerable patients with a certain degree of complexity in their care (polypharmacy, multi-pathological, palliative and/or residents in social and healthcare institutions). to ensure their continuity of care and safety in the use of drugs we applied a medication reconciliation process at admission, transition of care and/or hospital discharge. objective: to analyse the results of the medication reconciliation process of a fragile patient. setting and method: we developed a list of current medication with the following sources of information: medical history, clinical databases and information provided by the patient (interview). clinical case: -year-old woman admitted through emergency department due to severe dyspnoea. no known drug allergy. background: heart failure, chronic hypertension, hypercholesterolemia, hyperthyroidism, hyperuricemia, gouty arthritis, chronic kidney disease and cognitive impairment by alzheimer disease. exploration and complementary tests: echocardiogram and analytical control. clinical judgment: acute decompensated heart failure. acute myocardial infarction. prerenal acute kidney injury. main outcome measures: medication reconciliation made at admission with the detection of discrepancies and deprescribing criteria at hospital discharge. results: fragile patient (high-risk) with medicines as home treatment. patient was hemodynamically stable during the hospital stay. discrepancies were detected between the prescribed medication and the home treatment. discrepancies justified ( ): five by omission of medication (two new clinical situation, two therapeutic exchanges to adapt to the pharmacotherapy guide and one wrong drug) and two beginning of medication. discrepancies unjustified ( ): by omission of medication. to discharge: one antiplatelet therapy was. after the comprehensive review, we made the following recommendations of deprescription: suspend one non-steroidal anti-inflammatory drug-nsaid (by risk of bleeding in association with concomitant antiplatelet and antidepressant therapy) and one benzodiazepine (central nervous system-cns side effects); modify treatment: reduce doses of diuretics (blood pressure lowering effect). pharmacotherapeutic recommendations were accepted. conclusion: detection of discrepancies in the medication reconciliation and deprescription process are effective and safe strategies that allow optimization of pharmacotherapy in fragile patients. the use of drugs such as nsaids (gastrolesive effect), the combination of drugs with cns side effects and hypotensive action (associated with falls) in elderly patients constitute situations of risk that should be reviewed in fragile patients, as an essential part of the clinical evaluation. please specify your abstract type: descriptive abstract (for projects) background and objective: there are no positions for clinical pharmacists at the hospital, so we are dependent on projects to be able to show how pharmacists can contribute in the clinical team. our aim in this project was to introduce pharmaceutical knowledge by implementing medication reconciliation and medication review in different hospital wards. we wanted to show that many patients have discrepancies in their medication lists during hospital stay and that some of the drugs or doses given can cause drug related problems for the patient. our final goal was to get the physicians to be more aware of these issues when treating their patients. design: the method used was based on the two first parts of the integrated medicines management. the pharmacist conducted a standardized drug interview with patients who prior to admission were responsible for their own drugs. for patients who could not be interviewed or were not responsible for administering their own drugs, a current medication list from relevant care level was obtained. the medication lists obtained were compared to the documentation in the patient's drug chart and discrepancies communicated to the physician. during the hospital stay, a medication review and monitoring was also conducted by the pharmacist. results were presented to the patients physician and discussed. results: a total of patients were included and of these % had c discrepancy identified by the process. the most frequent type of discrepancy was the use of a drug that was not registered on admission (omission discrepancies). other discrepancies were wrong dose, dosage or formulation and registration of a drug the patient didn't use. drug-related problems were discovered in % of the patients and the most frequent were use of anticholinergic drugs in elderly, interactions, lack of treatment and monitoring and too high doses regarding kidney function. many of the detected drug related problems results in change in medication, other times the physician addresses the problem to the gp. the physicians were surprised of the high numbers of discrepancies in medication lists and drug related problems discovered. almost all the physicians considered that the pharmacist could be an important part of the treatment team and they wanted the participation of the pharmacist to be permanent. conclusion: the project led to increased awareness of the importance of medication reconciliation and medication review and showed the importance of pharmaceutical knowledge in the treatment team. unfortunately this was not sufficient to create positions for pharmacists in our hospital. new projects will focus on pharmacists teaching interns to improve the reconciliation at admission. please specify your abstract type: descriptive abstract (for projects) background and objective: we aimed to assess the quality of fluoroquinolones (fq) prescriptions at the toulouse university hospital emergency department as part of significant increase in consumption. design: retrospective mono-centric study of fq prescriptions written to adult patients managed at the emergency department (february th, -march th, . a pair consisting of a biologist pharmacist and a clinical pharmacist has analysed them using tools provided by the centre de coordination de lutte contre les infections nosocomiales (cclin). various criteria (pertinence of prescription, choice of antibiotic, dosage, duration of treatment, method of administration…) were faced with the guidelines issued by the société de pathologie infectieuse de langue française (spilf). results: about files were examined, contained fq prescriptions for systemic use. the most frequently prescribed antibiotic was ofloxacin ( %) and the most frequent indications were urinary tract infections ( %). among the prescriptions of fq, the establishment of fq was justified in % of cases and the antibiotic chosen was always the most suitable. nonconformities of dosage and/ or treatment time were found in a quarter of cases. overall, % did comply with guidelines. the prescriptions, due to the particularity of emergency were still permormed probabilistic. however, a reassessment of them was scheduled for two-third of outpatients. conclusion: this study highlights the conformity of less than half of the prescriptions. this demonstrates that there are still actions to ensure the accuracy of fq prescriptions. and it is in this sense that this audit should be registered under the impetus of the committee on anti-infectives. it will raise awareness among doctors in the proper use of this family of antibiotics. please specify your abstract type: descriptive abstract (for projects) background and objective: the number of persons suffering from end-stage renal disease (esrd) is growing worldwide, mainly due to the aging of the population. esrd incidence has been increasing by - % per year for years. it is estimated that worldwide, more than . million patients with established renal failure are being treated with haemodialysis (hd). water for haemodialysis must meet the physicochemical and bacteriological compliance standards defined by the european pharmacopoeia. as a medicine, this water is placed under the responsibility of hospital pharmacists. addressed to hospital pharmacists, this methodology guide will enable them not only to validate controls of haemodialysis water as well as drug prescriptions for dialysis patients, but also to familiarize themselves with the best currently existing dialysis techniques and medical devices. we have tried to simplify and synthesize existing circulars and guidelines so as to render them more readily understandable for the pharmacist in charge of a haemodialysis service, and thereby help to ensure optimally safe treatment of haemodialysis patients. design: the themes developed in this guide are: • a review of the different existing dialysis techniques, • a review of the different sampling points for controls of hd water, • a review of the physicochemical and bacteriological standards of these controls according to the latest recommendations of the european pharmacopeia, and of appropriate conduct for exceeding established thresholds, • a review of the main international recommendations with regard to clinical signs of chronic kidney disease: anaemia, mineral and bone disorders (ckd-mbd), high blood pressure. • a review of the various medical devices used in haemodialysis and haemodiafiltration. results: the recommendations of good practices summarized in this guide are integrated perfectly adapted to the concept of quality assurance and its role in the accreditation process. they are focused on improving patient safety by harmonizing pharmaceutical haemodialysis practices in different dialysis centres. conclusion: these types of recommendations may be transposable to other pharmaceutical fields and/or be used as a training tool for pharmacy students or young pharmacy school graduates. the format of this guide makes it convenient, easy to use every day. it will be revised regularly to ensure the sustainability of quality plans. please specify your abstract type: descriptive abstract (for projects) background and objective: combination antiretroviral therapy (cart) has strongly improved disease control in hiv-infected patients. however, aging and comorbidities are becoming a major problem in this group of patients. most hiv-infected patients are treated with five or more medications, and harms by polypharmacy increase proportionally with number of medications. possible risks include: poor medication adherence and consequently inefficient care, increased risk of drug interactions and adverse events, with prolonged hospitalization. the problem is worsened when patients are of nonnative language and so their comprehension and adherence to drug therapy can be very poor, compromising efficacy. the hivig study is designed to evaluate the impact of the interventions promoted by the clinical pharmacist in the optimization and comprehension to personal drug therapy, favouring compliance, in a cohort of patients, hiv infected with comorbidities like cancer; the cohort includes a high number of non-native italian language individuals. design: hivig is a randomised, parallel groups clinical trial. in april the study protocol was approved by the local ethical committee, aviano. the project is scheduled to start in autumn . main objective: evaluation of the impact of a series of tools-''drug therapy setting interventions'' (dtsis) applied by the clinical pharmacist on a cohort pf hiv-infected patients with comorbidities, afferent for care at cro aviano. the treatment arm will be submitted to dtsis. dtsis interventions (treatment group) consist in: motivational interview, sharing and delivery of printed, explanatory material in the patient's native language, reconciliation of patients medications at hospital admission and at discharge; identification of potential risks due to drug-drug interactions; monitoring of compliance to drug therapy, and finally detection of adverse drug reactions (adrs) occurring in the course of care. the control group will undergo only to scheduled standard medical visits at cro. results: we expect to recruit a total patients for a -months period of follow-up. statistical analysis will be performed by intention-to-treat and by protocol. at cro aviano, the italian cooperative group on aids and tumors (gicat) has studied malignancies in hiv-positive patients since and has a leading role for studies conducted in italy (vaccher, ) conclusion: previous collected data from the previous trial performed at cro aviano (target-vig), showed a positive impact in the optimization of individual drug therapy and in the reporting of adrs. hiv has an enormous impact on life of infected patients and represents a priority issue for the entire community. we consider the method of dtsi, combined with a close monitoring of patients by means of telephonic motivational interviews, the best added value performed by the profile of the clinical pharmacist in optimizing drug therapy and personal awareness about medicines. please specify your abstract type: research abstract background and objective: the world health organization reports that ''one in four people in the world will be affected by mental or neurological disorders at some point in their lives. around million people currently suffer from such conditions, placing mental disorders among the leading causes of ill-health and disability worldwide». to review the evidences published about the roles and the impact of pharmacists in psychiatry. setting and method: literature review. a literature search was conducted using pubmed and the following terms: pharmacists, clinical pharmacy, pharmaceutical services, pharmaceutical care, pharmacy, mental illness and psychiatry from january st until june th . manual search was also conducted using selected articles. the selection of articles was based on abstracts. selected articles were reviewed, analysed and entered in impactpharmacie.org website according a standard operating procedure. relevant key data were extracted for each article including the type and the description of pharmaceutical interventions and descriptive and outcomes indicators with their results. no statistical analysis was conducted. main outcome measures: proportion of outcome indicators associated to pharmaceutical interventions with a positive impact in psychiatry. results: a total of articles were included. described pharmaceutical interventions included patient-pharmacist relationship ( ), medication reconciliation ( ), patient care needs assessment ( ), drug therapy assessment ( ), patient follow-up ( ), interdisciplinary work ( ), knowledge transfer ( ), competencies maintenance ( ). the impact of pharmacists interventions was studied using a total of indicators from which ( %) had outcome measures. of these outcome indicators, ( %) were positive, neutral and negative (knowledge transfer strategy). positive impacts of pharmaceutical interventions were identified in the following areas: morbidity ( ), patient adherence ( ), patients or clinicians satisfaction ( ), side effects management ( ), medication errors prevention ( ), mortality ( ) please specify your abstract type: research abstract background and objective: the world health organization reports that . million people die each year from cancer, an estimated % of all deaths worldwide and that there is a % increase in new cases of cancer expected over the next two decades. to review the evidences published about the roles and the impact of pharmacists in cancer. setting and method: literature review. a literature search was conducted using pubmed and the following terms: pharmacists, clinical pharmacy, pharmaceutical services, pharmaceutical care, pharmacy, neoplasms from january st until june th . manual search was also conducted using selected articles. the selection of articles was based on abstracts. selected articles were reviewed, analysed and entered in impactpharmacie.org website according a standard operating procedure. relevant key data were extracted for each article including the type and the description of pharmaceutical interventions as well as descriptive and outcomes indicators with their results. no statistical analysis was conducted. main outcome measures: proportion of outcome indicators associated to pharmaceutical interventions with a positive impact in cancer. results: a total of articles were included. described pharmaceutical interventions included patient-pharmacist relationship ( ), patient care needs assessment ( ), drug therapy assessment ( ), drug compounding/dispensing ( ), patient follow-up ( ), interdisciplinary work ( ), knowledge transfer ( ). the impact of pharmacists interventions was studied using a total of indicators from which ( %) had outcome measures. of these outcomes indicators, ( %) were positive, ( %) neutral and ( %) negative. positive impacts of pharmaceutical interventions were identified in the following areas: morbidity ( ), patient adherence ( ), patients or clinicians' satisfaction ( ), side effects management ( ), medication errors prevention ( ), mortality ( ), costs ( ) setting and method: literature review. a literature search was conducted using pubmed and the following terms: pharmacists, clinical pharmacy, pharmaceutical services, pharmaceutical care, pharmacy, myocardial infarction, acute coronary syndrome from january st until june th . manual search was also conducted using selected articles. the selection of articles was based on abstracts. selected articles were reviewed, analysed and entered in impactpharmacie.org website according a standard operating procedure. relevant key data were extracted for each article including the type and the description of pharmaceutical interventions and descriptive and outcomes indicators with their results. no statistical analysis was conducted. main outcome measures: proportion of outcome indicators associated to pharmaceutical interventions with a positive impact in myocardial infarction. results: a total of articles were included. described pharmaceutical interventions included patient-pharmacist relationship ( ), medication reconciliation ( ), patient care needs assessment ( ), drug therapy assessment ( ), drug compounding/dispensing ( ), patient follow-up ( ), interdisciplinary work ( ), knowledge transfer ( ). the impact of pharmacists interventions was studied using a total of indicators from which ( %) had outcome int j clin pharm ( ) : - measures. of these outcome indicators, ( %) were positive, ( %) neutral and ( %) negative. positive impacts of pharmaceutical interventions were identified in the following areas: morbidity ( ), patient adherence ( ), side effects management ( ), mortality ( ) and others ( ). conclusion: the role and the impact of pharmacists have been studied in myocardial infarction and % of outcome indicators used in these studies show a positive impact of pharmaceutical interventions. pharmacists should pay attention to these evidences to improve their practice, contribute to prevention or insure treatment of patients with potential or found myocardial infarction. hp-pc : impact of pharmaceutical care in vaccination: a review of literature please specify your abstract type: research abstract background and objective: the world health organization reports that ''immunization is the process whereby a person is made immune or resistant to an infectious disease, typically by the administration of a vaccine and a proven tool for controlling and eliminating lifethreatening infectious diseases and is estimated to avert between and million deaths each year. it is one of the most cost-effective health investments, with proven strategies that make it accessible to even the most hard-to-reach and vulnerable populations». to review the evidences published about the roles and the impact of pharmacists in vaccination. setting and method: literature review. a literature search was conducted using pubmed and the following terms: pharmacists, clinical pharmacy, pharmaceutical services, pharmaceutical care, pharmacy, vaccination and immunization from january st until july th . manual search was also conducted using selected articles. the selection of articles was based on abstracts. selected articles were reviewed, analysed and entered in impactpharmacie.org website according a standard operating procedure. relevant key data were extracted for each article including the type and the description of pharmaceutical interventions and descriptive and outcomes indicators with their results. no statistical analysis was conducted. main outcome measures: proportion of outcome indicators associated to pharmaceutical interventions with a positive impact in vaccination. results: a total of articles were included. described pharmaceutical interventions included patient-pharmacist relationship ( ), medication reconciliation ( ), patient care needs assessment ( ), drug therapy assessment ( ), patient follow-up ( ), interdisciplinary work ( ), knowledge transfer ( ), competencies maintenance ( ). the impact of pharmacists interventions was studied using a total of indicators from which ( %) had outcome measures. of these outcome indicators, ( %) were positive, ( %) neutral and negative. positive impacts of pharmaceutical interventions were identified in the following areas: cost ( ), errors ( ), morbidity ( ), patient adherence ( ), patients or clinicians satisfaction ( ) please specify your abstract type: descriptive abstract (for projects) background and objective: evaluating the appropriateness and effectiveness of the patient's medications by analysing prescriptions is pharmacist side work. bedside drug administration and computerised drug administration traceability (cdat) in nursing care plan (ncp) are nurse's one. however, in order to check adherence, efficiency and tolerance of a drug, pharmacist has to ensure that the patient takes the medication appropriately. therefor ncp could be a useful tool. the aim of this study is to evaluate the effectiveness of cdat, and if not, define causes of divergences with real life situation. design: • comparison between unused drugs remained in individual patients' seven daily pill dispensers (considered as not taken) which come back from the evaluated service to the pharmacy, and their cdat status completed by nurses (taken, not taken or no status) • two recorded data, each collecting a three-week period, separated by a period of discussion with nurses: first results presentation, analysis of divergences by taking into account their feedbacks, and actions to raise their awareness about the importance of cdat. • pill dispensers' cdat is correct only if all returned drugs' status in ncp is ''not taken''. results: during the first period (n = pill dispensers), . % of pill dispensers had an incorrect cdat status. on average, . drug per pill dispenser didn't have an appropriate status in ncp (taken or no status). major causes of divergences were the lack of time and insufficient human resources, the fact that they often are interrupted in the middle of this task, a software which isn't ''user-friendly'' and a deficit of information about the issue. corrective actions were implemented, prior to the second recorded data period, targeting human factor of divergences (oral and written reminders about cdat with didactic memorandum on computers). after awareness actions, results (n = ) were . and . respectively. conclusion: efforts about cdat have been done but not enough to observe a significantly improvement in short terms. ncp's level of reliability is not optimal yet and still dependent on nurses' practices. this study allowed us to strengthen the relationship between clinical service and pharmacy, and opens the way for further works particularly through corrective actions targeting material and organizational causes of divergences. please specify your abstract type: descriptive abstract (for projects) background and objective: in , our teaching hospital has participated to a worldwide survey (global point prevalence survey (global-pps)) aimed to explore antimicrobial consumption and resistance in hospitals. because broad spectrum antibiotics have to be followed with the attention of resistance prevention, we focused our analysis on these antibiotics in our hospital (carbapenems, piperacillin/tazobactam and amoxicillin/clavulanic acid). design: the survey was performed by pharmaceutical team (senior and resident) with help of microbiologists and referring physicians. all wards of the hospital were included. hospitalized patients treated with antimicrobial agent (j , j , j a, p ab, a , j ah, p b from the atc classification) prescribed at a.m. on the day of the survey, were involved. from those who were treated by carbapenems, pip/taz or amx ac, following data were collected: age, gender, weight, doses, indications (probabilistic? documented? and if documented microbiological data), and mention of stop/review date of prescription. results: the survey was carried out from april to june in wards. among the patients included, patients ( . %) were treated with antimicrobial agents. patients ( . %) were treated with broad spectrum antibiotics: ( . %) with amx-ac, with pip-tz, with imipenem and with meropenem. the mean age of patients was . ± . and the weight was . ± . kg. their prescriptions were concentrated in three types of wards: ( . %) in icu, ( . %) in medicine, ( . %) in surgery. moreover, we observe that bsa were used to treat ( . %) community acquired infections, ( . %) nosocomial infections, ( . %) used as medical prophylaxis, or surgical prophylaxis (n = , . %). in relation to the type of treatment: were empirical treatment (including prophylaxes) and were targeted treatments ( bacteraemia, joint and bones infections, cardiovascular system infection, urinary tract infections, lower respiratory tract infections, skin and soft tissues infections and others infections). finally, extended spectrum beta-lactamase (esbl) producing enterobacteriaceae and third generation cephalosporin resistant enterobacteriaceae non-esbl producing were targeted by bsa regimen. conclusion: in this survey, use of bsa is globally compliant to french guidelines and we identified no improper prescription: multidrug resistant bacteria infections, several diseases and empirical treatments with limited duration of regimen. this shows that control of the proper use of antibiotics especially those with a broad spectrum is efficient in our hospital and has to be continued. this has been made possible due to a multidisciplinary approach including physicians, bacteriologists and pharmacists. please specify your abstract type: descriptive abstract (for projects) background and objective: in , our teaching hospital has participated to a worldwide survey (global point prevalence survey (global-pps)) aimed to explore antimicrobial consumption and resistance in hospitals. from these results, we observed that sulfamethoxazole/trimethoprim (tmp/smx) was largely prescribed in our hospital. we focused then our analysis on these results with the attention of check of its proper use. design: the survey was performed by pharmaceutical team (senior and resident) with help of microbiologists and referring physicians. all wards of the hospital were included. hospitalized patients treated with antimicrobial agent (j , j , j a, p ab, a , j ah, p b from the atc classification) prescribed at a.m. on the day of the survey, were involved. from those who were treated by tmp/smx, following data were collected: age, gender, weight, doses, and indications (probabilistic? documented? and if documented microbiological data), and mention of stop/review date of prescription. please specify your abstract type: research abstract background and objective: in france, benzodiazepine (bzd) is frequently prescribed in elderly people (ep). long-term efficacy is often questioned, and treatment has to be regularly re-examined, especially in ep. in our geriatric day-hospital for assessment of frailty, a multidisciplinary team evaluates the patients and gives them preventative measures against the loss of autonomy. medication evaluation is part of these measures. the aim of our study was to evaluate the impact of a standardized intervention on the optimization of bzd treatment. setting and method: after a short interview and the delivery of an information booklet about bzd, patients were proposed an optimization of their bzd treatment (dosage reduction, occasional medication, switch to a short half-life bzd, or total discontinuation). patients were followed up monthly by a phone-interview over a -months period. main outcome measures: the main outcome measure was the prevalence of bzd optimized treatments after a months follow-up. results: patients were included. among them, % have been taking a bzd for more than years, and % were prescribed a long half-life bzd, which can be qualified as inappropriate in ep. % of the subjects were frail and % pre-frail according to the fried criteria. at the end of the study, % of the patients had their bzd treatments optimized, including % of total discontinuation. conclusion: in frail or pre-frail elderly population, a standardized intervention can be useful to improve bzd treatment. an extension to this intervention would be the creation of an organisation tasked with routinely monitoring the patients withdrawal over a month period. hba c and weight were significantly reduced by . ± . %, p \ . and . ± . kg, p \ . , respectively; systolic bp ( . ± . mmhg, p \ . ), diastolic bp ( . ± . mmhg p \ . ) and triglycerides ( . ± . mg/dl, p . ).genital-and urinary tract infections were reported by . % patients. any diabetic ketoacidosis case was reported. conclusion: sglt- inhibitors added to other oral antidiabetic drugs or insulin in patients with uncontrolled t dm significantly improved glycaemic control, reduced weight, blood pressure and triglycerides, and was generally well tolerated. in conclusion, sglt- inhibitors, appears to be an important addition to the therapeutic options for the management of type diabetes, particularly when used as add-on therapy. ( ). treatment safety takes part of the decision to undergo bariatric surgery. during multidisciplinary team meetings, the clinical pharmacist must rely on guidelines to limit drug-induced iatrogenesis. this review aims at assessing influence of bariatric surgery on the clinical impact and pk of cardiotropic drugs so as to document pharmacists' notifications. setting and method: literature review on medline- to may -with terms: cardiovascular drugs and bariatric surgery or malabsorption syndrome. related articles were reviewed. main outcome measures: pharmacokinetic or pharmacodynamic data and clinical impact of cardiotropic drugs. results: a total of titles, and abstracts when necessary, were screened for eligibility. after reviewing process, studies were included: nine concerning digoxin, five beta-blockers (bb) and one amiodarone. published studies varied in methodology: five case report, seven case control and three cohort studies. studies reported variations of digoxin plasmatic concentrations among patients versus , suggesting liquid oral form are preferred. no clinical event was notified. more the bb is liposoluble (propranolol), the higher the toxicity is, such as heart rate and blood pressure decreasing, with potential fatal outcomes. a case of amiodarone-induced hyperthyroidism is described after bariatric procedure showing an increase plasma concentration adjusted to weight. conclusion: while the impact on narrow therapeutic range drugs is documented, others cardiotropic drugs may cause serious patient injury justifying their monitoring. therefore, risk must be identified for all patients undergoing bariatric surgery to setting up closely therapeutic monitoring. further studies are still expected to lead to recommendations about posology and treatment withdrawal to improve patient safety. please specify your abstract type: research abstract background and objective: the issue of non-compliance to prescribed medical treatment has been reported to be a crucial problem in psychiatric outpatients. the aims of this study were to assess the extent of non-compliance in a cohort of psychiatric outpatients in malta and to investigate the applicability of using a -day multi-dose pill box in terms of practicality, ease of use and impact on compliance within this patient group. setting and method: the study was conducted at mount carmel hospital, a psychiatric hospital in malta. twenty outpatients were recruited by convenience sampling. the study was divided into two phases. during phase , patient compliance was assessed using the medication adherence rating scale (mars) survey and patients were administered part a of a questionnaire entitled 'assessment of the -day multi dose pill box'. this questionnaire evaluated the patients' opinion regarding the -day multi dose pill box before and after its use. in phase , the chosen patients were given a demonstration on how to use the -day multi dose pill box and the device was given to them to use at home for one week. after one week, part b of the questionnaire was completed and compliance was re-assessed using mars . main outcome measures: evaluation of adherence before and after use of the compliance aid device. results: of the patients recruited, were male and were female. the mean age was years (range - ) and the mean number of daily medications (range - ). upon initial scoring using mars, patients were adherent and patients were nonadherent. a higher adherence was observed in patients taking or more medications daily. ten patients accepted to move on to phase of the study and took the device home to use for one week. out of these patients, felt that the way they take their medication improved following use of the device and out of patients would consider buying the device since they found it practical and easy to use. statistical analysis of mars score before and after use of the device showed no significant improvement in compliance (p [ . ). there was no significant association between level of adherence and type of psychiatric condition (p [ . ). furthermore, results did not indicate increased adherence in patients who have a carer in-charge of their medication administration or in patients using a compliance aid device (p [ . ). conclusion: the use of a compliance aid device in psychiatric patients is challenging due to difficulty in establishing patient communication and motivation. the pharmacist is in a position to identify patients who would benefit from the compliance aid device. adrien borowik * , anne fratta, fabien hernandez pharmacy, ap-hp, armand trousseau paediatric hospital, paris, france please specify your abstract type: descriptive abstract (for projects) background and objective: enoxaparin, a low molecular weight heparin, is the most prescribed anticoagulation treatment in paediatric indications. however, the marketing authorization mentions that due to the lack of data, the use of enoxaparin is not recommended to children nor anyone weighing less than kg. thus, expert recommendations described specific dosage for the paediatric use: we aimed to compare these with our hospital practices. ( ), sofosbuvir/ledipasvir ( ), ombitasvir/ paritaprevir/ritonavir ( ), ombitasvir/paritaprevir/ritonavir + dasabuvir ( ), simeprevir + ifn ( ) . twenty-six patients ( %) were treated for weeks ( week pay-back policy). twenty pharmacists' interventions were carried out with an acceptance rate of %. the interventions included treatment adjustments due to drug interactions ( ), inappropriate treatment according to genotype ( ), duration of treatment ( ) and switch to a more cost-effective therapy ( ). seven pharmacists' interventions concerning treatment switch were applied ( %) resulting in a cost saving of € , . . all assessable patients ( ) have a negative serum hcv rna weeks after the end of treatment (svr = %) while patient died during follow-up (due to the disease). conclusion: the hospital pharmacist, as an active member of the multidisciplinary team, has an essential role in guaranteeing optimal care for hcv patients at the best cost. monitoring has also shown to be fundamental to evaluate the real world effectiveness of these drugs approved with surrogate endpoints. hp-pc : are hospital pharmaceutical staff educated on the criticality of thermosensitive drugs? camille castel, guillaume saint-lorant * please specify your abstract type: research abstract background and objective: in the use of thermosensitive drugs, the safety of patient care involves compliance with allowed temperatures. having the right information at time of care is essential. the aim of this study is to assess, within a french university hospital, pharmaceutical staff knowledge on the criticality of thermosensitive drugs and to educate them accordingly, including associated patient risks. setting and method: an assessment of knowledge using a questionnaire was led in january among pharmaceutical staff in a -bed hospital ( pharmacists, pharmacy residents, pharmacy technicians). evaluation criteria were: storage temperature of refrigerated drugs and frozen drugs, thermosensitive drug retention period after removal from the refrigerator, highest risk situation for a thermosensitive drug (t [ °c or t \ °c) and action to be taken during a temperature excursion. main outcome measures: to determine shortcomings in the management of thermosensitive drugs in order to adapt appropriate tools. results: completed questionnaires were collected. collected questionnaires included % from pharmacists (n = ), % from pharmacy residents (n = ) and % from pharmacy technicians (n = ). regulatory variations in storage temperatures of refrigerated and frozen drugs are known in respectively and % of cases. % of pharmaceutical staff are aware of thermosensitive drug retention periods after removal from the refrigerator and % of the highest risk situation for a thermosensitive drug (t \ °c). the measures to adopt during a temperature excursion are understood in % of cases. conclusion: this study highlights the lack of knowledge on the management and criticality of thermosensitive drugs and the lack of information available to pharmaceutical staff. dissemination of data and questionnaire reponses have been beneficial for the pharmacy department and have reduced inequalities in available information among pharmaceutical staff. subsequent to the study, thermosensitive drug management procedures have been revised. the deployment of this questionnaire is continuing via the university hospital intranet in order to train all health professionals in good patient care. please specify your abstract type: research abstract background and objective: temocillin is a beta-lactam antibiotic exclusively active against gram-negative pathogens. its use can avoid that of broad spectrum antibiotics, such as carbapenems, for the treatment of infections due to extended-spectrum beta-lactamase producing enterobacteriaceae. however, the absence of recommendations by learned societies on temocillin use could lead to misuse and the emergence of resistance. the aim of this study is to identify the role of temocillin in a french university hospital arsenal in order to limit ecological risks. setting and method: a retrospective study was conducted in a -bed university hospital. all adult patients having received at least days of treatment between june and april were included. data collected for the study were: age, sex, treatment indication (type of infection, identified pathogen, dosage and treatment duration), previous antibiotics and therapeutic outcomes. main outcome measures: the indicators chosen were: treatment indication, prescribed dose and treatment duration. results: two patients were included. in july , temocillin was used in a year old female as first-line treatment of intraperitoneal haematoma infection due to multiresistant klebsiella pneumoniae. prescribed at a dose of g twice daily by an infectious diseases specialist, treatment was continued at the same dose for up weeks with therapeutic success. in august , temocillin was used in a year old male for the treatment of bacteraemia due to multiresistant enterobacter aerogenes. previously treated by imipenem/cilastatin, temocillin was prescribed as second-line treatment at a dose of g twice daily by an infectious diseases specialist. treatment was continued at the same dose for up weeks with therapeutic success. conclusion: the dissemination of antibiotic resistance among gramnegative enterobacteriaceae continues to be an increasing threat for healthcare worldwide. within this context, temocillin could be an interesting alternative. determining the role of temocillin in a therapeutic arsenal is essential. our hospital considers temocillin as a ''critical antibiotic'' although its use is not exclusively limited to the new drug application. therefore, temocillin prescriptions are monitored permanently by infectious diseases specialists, microbiologists and pharmacists in order to improve the good use of this antibiotic and to optimise patient safety. please specify your abstract type: research abstract background and objective: drinkable solutions are more susceptible to deterioration and can lead to a potential risk for patient care. having the right information at time of care is essential. the aim of this study is to assess nursing staff knowledge in a french university hospital on the management of drinkable solutions to elaborate tools to help health professionals and to enhance equality of information in order to optimise patient care. setting and method: an assessment of practice using a questionnaire was conducted in may among a share of the nursing staff in a int j clin pharm ( ) results: completed questionnaires were collected. % of nursing staff replied that the period-after-opening is the same for all of drinkable solutions. this period is estimated at month in % of cases, weeks in % of cases and days in % of cases. % of nursing staff do not know how to store drinkable solutions after opening. the date of opening or the date of expiry after opening are specified on the medicine bottle in respectively and % of cases. only % of nursing staff have tools pertaining to the management of drinkable solutions. these observations led the pharmacy to create and distribute appropriate tools. storage methods for the drinkable solutions available in our hospital were collected directly from pharmaceutical laboratories. this information has been made available to nursing staff via drug control software (pharma Ò , computer engineering, paris). conclusion: this study highlights the lack of knowledge on the management of drinkable solutions and the lack of information available to nursing staff. in our hospital, the dissemination of appropriate data reduced inequalities in available information between care units. data will soon be integrated within the drug prescription software (mc kesson usv Ò , crossway, san francisco) in order to homogeneously train all health professionals in good patient care. please specify your abstract type: descriptive abstract (for projects) background and objective: medication reconciliation (mr) is a process which allows prevention of iatrogenic injuries during patient's hospitalisation and transfers. since , a clinical pharmacist has been integrated into the orthopaedic surgery care. he has performed mr at patients' admission. the aim of this study was to evaluate the impact of medication reconciliation performed by a clinical pharmacist. design: a prospective monocentric study was conducted on patients admitted in an orthopaedic surgery care (elective or unplanned surgery), during months. the clinical pharmacist established the best possible medication history (bpmh) from at least three sources of information (including patient interview when possible). then, it was compared to the admission medication order (amo) (from anaesthetists when elective or orthopaedists when unplanned). unintended medication discrepancies (umd) detected were discussed with prescribers in order to be corrected. epidemiological data, number and type of umd, therapeutic classes involved and the percentage of corrected umd were collected and their potential clinical impact was assessed. results: in this study, patients were included during months. elective surgeries were concerned in % of the cases. at least one umd was identified in patients ( %) (median age: . years old; male/female ratio: . ). of these, ( %) were older than years old. finally, umd were detected, being . by patient. main therapeutic classes concerned cardiovascular system ( %), nervous system ( %) and digestive system ( %). of the umd detected by mr, there were % of omissions, % of inappropriate dosing and % of renewal prescriptions stopped by the patient. finally, % of umd were corrected. of these umd, % were major errors (i.e. causing potential harm), % were significant errors (i.e. monitoring or intervention potentially required to preclude harm) and % were minor errors (i.e. without potential harm to the patient). conclusion: medication reconciliation process performed by a clinical pharmacist allows detection and correction of umd on half of patients in surgery care particularly on elderly patients. the high proportion of umd can be explained by the multiplicity of actors involved in medication management. health information technology could help to focus mr on patients at high-risk of adverse drug events. please specify your abstract type: research abstract background and objective: darunavir plus ritonavir (drv/r) have shown optimized results in simplification strategies (monotherapy (mt) or dual therapy (dt)) for selected hiv + in randomized clinical trials and real life experience. recent introduction of one pill drv plus cobicistat co-formulation (drv/c) may be particularly suited for both mt/dt allowing once daily administration optimizing dosage and adherence. the objective of our study is to evaluate efficacy and security of drv/c in mt and dt. setting and method: all hiv + adults with antiretroviral change to drv/c in mt/dt at a reference hospital in the northwest of spain were included in this retrospective study. a statistical analysis was performed using the spss v. .software. main outcome measures: epidemiological, clinical, antiretroviral regimen, serum creatinine, lipids and inmunovirological data (rna-hiv and lymphocytes cd ) were compared previous and after change to drv/c. results: hiv treatment-experienced patients have received drv/c in dt ( ) or mt ( ). . % were men with a mean age of years. main risk factors were: . % heterosexual, . % msm, . % injection drug users, . % mother-to-child transmission, . % transfusion in haemophiliac patient and . % unknown. cdc category distribution was . % a, . % b, . % c and . % unknown. overall mean nadir cd counts were . ± . cells/mcl. mean time since drv/c prescription to discontinuation or until analysis was . days [range - ]. . % drv/c mt were prescribed to patients with prior drv/r mt in order to simplify treatment and the mean time of the duration of these prior therapies were . years. in case of dt, . % were prescribed on patients with prior drv/ r + tc with a mean duration of . years. serum creatinine increases ( . vs. . ; p \ . ) and cd decrease ( . vs. . ; p = . ) when patients move to drv/c. no significant change in the other analytical parameters and all patients maintained undetectable. patients discontinue drv/c due to intolerance and inability to swallow in each case. conclusion: this preliminary study concludes that drv/c in mt or dt is efficacy (no viral rebound) and safety. although an increase in creatinine was observed, it would not be considered clinically significant. of note, lymphocytes decreased significantly and it will be important closely monitored to check that maintain effectiveness during the follow up. hp-pc : developing clinical pharmacy in emergency department setting up a medication reconciliation process marion collignon *, , antoine gantier , florent lapacherie , hélène dewaele , laura foucault , anne-laure raso , emmanuel cirot , said laribi , xavier pourrat pharmacy, emergency department, chru tours, tours, france please specify your abstract type: descriptive abstract (for projects) background and objective: in emergency department (ed), if a drug related problem (drp) happens at the patient admission, the risk is the error remains until discharge. one part of drp may be avoided with using medication reconciliation (mr). the objective of this study was to evaluate the feasibility of setting up a medication history (mh) of patients in ed in an acceptable lap of time before they were transferred in another unit or discharged. design: a months prospective study was conducted in ed in a university hospital in france. two junior pharmacists coached by a senior pharmacist, after a months training for mr, were in charge of the data and mh collection. for all patients, we collected age, mh according to number of sources, discrepancies identified, adherence to treatment (according to the social security questionnaire), type of sources. mh were established according to community pharmacies, patients, previous electronic patient files, prescription sheets, patient's family, packs of pills and to the general practitioner (gp). for patients from long term care facilities (ltcf), the mh was established only by communication with the ltcf. then, the current prescription was compared with the home medication regimen. mh and discrepancies (omitting medication, incorrect dose, ambiguous name) were recorded in the electronic patient files to be available during hospitalization. because ed does not have a pharmaceutical review of prescriptions, only major discrepancies were transmitted to physicians. results: we collected mh ( from ltcf), with a sex ratio of . and a medium age of . years old. it represented an average of . mh per day or min per mh. among patients who did not come from ltcf, sources used by pharmacy students were patient's community pharmacy ( , % of cases), patient ( , %), previous electronic patient file ( , %), prescription sheets ( , %), call to gp ( , . %), gp mail ( , . %), patient's family ( , . %), packs of pills ( , . %), community nurse ( , . %). finally, patients ( %) had been hospitalized, others were discharged. we analysed mh for patients: at least one drp occurred for patients ( %). among patients, ( %) had an immediate pharmaceutical intervention because of the risk due to discrepancy. among patients who did not come from ltcf and who could communicate, were good adherent to treatment ( %). conclusion: this study highlights the great interest of the mh by pharmacists at ed, which avoids many drp. the presence of pharmacists in ed contributes to maintain a safe environment for medication and to assist prescribers in the continuity of treatment between home and hospital. spending min by mh, we identify one drp every min. nevertheless, it could be benefit to develop this activity because of the satisfaction of the emergency physicians. currently, mr is the first step to develop clinical pharmacy in the ed. please specify your abstract type: research abstract background and objective: emerging evidence in the literature suggests a high prevalence of suboptimal vitamin d (vitd) and an association between lower serum levels and higher mortality in cancer. the objective of this study was to quantify vitd deficiency in patients after surgery for head and neck cancer, and to determine the effect of one cholecalciferol intramuscular dose. setting and method: intervention study with a follow-up period of months (november -february ) performed on patients followed by the nutrition support unit after surgery for head and neck cancer. demographic and physiopatological data, including admission diagnosis, age, gender, calcium, magnesium and phosphate were collected. nutrition screening by conut index was carried out. a single intramuscular dose of . ui cholecalciferol (vitamine d bon Ò ) was administered to vitd-deficient patients and serum -hidroxy-vitamin d (s ohd) records after the administration, including primary carés records after discharge, were evaluated (reference range - ng/ml). main outcome measures: s ohd (\ ng/ml: deficiency; - ng/ml: insufficiency; c ng/ml: sufficiency). results: data from patients with a mean (sd) age of . ( . ) years were collected (males: %). the admission diagnosis was laryngeal squamosis cell carcinoma (n = ), glottis carcinoma (n = ) and nasopharynx, tongue and skull base cancer (n = ). at baseline, , and patients were considered have high, medium and low risk of malnutrition, respectively. the mean (sd) serum ohd was . ( . ) ng/ml (deficiency: patients; insufficiency: patient). despite the role of vitd in mineral balance, calcium, magnesium and phosphate mean (sd) serum levels were between the normal range . ( . ) mg/dl, . ( . ) mg/dl, and . ( . ) mg/dl, respectively. s ohd records were available week after the administration (mean (sd) = . ( . ) ng/ml). and patients still showed deficiency and insufficiency, respectively. primary care's records from patients were available after discharge ( . , . and . ng/ml). conclusion: poor nutritional status and high prevalence of suboptimal vitd in patients with head and neck cancer were found. a single dose of intramuscular cholecalciferol slowly raises s ohd. follow-up after discharge is essential to evaluate the achievement of the therapeutic objective. setting and method: this is a descriptive retrospective study. it took place in a teaching hospital. antifungal broad spectrum therapies (liposomal amphotericin b, caspofungin, micafungin, posaconazole, voriconazole) used between st january and st december were included. main outcome measures: indications, type of combination and patients specifications were analysed. results: only patients ( . % over all patients receiving antifungal therapy; n = / ) received an antifungal combination therapy during the study period. majority of patients presented risk factors: % of patients had an organ transplant (n = ), % suffered from malignant blood disorders (four acute myeloid leukaemia, two chronic lymphoid leukemia, one non-hodgkin's lymphoma, one hodgkin's lymphoma and two refractory anaemia with excessive blast), % suffered from solid cancer (one lung cancer and one breast cancer) and % suffered from chronic obstructive bronchopneumopathy (n = ). antifungal combination therapy was used against invasive aspergillosis in % of cases (n = ) among which complications such as brain and cardiac impairment were found in % of patients (n = ). the six remaining patients ( %) were co-infected with candidiasis for three patients and mucomycosis for three patients. voriconazole was logically the most used in combination, and just one patient received oral form. it was in majority prescribed with caspofungin ( %, n = ) and intravenous liposomal amphotericin b ( %; n = ). combination including liposomal amphotericin b and caspofungin (n = , %) or posaconazole with liposomal amphotericin b (n = ) were found in our study. five patients deceased during the hospitalization of the fungal infection ( %) which shows the gravity of these cases. majority of patients ([ %) was treated less than days with these combinations. conclusion: this retrospective study shows that patients who received antifungal combination therapy were mostly immunocompromised, co-infected or experienced a severe infection with severity factors. the antifungal combination was in majority initiated because monotherapy failed to cure the patient. all prescriptions were discussed with a mycologist who tried to shorter the combination treatment duration. this multidisciplinary approach is a major key in the process of these type of treatments. please specify your abstract type: research abstract background and objective: because of its broad spectrum and the risk of resistance mutation, delivery of posaconazole is nominative and controlled by hospital pharmacists. the aim of this work was to describe the use and pharmaceutical follow-up of posaconazole tablets over a -months period. setting and method: this is a descriptive retrospective study over a -months period from november to may in a teaching hospital. all patients who received posaconazole tablets were included. main outcome measures: indications and dosage were reported. results: patients were included in the study. posaconazole tablets were used for: fungal invasive infection prophylaxis in case of stem cell transplantation ( %; n = ), fungal invasive infection prophylaxis if a chemotherapy was started to treat a chronic myeloid leukaemia or a myelodysplasic syndrome ( %; n = ); treatment of invasive aspergillosis ( %; n = ); mycetoma ( %; n = ); zygomycosis or mucormycosis while patient had renal impairment ( %; n = ). all of these indications were approved for posaconazole (marketing authorization and local guidelines). only patients ( %) received a loading dose ( milligrams twice a day) as recommended in approval authorization. posaconazole blood levels were monitored by pharmacologists: % of patients (n = ) did not need dosage modulation which shows that variability is not so important. but three patients did not have any assay to monitor posaconazole blood concentration. patient received a loading dose and was switched to intravenous voriconazole after icu transfer. patients needed increase and/or reduction dose to obtain optimal posaconazole blood levels. conclusion: this study describes the use and the follow-up of posaconazole tablets during the first months after its approval in europe. all indications are approved for posaconazole but this analysis shows that pharmacist have to remind the necessity of a loading dose. dosage can be adjusted according to assays results. please specify your abstract type: research abstract background and objective: due to the acute, hectic environment in a fast-paced work-flow emergency department (ed) it is a challenge to verify the correct and updated medication list for the admitted patients. when performing medication reconciliation (mr) in this environment, these challenge has to be taken into account and prioritizing patients for mr could be necessary. the objective of this study was to identify risk factors correlated to clinical relevant medication discrepancies (crmds) among patients admitted to ed, and based on these revealed risk factors, develop a model for prioritizing patients for mr in the fast-paced work-flow at the ed. setting and method: patients continuously included at the ed, diakonhjemmet hospital (dh), oslo, norway. trained pharmacists and emergency nurse conducted mr. patient specific factors and revealed crmds, between hospital admission records and information about prehospital medication use, were recorded. binary linear regression was used to identify risk factors correlated to crmds. the prioritizing model was built using statics and clinical experiences. main outcome measures: what risk factors is correlated to crmds and how precisely do the prioritizing model classify the patients as high-and low-risk patients. results: % of the patients had c crmd. the following were identified as risk factors correlated to crmd and were suitable for inclusion in the prioritizing model; gender (woman), age (c ), c admission to hospital last months, admission causes; surgical, malfunction, cancer. the model correctly classified . % of the patients with crmds as high risk. further, . % of the patients with crmds were classified by the model as low-risk patients (false negatives). the model classified . % of the patients who did not have a crmd as high-risk patients (false positives). conclusion: the prioritizing model developed can be helpful in identifying what patients are at increased risk of having crmds in the fast-paced work-flow at the ed. identifying these patients will result in using the resources available in the ed in the most efficient manner and utilizing the full potential of the mr method. as a consequence of this, patient safety would be increased. hp-pc : intravenous potassium chloride: quick audit of prescribers knowledge and recommendations regarding safe practice and proper usage asmaa damou * , vincent zaugg, martine postaire please specify your abstract type: descriptive abstract (for projects) background and objective: our hospital has established methods that try to ensure the safe use of high alert medications. intravenous potassium chloride (kcl) was the subject of preventive measures: separation of different dosages (kcl . % vials reserved for paediatric services and kcl % vials reserved for adult services); creation of an advice record for doctors and nurses; specific labelling of storage areas; double-check the prescription and administration. the objective of this study was to evaluate the knowledge of the safe use of intravenous kcl by prescribers. design: multiple-choice questions were developed for prescribing recommendations established by our hospital with the collaboration of the doctor who is chairman of the central committee of vigilance and risk associated with care (cvris). a link to the online survey was sent by email to physicians practicing in departments (eight paediatric services and six adult services). the results were extracted and interpreted in excel Ò . results: % of physicians responded to the survey ( medicine residents, hospital doctors). in paediatric services, % of doctors know that only the kcl . % should be used. % know the unit of prescription to be used (mmol/kg or meq/kg), and % know that the maximum recommended infusion rate is . mmol/kg/hour (or mmol/kg/h in recovery unit). in adult services, the recommended maximum rate of infusion ( g/h) is known to all prescribers, but only % know that the concentration of kcl must be less than g/l. % of paediatric doctors say that their kcl prescriptions are checked by a second doctor, but the answers in the same service area are sometimes contradictory. in adult services, only % of physicians say that the prescriptions are double-checked. the information brochure available on the intranet of the hospital is known by % of prescribers. the response rate of physicians to the survey was satisfactory. therefore, the recommendations are rather well known by prescribers, except the value of the maximum concentration of infusion for adults. the results of this audit were returned to the doctors, accompanied by a reminder stating the need to double-check the prescription and the existence of advice records on the website of the hospital. conclusion: this audit is an approach to increase the safety of the use of high alert medications. it will be completed a second time, by an evaluation of prescriptions collected and the storage conditions of potassium chloride in the care units. please specify your abstract type: research abstract background and objective: data listed behind each unit dose of a primary packaging of a pharmaceutical product are essential for a safe identification for the patient. however, the last medical services of the lausanne university hospital where nurses remove the solid form drugs (sfd) from their blisters when they prepare in advance the week container were in the vaud's prisons. the aims of the study were: ( ), quetiapine ( ) and ibuprofen ( ) and were psychotropics ( . %). part . the four data identified as essential: brand name, dosage [mg], batch number, expiration date. the sfd unit doses were classified as green when the blister included four data, yellow with two or three and red with less than two. of the sfd in cupboards, were green ( %), yellow ( %) and red ( %); an infovigilance was sent to each manufacturers. part . potential barriers identified: trays' sizes and space in drug's cupboards; preparation time to cut versus to remove the blisters; risks of self/hetero-aggression with pre-cut blisters; drugs packaged in bulk; multidose liquid medications. using containers larger than is usual was rarely necessary; space in cupboards was sufficient. the preparation time gradually decreased during the study. ingestion or aggression with pre-cut blisters was considered as limited, based on literature and experiences of two others prisons (geneva; lyon). for bulk sfd and multidose liquid drugs: proposals to the pharmacy to store some alternatives blistered sfd; blistering expensive bulked drugs; availability of the entire package delivered to inmates. the pilot phase was initiated in may . conclusion: a majority of inmates takes a drug treatment. half of sfd unit dose is identifiable (trade name and dosage) but an effort from manufacturers would better secure the drug supply chain. the study of the barriers helped to further implement the pilot phase. since early , none of the five prisons medical wards are removing the blisters and no incident was reported. please specify your abstract type: research abstract background and objective: nefopam is a widely used antalgic in hospital. its use is contraindicated in the epileptic patient as it results in lowering the epileptogen threshold and is likely to trigger epileptic seizures. the clinical pharmacist should systematically warn the prescriber against this contraindication when analysing prescriptions. following the onset in our establishment of an epileptic condition in a patient treated with nefopam, who had not been subject to any pharmaceutical intervention (pi), we set about analysing the validation practices regarding this contraindication and possibly implementing actions designed to improve those practices. setting and method: retrospective collection over a period of months of prescriptions for patients hospitalized in hospital beds with clinical pharmacy service (associating med-reconciliation, checking prescription according to medical file and participation to medical rounds): orthopaedic surgery, hepatic-gastro-enterology, general surgery, liver transplant and chest surgery. records of patients with nefopam prescription associated to medication belonging to the therapeutic class of antiepileptics were consulted with a view to finding cases of epilepsy. the pharmaceutical alerts were extracted from the pharmaceutical software. main outcome measures: number of epileptic patients treated with nefopam, number of pharmaceutical interventions issued when prescribing nefopam in epileptic patients. the study focused on , patients. ( . %) of them were prescribed nefopam, and ( . %) of them were prescribed nefopam associated to medication belonging to the therapeutic class of antiepileptics. after analysis of the patients' records has shown that of them were really epileptic. only pi's were effected ( . % of problematic prescriptions), and ( %) of them had an immediate prescription change. . % ( / ) of the patients have a pi in medicine services compared to . ( / ) in surgery services (p \ . ). the results of this study show that % of the contraindications related to the use of nefopam in epileptic patients are not reported to the prescriber. these results will be presented to our pharmacists so they can take them into account. subsequently a new study will be conducted to measure the relevance and efficiency of this program. hélène dewaele * , anne-laure raso, emmanuel cirot, marion collignon, laura foucault, xavier pourrat please specify your abstract type: descriptive abstract (for projects) background and objective: medication reconciliation (mr) has been demonstrated to reduce drug-related problems in inpatients. in our university hospital, mr has been performed for beds for years at the same time as prescriptions review. the aim of this study was to assess the impact of mr on pharmaceutical interventions (pi) during prescriptions review. design: a -month prospective study in orthopaedic surgery, hepato-gastro-enterology, general surgery, liver transplant and chest surgery was conducted. during medication review all pis were collected and those related to mr (rpi) were identified. thereafter for each patient we collected age, type of hospitalization unit (med or surgery) and for pis the drug associated and its acceptance by the medical team. results: during the study patients had a daily prescription review. patients ( %) had at least one drug-related problem. lines of prescriptions were mentioned to have at least one rpi. rpi represent % of drug-related problems. ( %) discrepancies were corrected by prescribers. the age of the patient was significantly different between patients with rpi (mean age: years old) and with pi (mean age: years old; p \ . ). the type of unit did impact the percentage of prescriptions with drug-related problems (medicine: . ; surgery: . ; p \ . ), the rate of corrected pi (medicine: %, surgery: %, p \ . ), but did not impact the rate of corrected rpi (p = . ). in surgery units the rate of corrected rpi ( / ) is significantly higher than corrected pi ( / ; p \ . ). medicines belonging to the four classes of: digestive and metabolism system, blood and blood flow, cardiovascular system, neurological system represent more than % of all the medication concerned by a resolved pi or rpi. the proportion of medicines from the digestive and metabolism class is the only class among those four that is not significantly different between resolved pi and rpi. conclusion: mr highlights a large number of discrepancies in inpatients. a modification of prescriptions due to mr occurs in % of the patients. in surgery units, these rpi are more frequently taken into account than drug-related problem warned by pis. indentifying patients for whom mr has the bigger impact could help us to reinforce our actions. please specify your abstract type: research abstract background and objective: diabetes is very frequently causing cardiovascular complications, thus impairing various systems and organs. therapy for these multiple conditions has to be revised and improved constantly. the aim of this closed retrospective study lead in bucharest emergency clinical hospital was the assessment of some of the diabetes mellitus (dm) complications and the related medication. setting and method: data was collected from cardiology, neurology, gastroenterology, internal medicine wards from bucharest emergency clinical hospital. only patients diagnosed with type dm were included in the study. there were analysed records from patients aged - of whom were men, following the presence, signalling and monitoring of diabetic nephropathy and arteriopathy. main outcome measures: we investigated the relationship between diagnosis and/or biochemical signs of kidney disease (serum urea, serum creatinine levels), diagnosis of arteriopathy, and the drug therapy administered in the respective cases. we also assessed the sex and age distribution of the patients diagnosed with diabetes mellitus and facing at least one of its complications. results: kidney disease, as a dm complication, was present in % of cases, patients aged - , of whom % were men. patients received diuretic treatment, of them being given hydrochlorothiazide, contraindicated in dm because of its hyperglycaemia-inducing effect. of the patients, had high serum urea levels ([ mg/dl), had high levels of serum creatinine ([ . mg/dl), and presented risen levels for both, but only were also diagnosed with kidney disease. patients with kidney disease were given furosemide, known for altering the renal function. circulatory failure was found in % of the patients, aged - and % of subjects, aged - , had both diabetic complications. conclusion: the present study emphasizes the role of the clinical pharmacist in adapting the medication of the diabetic patient, an inappropriate pharmacotherapy worsening dm complications. this is essential especially for elders, where polypathology and polymedication lead to a significant increase of dm complications risk. hp-pc : epileptic seizure after treatment with thiocolchicoside: discussion about a case report valérie dobremez *, , adeline martin-dupray , jacqueline berlioz , pierric giraud pharmacy, neurology, centre hospitalier annecy-genevois, metz-tessy, france please specify your abstract type: descriptive abstract (for projects) background and objective: thiocolchicoside is a semisynthetic derivate of naturally occurring colchicoside, which is largely used in humans as a centrally acting muscle relaxant. this compound also has anti-inflammatory and analgesic effects. the objective of this work is to report a recent case of serious adverse effect of thiocolchicoside occurring in context post traumatic brain damage without sequalae. design: a -year-old woman suffered from headaches and neck pain since days, she was treated with thiocolchicoside. she took mg in the evening and mg the next morning. five generalized tonic-clonic seizures, without recovery of normal consciousness between seizures, have occurred suddenly - min after the second administration. the patient was admitted to intensive care unit in order to control the epileptic seizures. a status epilepticus was diagnosed requiring intravenous drugs with clonazepam, phenobarbital and propofol. the patient was controlled and transferred in neurologic unit in order to complete paraclinical investigations. its main antecedent was a severe head injury at the age of years following a public road accident. the brain scan revealed an old frontal hypodensity. rest of etiological assessment was negative (lumbar puncture, no infectious disease), numbers were normal. the definitive diagnosis was a status epilepticus on post-traumatic sequelae, sensitized by taking a proconvulsant drug. a treatment with levitiracetam was initiated at mg twice a day. outcome was favourable with no recurrence months later, a recommendation was requested to pharmacovigilance. results: the muscle relaxant activity of thiocolchicoside results of an agonist action on glycinergic receptors located primarily in the brain stem and spinal cord. however, thiocolchicoside also acts as an antagonist of the gaba-a receptor (mainly located in the cerebral cortex), this pharmacological action can cause a proconvulsant effect. epilepsy is a very rare adverse effect, only few cases have been reported in literature. the epileptogenic activity of thiocolchicoside occur mainly in patients with a history of epilepsy, acute brain injury or possible blood-brain barrier disruption. the chronology is consistent with the responsibility of the drug as a promoting factor. pharmacovigilance retains after analysing drug causality. conclusion: the case history indicates that thiocolchicoside has a powerful epileptogenic activity. thiocolchicoside can precipitate seizures in predisposed patients, and that its use should be avoided in patients with brain diseases (and therefore lower seizure thresholds) or blood-brain barrier disruption. pharmacists could warn physicians and should verify the absence of notable history before dispensing thiocolchicoside. hp-pc : acute exacerbation generalized myasthenia after red yeast rice use: a case report valérie dobremez *, , amélie serra , déborah grosset-janin , jacqueline berlioz , aymeric dopter , jean-henri ruel pharmacy, neurology, centre hospitalier annecy-genevois, metz-tessy, nutrivigilance, french agency for food, environmental and occupational health and safety, paris, france please specify your abstract type: descriptive abstract (for projects) background and objective: many drugs can induce acute exacerbations or reveal myasthenia gravis. self-medication or complementary and alternatives medicines expose patients. the objective of this work is to report a recent case of acute exacerbation of myasthenia gravis because of a dietary supplement use. design: intermittent vertical diplopia and ptosis of the left eye settled in a -year-old man. its main antecedent is hypertension treated with perindopril. the neurovascular origin was ruled out. the electromyogram (emg) found a significant decrement ( %) of a postsynaptic block in the tongue and right orbicularis muscle. acetylcholine receptor-antibodies were positive. myasthenia gravis was diagnosed (osserman score / ) and the patient was treated with pyridostigmine. the identification of carotid atheroma required a treatment with a statin that the patient refused. he preferred a cholesterol lowering dietary supplement, containing red yeast rice. six days later, he was hospitalized for an acute decompensation of myasthenia with bilateral ptosis, oculomotor paresis, drooping head, int j clin pharm ( ) : - chewing trouble and dysphagia (osserman score / ). the patient is treated with high-dose intravenous immunoglobulins then corticosteroids. the dietary supplement is stopped. an opinion was requested to the clinical pharmacist of neurology. the osserman score gradually increases to / . results: red yeast rice contains a range of compounds known as monacolins, of which monacolin k-renamed lovastatin, which was found to be an inhibitor of cholesterol synthesis and the progenitor of the statin family. a literature review has highlighted the responsibility of statins in acute exacerbations or reveal myasthenia gravis occurrences. in this case, the chronology is consistent with the responsibility of red yeast rice. the case was reported to the french system of nutrivigilance, which retained after analysing a probable intrinsic imputability score. conclusion: dietary supplement with red rice yeast are not recommended in case of myasthenia gravis. this is the first case of acute decompensation of myasthenia recorded with red yeast rice in the french system of nutrivigilance. multidisciplinary collaboration (neurologists, clinical pharmacist) has optimized the patient management. fanny durand * , camille lambert, antoine dupuis please specify your abstract type: descriptive abstract (for projects) background and objective: development of computerized prescription highlights the need to harmonize pharmaceutical analysis practices. the aim of this study is to analyse the antibiotics prescriptions in the treatment of urinary tract infections, to develop a pharmaceutical validation tool. design: a prospective observational study was conducted for one week, in care units. pharmacists, interns, and pharmacy students were trained on spilf (french society of infectious pathology) recommendations, on pharmacist's role in the management of urinary tract infections, and on the data collection. all patients with antibiotic prescription for urinary tract infection were included. some data were collected: reason for hospitalization, clinical signs, results of susceptibility testing, risk factors for complications (organic or functional abnormality of urinary tract, male, pregnancy, elderly, severe immunodeficiency, severe renal impairment) and signs of severity (severe sepsis, septic shock, interventional surgical drainage). then, the treatments prescribed to the patient, probabilistic on the one hand and documented on the other hand, were compared to spilf recommendations. finally, during a multidisciplinary meeting (pharmacist, expert in infectious diseases), we selected the relevant pharmacist interventions. results: twenty-three patients were included ( women, men), % had a urinary catheter. . % of prescriptions were concordant with spilf recommendations: probabilistic and documented treatment, and duration. among the non-conforming prescriptions, nine pharmacist interventions have been formulated: four prescriptions did not specify the duration of treatment, one antibiotic was prescribed on an insufficient period, two cases of severe acute pyelonephritis without prescription of aminoglycoside, one prescription was not reassessed according to results of susceptibility testing, one pregnant woman with urinary colonization without clinical signs, was treated before obtaining results of susceptibility testing. three cases of poor management are identified: two cases which treatment began only after results of susceptibility testing (a urinary tract infection linked to care, an acute pyelonephritis with complication risk), and a cystitis treated with nitrofurantoin while the germ was resistant. conclusion: a synthetic tool was created. there are three elements for helping pharmaceutical analysis: the questions to ask oneself facing a prescription of antibiotic for urinary tract infection, a flowchart to identify the recommendation adapted to the case, and finally a summary table showing spilf recommendations. this tool will be distributed and evaluated. hp-pc : off-label use of rituximab in refractory antisynthetase syndrome (as) through a long-time experience in a neuromuscular diesases center lise durand * , carole metz, patrick tilleul, helga junot pharmacy, gh pitié salpêtrière, paris, france please specify your abstract type: descriptive abstract (for projects) background and objective: as is an idiopathic autoimmune inflammatory myopathy, characterized by presence of antisynthetase antibodies: anti-jo , anti-pl , anti-pl . patients are usually first treated by corticosteroids (cs) or immunomodulating drugs. rituximab (rtx) has become another option for refractory as, supported by few uncontrolled studies . because of its off-label use, our hospital pharmacy has implemented a controlled drug delivery. this work assesses a -years follow-up of patients treated by rtx and the resulted drug costs. design: patients registered in our database since december who received c injections of rtx to treat as, were analysed to describe their eligibility criteria, conditions of management and the clinical and biological effects of the treatment (creatine kinase (cpk) used as biomarker). patient files were consulted to collect all individual data and pharmaceutical software was used to review deliveries. drug costs were also reckoned based on prices from french health insurance. results: for months, patients (median age (min-max): ( - ), % women) have been treated with rtx for refractory as, the majority with anti-jo antibodies ( ). all patients suffer from muscular and lung affections, particularly interstitial pneumonia. many are also living with arthropathies ( ) or cutaneous disorders ( ). cardiac involvement is seldom ( symptomatic patients). the mean age of diagnostic is . years and the mean treatment period is . years. the common treatment is g at day (d ) and d , then g all months. before rtx treatment, seven patients received c other drugs such as cs ( %), azathioprine ( %), methotrexate or mycophenolate mofetil ( %). prednisone and azathioprine are also prescribed with rtx respectively for and %. treatment is associated with cures of intravenous immunoglobulins for four patients. to date, median number of administrations per patient is ( - ), d and d included. all patients have presented positive effects on both clinical and biological markers, mainly during the first months after treatment induction. wilcoxon tests show a significant difference in cpk level between d and m , also between d and the last known result. today, three complete remissions are specified in patient file; only one hepatitis b virus reactivation is reported. since , budget impact due to drug cost amounts to €. conclusion: whereas the use of rtx is controverted for treatment of all types of myopathy, as could have one of the best response . our cohort shows real clinical results and positive effect on usual biomarker. our experience demonstrates the safe and successful use of repeated administrations in refractory as. however, there is a need for further controlled studies to assess the efficacy/safety of rtx and to define its place in the strategy in view of its cost-effectiveness ratio. the pharmaceutical controlled drug delivery has to be continued to supervise, support and document its proper off-label use. please specify your abstract type: descriptive abstract (for projects) background and objective: as a part of the national patient safety program, the northern norway regional health authority are implementing new procedures for medication reconciliation (mr) in hospitals in the region. the procedure defines that mr is the doctor's responsibility and describes how it should be performed. the aim of this study was to investigate whether the implementation of the procedure reduces medication discrepancies (mds) in the charts at bodø hospital. and . % ( / ) of the patients died before discharge. parenteral nutrition was administered an average of . days ( % ci . - . ), of which . ( % ci . - . ) were with ipn. previous spn had been administered in . % ( / ) of the patients. before beginning ipn, the average triglycerides level was . mg/ dl ( % ci . - . ) but at the end of the ipn it was . mg/ dl ( % ci . - . ), which lead to a mean reduction of . mg/dl ( % ci . - . ; p = . ). regarding to the total amount of lipids provided with parenteral nutrition, with ipn there was a mean reduction of . g ( % ci . - . ; p = . ) comparing to those administered with spn. conclusion: usage of ipn in critically ill patients with htg permits to adjust parenteral nutrition formulations to meet specific nutrition needs, enables to reduce the total amount of lipids administered and, therefore, it allows to significantly decrease triglycerides levels. jennifer a. esteban gonzález * , elisabet nogué pujadas, angels andreu crespo, xavier bonafont pujol, nuria romero pascual please specify your abstract type: descriptive abstract (for projects) background and objective: the incidents involving patient misidentification (pm), or wrong patient medical errors (wpme), are medication errors (me), near-miss or close-call situations which can pose a considerable threat to patient health. pm may be under-reported due to the unawareness of the error or the difficulty of identifying them. the aim of this study is to describe the incidence and categories of wpme in a university hospital. design: observational, retrospective analysis of the voluntary reported wpme in the pharmacy database since march until june . these were classified in prescription, transcription, dispensing, administration and drug system errors. in addition, the national coordinating council for medication error reporting and prevention (nccmerp) taxonomy was used for classifying me according to the severity of the outcome. results: of me registered, of them were wpme ( . %). . % of them were due to prescription errors, which consist on wrong labelled medical orders, intermingled patient prescriptions or patient misidentification in computerized physician order entry (cpoe). the administration errors supposed a . % of the total amount of wpme and dispensing errors were . %. % of wpme were transcription errors, which occurred previously to the implementation of cpoe, and the remaining . % were system errors after cpoe. the wpme reported took place in the hospitalization wards ( . %), pharmacy ( . %), outpatient services ( . %), intensive care unit ( . %) and day-care hospital unit ( . %). . % occurred at working days and . % at the weekends. wpme were notified by pharmacists ( . %), nurses ( . %) and physicians ( . %). referring to the classification according to nccmerp, . % of wpme didn't reach the patient (category b) whereas . % reached the patient but didn't cause harm (category c) and . % required patient monitoring (category d). the remaining wpme ( . %) caused harm to patients and required medical intervention (category e). finally, in int j clin pharm ( ) : - more than half of wpme ( . %), reporters suggested measures to prevent these errors. conclusion: wpme represents near % of total me reported in our hospital. given that more than % reached the patient, safety measures must be implemented to reduce the risk of hazardous events. additionally, further encouragement in notification is necessary in order to improve patient safety. results: two men diagnosed with rrms aggressive evolution were included in the study. age: and . both of them without any treatment by the time they started being treated with alemtuzumab (previously one of the patients had been treated with fingolimod, suspended by inefficiency). the protocol design for the elaboration and control of alemtuzumab in the pharmacy service ensures greater safety and represents a saving strategy. in addition, the development of the protocol in the electronic prescription system (silicon Ò ) facilitates the prescription, proper administration and standardization of treatment among patients. the protocol includes daily alemtuzumab infusion for days and other necessary medications including premedication (metylprednisolone, omeprazole, paracetamol and metoclopramide) and anti-infective prophylaxis (aciclovir). developed adverse effects during infusion were skin erythema, pruritus and fever. it was not necessary to stop the alemtuzumab infusion in any patient. during treatment, one patient developed a severe lymphopenia and upper respiratory tract infection (influenza a). conclusion: the role of the pharmacist is critical at various stages, from the preparation and the administration guidelines, to detection, monitoring and reporting of adverse effects. alemtuzumab is presented as an alternative for those patients who do not respond to standard therapies or who have rapidly evolving severe rrms. because of its mechanism of action it is important to closely monitor patients, with particular emphasis on prophylaxis of possible infections. hp-pc : descriptive analysis of patients receiving oral anticoagulation following acute coronary syndromes sadeer fhadil * , paul wright, sotiris antoniou please specify your abstract type: descriptive abstract (for projects) background and objective: triple therapy with concomitant anticoagulant and dual antiplatelet therapy (dapt) following acute coronary syndrome (acs) increases bleeding risk by % compared to patients on dapt. bleeding post acs increases mortality and reinfarction risk; balancing ischemic and bleeding risks is particularly challenging in this population. european society of cardiology (esc) produced a consensus document, providing guidance for patients presenting with acs requiring concomitant anticoagulation; however optimal duration of triple therapy and safety and efficacy of novel oral anticoagulants (noacs) and more potent antiplatelet agents requires further evidence. design: a registry was collated of patients presenting with acs requiring concomitant anticoagulation. baseline characteristics, bleeding and ischemic risk scores, periprocedural treatment and antiplatelet/anticoagulant choice and duration was recorded and analysed for trends in prescribing. results: patients have been included in the registry between oct and june , of which ( %) were naïve to anticoagulation prior to admission, ( %) were taking warfarin and ( %) were on noacs. atrial fibrillation (af) accounted for ( %) cases, (average chadsvasc score of , hasbled score of ), and ( %) were for lv thrombus. of those naïve to anticoagulation, ( %) were initiated on warfarin and ( %) on a noac (last patients all received noacs). of those on a noac for af, ( %) were dose reduced on triple therapy; apixaban being the most commonly prescribed ( % apixaban, % rivaroxaban, % dabigatran). background and objective: solid oral formulations are more convenient than liquids to manufacture, store and administer for most adults. given this superiority, one would think that children were prompted to use solid formulations when available in an eligible dose. there are indications, however, that the conversion from liquid to solid formulation in children is influenced by characteristics of the liquid medication, rather than the child's ability to swallow solid medications. the aim of this study was therefore to explore if the proportion of oral liquid formulations differed between antibiotics commonly used for upper respiratory tract infections (urti) in hospitalized children. setting and method: we collected the sales data for for the children's department of the five university hospitals in norway. the three most common oral antibiotics used for urti in children were included: penicillin v, amoxicillin and erythromycin. the proportion of oral liquids was calculated by dividing the number of defined daily doses (ddd) of liquids by the total oral ddds for each substance. main outcome measures: the proportion of ddds of oral liquid antibiotics. results: a total of ddds of common oral urti antibiotics were sold in , distributed as % erythromycin % amoxicillin and % penicillin v. amoxicillin had the highest proportion of liquid with %, followed closely by erythromycin at %. in contrast, only % of the ddds sold of oral penicillin v were liquids. conclusion: higher proportions of liquid amoxicillin and erythromycin compared to penicillin v were sold to children's departments in hospitals. there are several limitations regarding the quality of sales data, as we lack information of the administered doses as well as the child's age, gender, infection and specific needs. infections in hospitals often require initial intravenous treatment, and oral switch will often be based on the initial treatment. despite these limitations, the results fit well with earlier findings which indicate that children prefer liquid amoxicillin and erythromycin to penicillin v. hp-pc : proactive medication reconciliation: a preliminary study to identify barriers before its implementation in surgery departments laura foucault * , marion collignon, hélène dewaele, anne laure raso, emmanuel cirot, xavier pourrat please specify your abstract type: descriptive abstract (for projects) background and objective: it's well known that medication reconciliation (mr) decreases drug-related problems at patient admission (pa). in surgery departments, for planned hospitalizations, mr is performed - h after the pa (pourrat x and al, ). during this period, some chronic treatments are unintentionally not prescribed to patients. the aim of proactive mr (pmr) is to anticipate the pa by collecting their medication history before their hospitalization. the objective of this study was to identify the barriers preventing pmr implementation in our hospital. design: one week prospective study in digestive and orthopaedic surgery units in a beds' university hospital. the main outcome is to identify which barriers prevent the collection of mr before pa including the evaluation of time required to collect the relevant information, reconcile any discrepancies after the pa and identify the right sources from which to perform the mr. results: eighteen patients with a median age of years old ( - ) were contacted by phone one week before their scheduled surgery. these calls were conducted by pharmacy residents mainly between and p.m. (a more practical time for patients and at the end of pharmacist's routine tasks). an average of . ( - ) calls per patient were conducted. one patient was unreachable by phone. the average duration of the calls was min ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . twelve community pharmacy (cp) were contacted. in all cases, cp have accepted to share information about the patient's prescriptions by phone and sending it by fax during the day. five pharmacists were not contacted because patients had no chronic treatment and consequently no regular cp. on lines of prescriptions, discrepancies between the patient's information and prescriptions were identified and between prescriptions and the anaesthesia records. drug history was reported in the patient's records by pharmacy students on the day of pa in order to be used immediately by prescribers. surgery was cancelled for one patient. conclusion: the first step of an mr is made by a hospital anaesthetist some weeks before hospitalization but we have demonstrated that this step is not able to avert all potential errors. our study highlights that the time necessary to perform an mpr appears to be shorter than for an mr. in fact, it's sometimes difficult to properly interview patients during hospitalization (patient in operating room, drug-induced drowsiness). additionally, a key hurdle is to obtain any necessary modification of the prescriptions by surgeons. pmr can be expected to produce time saving efficiencies given that at pa, prescribers will have their full medication history. this study also allowed us to highlight the good cooperation between patients, cp and the hospital. it is worth noting that efforts were made to accommodate the schedules of a majority of working patients. however, as we would expect pharmacy student to perform the pmr, they will most likely attempt to contact patients during standard working hours which may impact the number of patients they are able to reach. laura foucault * , hélène dewaele, marion collignon, emmanuel cirot, anne laure raso, xavier pourrat please specify your abstract type: descriptive abstract (for projects) background and objective: the french legislation has clearly defined and integrated the therapeutic education of patient (tep) for healthcare professionals. the pharmacist is invited to get involved in tep as a caregiver around the patient. in our study, we are investigating how the pharmacist's role is viewed by patients with chronic diseases that are included in a tep program. design: prospective study on patients included in a tep program (chronic inflammatory bowel diseases, rheumatoid arthritis, ankylosing spondylitis) between september and april . in july , the participants of group sessions (gs) conducted with health professionals, including a pharmacist, were interviewed on the phone. the principal outcome of the interviews was to evaluate how their view of the involved health professional's roles evolved before and after gs; to evaluate if they would consider being followed by their pharmacist for individual sessions (is) in a community pharmacy (cp); and if the information supplied by the pharmacist during gs was understandable. to health care. however, discussions between patients appear to be essential to facilitate their acceptance of a chronic condition. some patients also questioned the cp's skills and knowledge when it comes to their particular disease. nevertheless, . % of patients have found that the vocabulary and documents used by pharmacist during gs was adapted and that the information supplied was very useful. conclusion: this study highlights that although the pharmacist is the drug's specialist, a majority of patients will more likely ask their physician about medication. their participation to the gs hasn't changed their habits even if the pharmacist intervention was relevant and understandable. the fact that the pharmacists took into account the level of health literacy of each participant was an appreciated aspect. cp should be more proactive in their relationship with the patients in order to highlight their skills and the assistance they can provide in a chronic disease. however, it's important to take in consideration that in some cases, patients have lived with their disease since childhood. the role of is is likely to be much more limited than in other situations given their key need is to interact with patients afflicted with the same condition. hp-pc : use and safety of trastuzumab emtansin in her + metastatic breast cancer in a tertiary hospital c. chaguaceda galisteo * , alba manzaneque gordon, héctor josé del río torres, natália creus baró please specify your abstract type: descriptive abstract (for projects) background and objective: novel anti her drugs have changed the management of her + metastatic breast cancer patients. the aim of this study is to describe the use of trastuzumab emtansin (tdm- ) in clinical practice in a tertiary hospital and to evaluate its safety profile. design: we performed a retrospective study of patients who started tdm- between january and december . we recorded demographic data, clinical and treatment variables, number of doses received, reasons for discontinuation, progression-free survival (pfs) and adverse effects (aes). data were obtained from the chemotherapy prescription program and medical records. aes were classified according to the common terminology criteria for adverse events version . of the national cancer institute. results: eleven female patients with a median age of . years [ . - . ] and an ecog ( / ) were included. tdm- was prescribed as a third or further line treatment in / patients and as firstline in one patient who develop disease recurrence within months of completing adjuvant therapy. median number of tdm- cycles was . . all treatments discontinuations were due to disease progression ( / ). pfs was . [ . - . months] (patients that received less than three cycles were excluded (n = )). most frequent aes were plaquetopenia, neutropenia and transaminitis but only grade in three patients (two transaminitis and one neutropenia). conclusion: the lower pfs obtained comparing to the pivotal study ( . vs. . months) could be explained by the later use of tdm- in clinical practice ( / patients received tdm- as third or further line while % in the pivotal study were first or second line). tdm- safety profile was according to the summary product characteristics. few data are currently available regarding the use of tdm- in clinical practice. further data are required to position this drug in clinical practice. please specify your abstract type: descriptive abstract (for projects) background and objective: the hospital pharmacist for their specialized training in the area of medicines, possess a greater responsibility in the detection and reporting of adverse drug reactions (adrs), as well as other problems related to treatment, which may be subject to monitoring and reporting to the regulatory authorities and the respective laboratories. thus, the pharmaceutical services of the cuf infante santo hospital has implemented a pharmacovigilance program, with two main objectives: . optimization of the detection and reporting of problems related to therapy; . implementation of corrective and/or risk minimization measures. the pharmacovigilance program is based on the following methodology: . detection of adrs/problems related to therapy/medical device: the detection can be performed by the pharmacist or other health professional that guides the process to the pharmaceutical services. . information processing by the pharmaceutical services and realization of spontaneous reporting: the notification is performed both for the portuguese regulator (infarmed) as to the appropriate laboratory (if applicable). after evaluation by both entity, the conclusions are communicated to the pharmaceutical services, which has the responsibility to share it with all the other hospital services. . report of the event in the internal risk management platform: when applicable, the pharmaceutical services internally report the adverse event to the hospital's risk management department, leading to an internal evaluation of the current process. . completion of the process and implementation of corrective measures: when the regulatory authority and/or the laboratory sends the report/technical advice about the notification, the pharmaceutical service in partnership with the risk management team perform a reassessment of the whole process. if needed, corrective and/or monitoring measures are implemented. . monitoring of implemented measures: after the implementation of corrective and/or monitoring measures there is a period of evaluation. results: the implementation of this program for the period of year, has led to a total of fourteen spontaneous reports. from all of these notifications, seven were related to quality defect of medicines, four were of adr, one was due to suspected lack of therapeutic efficacy, and lastly, one of the notifications was medication error derived. conclusion: the obtained results, over a year period, by the pharmacovigilance program were satisfactory but the aim of the pharmaceutical services is to consolidate and optimize the same program with a view to achieving better results. the pharmaceutical services will continue to take responsibility for the pharmacovigilance circuit management in this hospital, by promoting a proactive approach to monitoring the safety, quality and efficacy of medicines, which possess the primary objective to patient safety assurance. please specify your abstract type: descriptive abstract (for projects) background and objective: for prematures, parenteral nutrition (pn) is essential for medical care but is complex (specific needs, daily change of intakes…). now, the software logipren Ò , developed by the french society of neonatology, allows the prescription of pn as well as all the childish therapeutics. it is also in link with our production robot (baxa pomp) for individual pn bags. our objective was to integrate this software while optimizing our pharmaceutical validation process. design: the software implementation was lead by a physician/ pharmacist collaboration with several preliminary steps: • identification of pharmaceutical validation settings (pertinence of individual pn vs. industrial bags, parenteral approach, elements…). before the life-sized use of logipren Ò , a base test has been experimented to identify possible difficulties and to realize some correctives actions of the software or our process. results: logipren Ò leads us to a change in our pharmaceutical validation process, by introducing new elements: • the pharmaceutical validation of pn bags is done in collaboration with the physician, during the prescription step. • all the therapeutics are known, which allow the pharmacist to take into consideration all the intakes (micro-nutrients, vitamins…). • remove the transcription step of pn bags in our production software (abacus Ò ) thanks to an interface with our production robot. • less production problems because of the coverage of those pharmaceutical aspects during the prescription. since months, this reorganization helped us to propose pharmaceutical notices for prescriptions: • omissions (remove lipids, levocarnyl Ò , micro-nutrients, electrolytes, remove industrial bags…) • modification (reduce proteins according to urea level, micronutrients and electrolytes posology, duration of lipids infusion…) conclusion: the implementation of logipren Ò enabled us to reorganize of the pharmaceutical validation process with a consolidation of the role of the pharmacist during the prescription step, in the paediatric ward. it had a beneficial aspect by the reduction of the validation and production time, a decreased risk of error (suppression of job interrupts and better communication) and an improved production by the end of transcription step to abacus Ò . furthermore, during our experimentation, we could bring to the software editor new ways to improve it and make it more efficient. % ( . % in ) , difficulties in swallowing/psycho-behavioural distress in . % ( . % in ) , and rejection of oral drug in . % ( . % in ) . physicians and nurses indicate the reason in the medical record in . % of case versus % last year. this year, drug were crushed versus drugs in : % concerned nervous system group (vs. % in ), % concerned cardiovascular system group (vs. % in ) , and % concerned alimentary tract and metabolism group (vs. % in ) . nurses use guideline in % of cases versus . % last year. as the previous year, in % of cases, washing hands before preparation and after administration are met. last year, none of them was wearing mask and gloves during this operation while this year, % was wearing mask and gloves. finally, in the two assessment, for each patient, drugs are systematically crushed together and then mixed with the patient's meal. conclusion: this study shows that crushing drugs is still problematic in our units. however, best practices were observed, such as the indication of the reason of crushing in the medical record, or the consultation of guideline. a new training for nurses will be conducted to create awareness about risks of crushing drug. please specify your abstract type: research abstract background and objective: in invasive candidemia, three echinocandins are indicated: caspofungin, mycafungin and anidulafungin. the aim of this work is to establish which echinocandin to prescribe in a french university hospital, given the scarcity of available clinical data in the literature regarding obese patients. setting and method: in a french uhc with beds, a multidisciplinary working group composed of a microbiologist, an infectious disease specialist and a pharmacist has been set up to analyse the various therapeutic options. main outcome measures: analysis of the literature, pharmacoeconomic study. results: four medications have been identified as possible therapeutic options. their adverse effects are similar and their administration rhythm is the same. according to recommendations by the esmid ( ) and the idsa ( ), the level of evidence for these three echinocandins in initial treatment of candidemia is equivalent. concerning obese patients, no weight limit is mentioned, int j clin pharm ( ) : - despite recommended dosage adjustment. caspofungin must be prescribed at a dose of mg/day for patients weighing over kg. micafungin must be administered at a dose of mg/day regardless of patient weight. in the case of persistence of cultures or if clinical condition does not improve, the dose may be increased to mg/day. anidulafungin, which is not referenced in our establishment, must be prescribed at the same dose regardless of patient weight. from an economic point of view, in our hospital, micafungin at a dose mg/day remains the least costly therapy. however, if its posology is doubled as indicated, caspofungin then becomes the most economic therapy. amphothericin b, an optional treatment, is never the most economically advantageous therapy. conclusion: as a result of this study, the chosen prescribed therapy for obese patients is caspofungin at a dose of mg/day. this work has improved access to healthcare for obese patients. pharmacokinetics and survival data must be collected on the basis of various patient weights in order to predict clinical efficacy. kristin f. heier * , liv czynski please specify your abstract type: descriptive abstract (for projects) background and objective: the aim of this study was to develop a system to prioritize patients for medication reconciliation by pharmacists in the emergency department. it also proved a useful setting for evaluating how other health care professionals perceived the role of the pharmacist performing medical reconciliations within the emergency department. design: the study was located in the Østfold municipal hospital, located in kalnes, norway. pharmacists used a prioritization model to identify ''high-risk patients'' having clinically relevant prehospital medication discrepancies between hospital admission records and the information obtained via medication histories, general physician referrals and nursing homes. pharmacists registered patient information such as age, gender and drug-related problems (drps). seventeen physicians and thirty nurses in the emergency department answered structured questionnaires anonymously. main outcome measures: • number of patients with medication reconciliation performed by a pharmacist. • number of drug-related problems denoted in the electronical journal and presented to the physician. • the overall experience physicians and the nurses had with pharmacists when located in the emergency department. results: pharmacists performed medication reconciliation for patients, identifying drps and potential drps in total. fourteen of the physicians had read the journal notes from pharmacist and found them helpful (n = , %) or greatly beneficial (n = , %). most physicians (n = , %) and nurses (n = , %) reported a good cooperation with the pharmacist in the care of the patients. some of the physicians (n = , %) and most nurses (n = , %) wanted more information about the pharmacists work in the emergency department. the majority of ed staff ( % of physicians and % of nurses) found pharmacist as a good academic resource in the emergency department. conclusion: the physicians reported an improvement regarding the quality in the medication reconciliation made by pharmacists in the emergency department and both physicians and nurses expressed a need that pharmacists work in the emergency department on a more permanent basis. more information in general and especially better communication with nurses regarding the care of the patients are important actions need to optimise collaboration with pharmacist in the emergency department. results: a total of patients were included in the study, median age was years and % were males. they used in average four drugs regularly (range - ). almost three-quarters ( %) of the patients reported high or moderate adherence to all their regularly used drugs (mmas- c (max )). of the patients using oral spasmolytics, % reported high or moderate adherence to these drugs. the majority ( % of the patients) had high perceptions of necessity to their treatment (bmq [ . (max )), and % had a high level of concern (bmq [ . (max )). logistic regression analysis showed that there was no association between adherence and pain, nor between adherence and spasticity. younger age was found to be associated with higher risk of nonadherence. conclusion: even though overall adherence was high, the patients were more concerned to take their medicines compared to other patients with other chronic conditions. further studies are required for understanding adherence and attitudes toward medication in this population, and to help the patients feel safe about their medication regime. please specify your abstract type: descriptive abstract (for projects) background and objective: errors in medication lists often emerge in transition between health care levels, and there is need for strategies to communicate medication information. therefor we aimed to describe reasons why medication discrepancies (md) occurs in the transfer of patients between hospital and primary care service. design: in conjunction to a study based on use of structured medication report at transition from hospital to primary care service, we observed different reasons to why mds occurs. our observations and experiences linked to communication between health care levels is outlined. results: we observed that many md's disclosed at discharge could most likely be attributed to lack of medicines reconciliation at admission to hospital. for instance, several medicines were prescribed in primary care service prior to admission, but not at admission to the hospital. in addition, at admission, some medicines were listed as prescribed medications although not found in the medication lists in primary care service. we also observed that newly started and discontinued medicines were documented in the hospital discharge letter, but not implemented in primary care service. according to health care personnel in primary care service, insufficient communication about the patients' medications at discharge from hospital, led to corrections in the medication lists based on their previous knowledge about the patients. in addition, justified medication changes at discharge from hospital were not always implemented in primary care service due to professional disagreement. some stated that lack of trust was one reason for not always taking changes into account, often based on earlier experience. conclusion: these observations indicated that mds occurred both with and without intent when patients from primary care service were admitted to hospital and returned back due to poor communication. medication errors during hospitalisation and unproven intentional changes may be the consequences. due to this, it is important to improve the communication and confidence between professionals in the hospital and primary care service in order to reduce the number of mds and to enhance patient safety. please specify your abstract type: descriptive abstract (for projects) background and objective: intravenous human immunoglobulins (iv igs), plasma protein products, may cause in patient to a range of adverse side effects (headache, skin rash, kidney failure, thromboembolic event). in the framework of securing medicinal care, an assessment of professional practices has been conducted within our university hospital. the overall goal of this study is to evaluate the process of intravenous administration of human immunoglobulins done by the nurse staff. design: this prospective study has been carried out in three departments of neurology. an observation grid was established on the basis of guidelines on good practices. all in all, criterions have been examined resuming: prerequisites before administration, patient setup, iv igs administration, monitoring, traceability of drug delivery and management of adverse side effects. results: during the course of this investigation, administrations were observed. only % of nurses deliver information about the treatment to their patients before administration and % question patients about previous hypersensitivity reaction. the presence of spontaneous diuresis is verified in % of cases. emergency cart is not reachable in % of all cases. % of nurses ask patients to decline their identity. the use-by date on the bottles is checked in % of cases. at the time of preparation of perfusion, labelling does not mention either patient's name ( %) or date and hour of perfusion ( %). int j clin pharm ( ) : - during perfusion, only % of nurses follow diuresis and % watch rate of administration. hydration is not always kept min after the end of perfusion ( %). patient monitoring varies between min and h after perfusion's end. in % of cases, diuresis is monitored after the end of administration. % of nurses explain to patients side effects that may occur remotely. finally, administration traceability is was conform in % of all cases and in the event of adverse side effects, statement was made in % of cases. conclusion: best compliance scores have been achieved in myology department where patients are fewer than in the two others departments ( vs. and ). a presentation of those results will be given in theses three departments in order to improve patient management and securitization of iv igs administration. this audit will be carried out soon in other departments. please specify your abstract type: descriptive abstract (for projects) background and objective: a new human polyvalent immunoglobulins dose ( g) for intravenous administration is available on our establishment since . in order to secure the administration, this new dosage was initially reserved for the healthcares using administration pumps (being four health-care). the aim of this survey was to evaluate the satisfaction of the nursing staff already user of the new g dose and to estimate the motivation of the nonuser nursing staff by the audit date. design: this satisfaction survey was carried out with the most igiv consumer services (being internal medicine, neurology, cardiology and haematology). the questionnaire was structured in two sections: the first section regarding igiv in general, the second section concerning the new g dose. the survey included multiple choice questions or questions with answers based on a four levels evaluation scale (not satisfied, mildly satisfied, satisfied, and very satisfied). results: the audit was realized on eight health-care, involving nurses. among the interviewed, ( %) have already used the g dosage. in % of cases, users were very satisfied and % were satisfied. the most positive points noted were: gain of time provided ( . % of satisfaction), less manipulation needed ( . % of satisfaction), and reducing of infectious risk ( . %). moreover, the influence of the injection technique on users' satisfaction was further reported. indeed, according to nurses interviewed, the use of an injection pump is safer and improves the job comfort of nursing staff, unlike the injection by gravity (used in % of cases), which seems to slow down the use of this new dosage. in two cases, a positive opinion given by patient was also reported. finally, negatives points noted were related to administration instruments (use of pump or not) and to less flexibility in daily dose regulation. among the % not-user of this new dose, the % showed a strong interest for the product apart from services making the igiv administration by gravity. conclusion: in light of these results, the use of g dose will be spread to other services. the general diffusion of this dosage will provide a gain of time also at the pharmacy, during the unitary delivery and the computer-based administration of every units. a second survey will be soon effected within patients involved in the switch g/ g. the capital region pharmacy, clinic of neurology, rigshospitalet, blegdamsvej, copenhagen, denmark please specify your abstract type: research abstract background and objective: the clinic of neurology, rigshospitalet, copenhagen, denmark experience continuous medicine-related patient safety incidents (psi) related to newly admitted patients and patient transfers between wards. in order to prevent drug related problems (drp), the pharmacists increased their focus on these patients and provided systematic medication reconciliation. thus, the objective of this pilot study was to investigate if the intervention would help identify drug discrepancies (dd) and prevent drp. four wards were included in this study; two neurological, one neuro-anaesthetic (icu), and one neurosurgical ward(s). three wards use electronic medication module (epm), whereas the icu uses critical information system (cis). furthermore, all patients' prescriptions are registered on shared medication record (smr), which provides an overview of prescribed medicine. prescriptions cannot be transferred from smr and epm to cis and vice versa. we suspected that psi resulted from these system incompatibilities. setting and method: patients admitted or transferred from may nd to june rd were included. medication reconciliations using smr, epm and cis were conducted by a pharmacist on weekdays. dd were presented to a physician orally and documented. only dd accepted by physicians led to drug prescribing change. main outcome measures: number of identified dd. results: the study included patients, of which ( %) were newly-admitted. patients ( %) were transferred between wards. of the transferred patients, ( %) were transferred from the icu to other wards and ( %) were transferred from other wards to the icu. of the newly-admitted patients, ( %) were admitted to the icu and ( %) were admitted to other wards. the pharmacists identified dd; dd ( %) in the transferred and dd ( %) in the newly-admitted patients. in the transferred, dd were all related to the icu. in the newly-admitted, dd ( %) was related to the icu and dd ( %) to other wards. of the dds, ( %) were accepted by the physician. an example of a severe dd identified was an omission of prednisolone to a patient admitted to the icu. conclusion: most dd were identified in patients admitted to or transferred from icu, which uses the incompatible system cis. pharmacist systematic medication reconciliation helps identify these dd and prevent drp. please specify your abstract type: research abstract background and objective: antibiotic related drug interactions are more likely in intensive care unit patients due to common polypharmacy and antibiotic usage. the aim of this study is to determine the antibiotic related drug interactions with three different online databases (micromedex-paid, medscape-free and drugs.com-free) and to evaluate these interaction information by clinical pharmacist. setting and method: a retrospective, descriptive study was set up in hacettepe university hospital's intensive care units, between november and december , . patients who use at least one antibiotic were involved in this study. all drugs were assessed by each three databases and only antibiotic drug interactions were evaluated. clinical significance of identified drug interactions were evaluated by clinical pharmacist. main outcome measures: clinical pharmacist's assessment in significance of drug interactions indicated by three online databases. please specify your abstract type: research abstract background and objective: an implementation of clinical pharmacy practice by postgraduate students in intensive care units is a new way of learning in postgraduate education which creates opportunities in multidisciplinary collaboration in clinical pharmacy research, and also has influence on clinicians' routine patient care process. this system in educational program was ongoing in the department of clinical pharmacy since . as a part of this educational program, drug related problems in intensive care units were described and analysed, an influence of clinical pharmacy postgraduate students on patient treatment process was sought. setting and method: a prospective, cross-sectional study was performed between the march-june in hacettepe university hospitals, department of internal diseases intensive care units which consists of beds. three postgraduate pharmacy students from the department of clinical pharmacy, faculty of pharmacy conducted medication reconciliation in order to identify any problems in patients' medical orders. drug related problems (drps) were identified by the students and recommendations for management were approved by a supervisor of clinical pharmacy department before they were directed to physicians for approval. the students were not authorized to undertake any action in patient care process, therefore all required interventions for drp were undertaken by physicians and the acceptance ratio of the interventions were recorded. the pharmaceutical care network europe foundation classification system (v. . ) was used to asses drps. main outcome measures: determination of drps by pharmacists and evaluation of their interventions' acceptance by physicians in intensive care units. results: during the study period, patients were admitted to the intensive care units. each patient's medication orders were evaluated and interventions were recommended by postgraduate students. the number of interventions per patient was . . the acceptability rate of interventions by physicians was . %. in addition, physicians were provided drug information on seven different occasions. recommendations regarding drug therapy were mainly related with treatment effectiveness and adverse reactions. the common causes of drps were requiring dose adjustment due to pharmacokinetic problems ( . %), no therapeutic drug monitoring ( . %), inappropriate timing of administration and/or dosing intervals ( . %), requiring dose adjustment due to deterioration/improvement of diseases ( . %), inappropriate drug selection ( %) and new indication for drug treatment presented ( %). the most common drugs responsible for drps were ranitidin, levothyroxine, allopurinol, pantoprazol, piperacillintazobactam and vancomycin. the study showed that the most common drps was dose-related, therefore close monitorisation of the intensive care unit patients by students in clinical pharmacy postgraduate program can help physicians in terms of detecting, preventing and minimizing drps in order to improve patients' health outcomes. please specify your abstract type: research abstract background and objective: antibiotic stewardship is the process of salvaging important antibiotic agents from becoming ineffective due to bacterial resistance. this is important because throughout the world antibiotics continue to be one of the most important classes of therapeutic agents due to their vital role in saving patient lives. key goals of antimicrobial stewardship are to improve clinical outcomes, prevent antibiotic resistance, promote patient safety, and reduce health care cost. pharmacist are in the frontlines because they perform antibiotic stewardship activities, such as selecting the most optimal antibiotic agent, adjusting drug-dosage, and stopping use of unnecessary antibiotics. as a result of the continuous rise in antibiotic resistance and decline in development of new antibiotics, antibiotic stewardship programs are proving to be indispensable in a health care settings. setting and method: adult and paediatric inpatients receiving antibiotic therapy in the hospital medipol university has been evaluated. patients were selected randomly in the hospital system. patients were evaluated for antibiotic susceptibility results and compliance with antibiotic management guidelines. main outcome measures: to evaluate the antibiotic therapy in patients with culture results and to determine according the treatment guidelines. results: it was observed different pathogens in blood culture results of inpatients out of patients who were treated with antibiotics in hospital. antibiotic susceptibility results for acinetobacter spp, staphylococcus spp, enterococcus spp, pseudomonas aeruginosa, klebsiella spp, e. coli spp, streptococcus spp, corynebacterium spp, streptococcus pneumonia and enterobacter spp are evaluated in the study. klebsiella spp was the most isolated pathogen at total of culture results. most frequently resistance were int j clin pharm ( ) results: a total of ( %) questionnaires were completed. of these, approximately % were answered by hospital nurses, the remaining mainly by physicians ( %) and % ''other''. on the question ''what is your general perception of the benefit of the clinical pharmacy service; for collaborating health professionals? for the patient?'' the total benefit was ranked . and . respectively (scale from (''no benefit'') to (''beneficial to a very large extent''). the open questions: ''what disadvantages/advantages have you experienced by the introduction of clinical pharmacist into multidisciplinary teams?'' received / comments respectively. physical obstacles regarding office space, interference with the decision making process, more time consuming processes and the issue of relying too much upon the advices given was reported as possible disadvantages. respondents answered ''none'' to this question. the comments regarding advantages dealt mainly with general increased patient safety and quality assurance. in addition, advantages as work-load relieve, time saved, collegial support, practical help, and learning interchange between professions, were highlighted. conclusion: health-professionals assessed the clinical pharmacy service as highly beneficial. the advantages outlined were higher patient safety and quality regarding medication, in addition to collegial support, practical help and learning interchange. please specify your abstract type: descriptive abstract (for projects) background and objective: in june , the french health authority, the «has», published an index resuming the recommendations of benzodiazépines (bzd) prescriptions and proposing an approach to stop using it. indeed, it has been established that there is a too high and too long consumption of bzd in france. a study of prescriptions' prevalence has been done in our hospital centre. the aim of this study was to know our situation regarding the use of bzd in order to set up some improvements and take part in their proper use. design: a prospective study has been done on a months period in different services: geriatric, post-op and rehabilitation facilities, endocrinology, internal medicine, pneumology and cardiology services. the data were raised on a given day in each services and recovered thanks to the prescriptions software but also through interviews with the patients and their doctors. it was examined whether there was a bzd prescription (hypnotic or anxiolytic), whether the duration was superior or not to the duration of the amm and whether the prescription was done in our hospital centre. if the prescription was already part of the patient treatment, we looked if it was possible for the patient to stop using it, according to the has criteria. on their discharge, the letters and bzd prescriptions were also analysed and some patients' general practitioner were contacted after their discharge. results: patients (median age years old) were included from november to march . . % ( / ) of the patients had at least one bzd prescription the day we collected the data. we found only one bzd in prescriptions ( . %) and among them . % ( / ) were anxiolytic bzd. among those prescriptions, . % ( / ) already existed before the hospitalization and . % ( / ) were given during the hospitalization ( were prescribed automatically). . % ( / ) of the prescriptions did not respect the legal duration of the amm ( pieces of data were not found). . % ( / ) already exceeded this duration limit. among the patients who already had a bzd treatment before going to hospital, . % ( out of ) could consider stopping their use of bzd. by the end of this study, patients were discharged from hospital, among them . % ( / ) with a prescription of bzd. . % ( / ) of the prescriptions established during the hospitalization had been renewed when the patient came out of the hospital, we managed to contact ten general practitioners (approximately . days after their discharge), nine patients carried on their bzd treatment, among them one patient had reduced his consumption. conclusion: this study is an example of the high proportion of bzd prescriptions in france which the majority doesn't respect the legal length of the amm. the prescriptions of bzd in the hospital are generally systematically renewed by the general practitioners. the patients must be informed about the risks of using those molecules. in order to ameliorate this practice in the hospital, a proper use leaflet, reminding the prescriptions of bzd, has been created and distributed in each services to make people aware. main causes of admission were infections ( %) (respiratory disease ( %) and other ( %)), hepatic disease ( %) and neoplasias complications ( . %). patients died during their admission; due to hepatic disorder, due to neoplasia, and due to infections. conclusion: last diagnosis of hiv or no art treatment are causes of admission. immunovirological situation is related with their adherence but isńt with admissions. coinfection with hcv or hbv or others infections are risk factors for admission. center for psychopharmacology, diakonhjemmet hospital, oslo, norway please specify your abstract type: research abstract background and objective: complex medical history and treatment can potentially cause problems. the objective of this study was to investigate the prevalence of drug-related problems (drps) and medication discrepancies in internal medical patients with complex treatment at hospital admission. further, to investigate to which extent drps were identified as a result of medication reconciliation, and to which extent drps could be associated to the hospitalization. setting and method: patients with at least four regular medicines from two different therapeutic groups were consecutively included at admission to an internal medicine ward at a university hospital in norway in the period . . - . . . pharmacists used the integrated medicines management (imm) model for medication reconciliation and medication reviews at admission. a medication discrepancy was defined as any discrepancy between the recorded medication list at admission and the patient's actual use of medications, as revealed by medication reconciliation. the patients' actual use of medications, medical journal and laboratory results, were used to perform a medication review at admission time and identify drps. the proportion of drps revealed due to medication reconciliation was calculated. moreover, the project group retrospectively assessed possible drp-induced hospitalizations based on clinical history, cause(s) of admission and identified drps. main outcome measures: the main outcome was the median number of drps per patient at admission. the proportion of drps revealed due to medication reconciliation, the proportion of patients with drps possibly associated to the hospitalization, and the median number of medication discrepancies, were included as secondary outcomes. results: patients were included, . % women. median patient age was (range - ) and most of the patients were home-living before admittance ( . %). in total drps were identified at admission, with a median number of (range - ) per patient. drps ( . %) were identified due to medication reconciliation. for patients ( . %) a causal relationship between the hospitalization and the drps was assessed as ''possible''. medication discrepancies were revealed in of the included patients ( . %), with a median number of (range - ) per patient. conclusion: internal medical patients with complex drug regime are frequently exposed to drps and medication discrepancies at hospitalization. medication reconciliation could be essential to identify drps, which is likely a common cause of hospitalization in the studied patient population. hp-pc : assessment of oral anticoagulant prescriptions and pharmaceutical analysis at the hospital by regional audit damien fuss *, , clélia monchablon , anaïs breteau , marie lefebvre-caussin , rémi varin , jean doucet , mikael daouphars , doreya monzat omedit normandie -chu rouen, chu rouen, please specify your abstract type: descriptive abstract (for projects) background and objective: oral anticoagulants (oa) are the most common drug class associated with preventable adverse drug events in hospitalized patients that require optimizing the pharmaceutical analysis (pa) process. in this context, a regional audit was conducted on pa of prescriptions oral of oa. the aim of this study is to provide an overview of the treatment by oa in the hospital by evaluating the consistency of the oa prescriptions compared with national and european guidelines and evaluate the pharmaceutical interventions. design: this study is based on the collection of pa data (demographics, indication, posology, drug interactions, monitoring) as well as the collection of pharmaceutical interventions and discordance int j clin pharm ( ) : - between guidelines recommendations and clinical practice. the inclusion criteria were any patient treated with oa (vitamin k antagonists (vka), non-vitamin k antagonist oral anticoagulants (noacs)). included patients were followed minimum months. the primary outcomes include description of baseline characteristics of patients, the number of inappropriate prescriptions compared to the different clinical recommendations, the number of pharmaceutical interventions, the number of adverse drug reactions (adrs) related to oa use and the assessment of patient monitoring. results: during the -months study period, patients were included in six health institutions. the average age was years ( % of patients over years old) and % of the patients were women. % of patients had renal impairment. % of patients were treated with vka, and % with noacs. it was the first prescription of oa for % of patients ( % with vka; % with noacs). the most common indication was the non-valvular atrial fibrillation ( %). in this indication, % of patients had cha ds -vasc score c , and nearly % had a high risk of bleeding (has-bled score c ). drug interactions were observed, and adrs occurred related to oa. % of patients with an adrs had a has-bled score c . . % of prescriptions were considered inappropriate, including % noacs (no monitoring renal function in % of patients over years initiating treatment, inappropriate posology in %, and % of contraindications). the rate of pharmaceutical interventions was %. nearly % of the prescriptions were already adapted when the pharmacist was starting analysis. conclusion: prescribers are sensitized of the risks on the oa prescriptions, which explained the delay upon pa and low rate of pharmaceutical interventions. however, the high number of inappropriate prescriptions shows the necessity to improve the pa process on these drugs, particularly by actions on therapy initiation and patient monitoring, especially for noacs. for this class, the impossibility of assess the level of anticoagulation by laboratory monitoring requires appropriate initiation and monitoring, especially an assessment of baseline renal function. please specify your abstract type: descriptive abstract (for projects) background and objective: the development of bacterial resistance these last years is a public health major problem in the world and needs to implement actions. in france, the national drug safety agency has defined a list of ''critical antibiotics''. this list includes antibiotics particularly generator of bacterial resistance (amoxicillinclavulanate, cephalosporine, fluroquinolone) and antibiotics called ''last resort'' (antibiotics against gram-positive cocci, car-bapenem…). at our regional level, an evaluation of prescription of these critical antibiotics was proposed to all medical centers. the aim was to evaluate the quality of prescription of these critical antibiotics. design: the regional working group (pharmacists, infectious diseases physicians and biologists) had developed a collection grid including data on patients, antibiotics and four criteria: adequate molecule, compliance with medical prescriptions, duration of antibiotic therapy and reassessment at h. this is a prospective study proposed to all health institutions (public and private), which had to be completed on a given day in all care units and had to be conducted by a team of multi-professional evaluators. the study included a quantitative part (number of patients hospitalized in the audited units, number of patients receiving antibiotics and number of critical antibiotic prescriptions) and a qualitative part (adequate to the four criteria). results: response rate was of %. the study investigated on patients hospitalized in the audited units, including patients ( %) receiving antibiotics. among the patients, % were hospitalized in medical, surgery or obstetrics units we recorded prescriptions of ''critical antibiotics particularly generator of bacterial resistance'' ( % amoxicillin-clavulanate, % ceftriaxone, % fluoroquinolone and % other third-generation cephalosporine) and prescriptions of antibiotics called ''last resort'' ( % carbapenem). the average age of the population was . years (± years). sex ratio was . . % corresponded to curative use and % to prophylactic use. the expertise of infections diseases physician was requested in only % of the cases. the antibiotics were prescribed in majority to treat bronchopulmonary infections ( %), urinary tract infections ( %) and intraabdominal infections ( %). ninety-two percent of the prescriptions had a proper indication. % of the prescriptions complied to the guidelines. the duration of antibiotic therapy was adequate in % of the cases. only % of the prescriptions were correct according to these three criteria. forty-four percent of the prescriptions were reassessed and adapted by the physician. conclusion: this study is original because of its regional dimension and antibiotic analysis. the number of analysed prescriptions was significant with an overall proper prescription in adequate with the guidelines. however, actions must be implemented on duration and reassessment and adjustment of treatment. these results were presented to the participating hospitals. these three points will be reevaluated during a new regional audit. the criterion «no more psychotropic drugs» has been met in . % of assessment. otherwise, or more psychotropic drugs are prescribed in . % of assessment from the point of admission. the criterion «no more a benzodiazepine drug» has been met in . % of assessment. otherwise, more than one benzodiazepine drug is prescribed in . % of assessment from the point of admission. no contra-indication is detected in . % otherwise, a contra-indication between two drugs causing torsade de pointes is detected from the point of admission in this department. no more anticholinergic drug is prescribed in . % of assessment. according to the french criteria, one or more inappropriate drug is prescribe in . % of assessment. the most common inappropriate drug group prescribed was alimentary tract and metabolism drug ( . %) (the hospital at home team needs these class of drug) followed by nervous system ( . %) (prescribed at the point of admission) and by cardiovascular drugs ( . %) (prescribed at the point of admission). finally, the criteria «no more one non-steroidal anti-inflammatory drug» and «no illogical association» have been met in all cases. conclusion: this analysis shows that most of criteria for «assessment of prescription among elderly in a «hospital at home» department have been met. when one has not been met, either the hospital at home team needs the drug prescribed, or this drug have been yet prescribed from the point of admission in this department. this study could be used for the next certification. hp-pc : access to health care: case of autologous serum eye drops batiste martel, fabien lindenberg, camille castel, guillaume saint-lorant * please specify your abstract type: research abstract background and objective: autologous serum eye drops (ase), prepared from patient's serum, are indicated in the treatment of severe dry eye syndrome and defective epithelial healing. its in-hospital preparation within a controlled-atmosphere zone unable it to be dispensed by non-equipped hospital pharmacies. the aim of this study was to implement security measures to allow transport towards distant hospital pharmacies and all patients even those residing far from a regional university hospital (uh). setting and method: this study was conducted in a -bed french university hospital. patient blood samples were taken within the university hospital every weeks. serum was then biologically controlled (negative tests for hiv, hbv, hcv, tpha, vdrl). preparation was conducted days after blood sampling. sterile preparations were then stored at a temperature of - °c. studies showed that eye drops were stable days after being thawed. transport of eye drops to distant hospital pharmacies requires to be conducted under controlled temperature i.e. below °c, to ensure the stability of eye drops. these pharmacies are located close to patient's homes. the entire process was examined by a pharmacy team in order to study and secure each step, transport in particular. main outcome measures: validation of each step of the autologous serum eye drop dispensing process, from sampling to receipt by different hospital pharmacies, transport in particular. results: patients benefitted from the preparation. all patients resided more than kilometres from a uh. a follow-up form was completed to qualify dispatching and to trace each step during transport. a temperature sensor was placed inside the box. the receiving agent was required to stop and control the sensor. a double retrospective control was performed by a pharmaceutical team via the recording of temperature sensors. a second follow-up form was drafted in order to track dispensation reviews, ongoing dispensation and future planning. a patient information booklet was distributed to hospital pharmacies to inform patients about good practice concerning eye drops. conclusion: technological necessities concerning autologous serum eye drop preparation and transport limit access to health care. in this study, the role of the pharmacist consisted in reducing inequalities among patients residing at a distance from the only regional uh. the role of the pharmacist is to ensure absolute quality of preparation between the uh and the patient. hp-pc : computerized medication reconciliation: overview of pharmaceutical software used and support for development of integrated modules julie mocquard, anaïs berthe, elise rochais * , nicolas prévost, jean-claude maupetit, on behalf of centre de ressources régional en conciliation médicamenteuse omedit pays de la loire, nantes, france please specify your abstract type: descriptive abstract (for projects) background and objective: medication reconciliation aims to improve continuity of care for patients. in , a national survey identified barriers for implementation of this activity in france, among which computerized systems were judged unsuitable for hospital practices. in the absence of appropriate hospital information systems (his), medication reconciliation remains a time consuming process implying manual transcriptions, potentially leading to a lack of traceability and medication errors. the objective of the study was to assess the current his used in a french region including the integration of medication reconciliation into the software and to define courses of action to assist this integration. design: an online survey conducted in may was addressed to head pharmacists of the health facilities in the region, giving a total of head pharmacists concerned. it included questions on the software used by the health facility, the development of medication reconciliation and its traceability, formulation of operational requirements to the editors of software and availability of a module integrating medication reconciliation provided by the software. results: seventy-eight pharmacists ( %) participated in the study, with all types of health facilities represented: public hospitals, clinics, home health agencies, haemodialysis structures and after care and rehabilitation facilities. thirty different software were identified in the region. ( %) pharmacists planned to develop medication reconciliation in their health facility and ( %) were already carrying out this activity. within these %, medication reconciliation was conducted on paper only for ( %) of them, while ( %) were using a computerized system (patient file, pharmaceutical software, other) for traceability. the most widely used software in the region contains a module enable for computerized medication reconciliation, and three other editors are currently developing one. no development is scheduled for another three editors nonetheless commonly used in the region. ( %) pharmacists had contact with the editor of the software, and had given thought to the preparation of requirement specifications to the editor to develop an integrated module of medication reconciliation. conclusion: despite the interest attributed to medication reconciliation and despite the need of a fully integrated module of medication reconciliation to his, only a few health facilities of the region possess an appropriate computerized system to develop this activity. this study underlines the approaches already made by pharmacists to editors in order to integrate medication reconciliation to the his. subsequently, retrieving these approaches and writing specifications common to all health facilities is scheduled, in order to assist them in providing a strong incentive for the editors to integrate medication reconciliation to existing his. please specify your abstract type: descriptive abstract (for projects) background and objective: medication reconciliation (mr) is an interactive and multiprofessional process that ensures the continuity of care by integrating the ongoing treatment to the new hospital prescription. this helps securing the patient's care pathway particularly at transition points. the objective is to initiate the mr process in our medical institution with a pilot study in the department of internal medicineemergency downstream to validate a methodology and adapted tools. design: the mr takes place in three steps performed by a pharmacy student: ( ) realization of best possible medication histories (bpmh), combining at least three sources of information and using sources' collection form. this research begins with a patient interview done in pairs with a medical student using an interview guide. ( ) comparison bpmh with the initial hospital prescription in the department (after passing through the emergency department) on the treatment reconciliation form. a status is assigned to each line of drugs and then the differences are identified (stopped, changed or added). these two steps are validated by a pharmacist. ( ) discussion and characterization of observed differences (intentional/unintentional and documented/undocumented) with the senior physician. results: twenty-six mr were performed over weeks in . the mr is performed within days after admission. on average, . information sources per patient were used for the bpmh: mainly drug prescription (dispensed in pharmacies community); analysis of emergency medical records and patient interview. for the patients included, drugs were listed. discrepancies were observed and were studied (status stopped or changed only): one documented intentional discrepancy, undocumented intentional discrepancies and unintentional discrepancies (ud). these uds affected patients ( - medication errors per patient) and corresponded to a non-prescribed drug in % of the cases. vitamins, antihistamines, anti-reflux and proton-pump inhibitors were involved in % of cases; cardiovascular drugs in % and antiinfectious in %. through this pilot study, the methodology was validated: (a) need to have a minimum of three sources to achieve a relevant bpmh and to confirm each information with two sources; (b) need for a dedicated time with trained staffs; (c) development of tools to improve the traceability of information obtained from each source and traceability of medication reconciliation activity. conclusion: the mr establishment in the internal medicine department was helpful in identifying medication errors that have been corrected. it is proposed to archive the treatment reconciliation form in the patient file to contribute to the traceability of information on treatment. this study strengthens the deployment of this method and mr tools to other services of the hospital. alma mulac * please specify your abstract type: research abstract background and objective: clinical pharmacists have an important role in improving healthcare services. there is lack of knowledge of clinical pharmacists' experiences in interprofessional collaboration. our objective was to explore the challenges and barriers experienced by clinical pharmacists in interdisciplinary teams in norway and incorporation of expanded pharmacist roles in hospital settings. setting and method: this qualitative study was conducted using semi-structured interviews. a total of clinical pharmacists from four (government) hospitals were included in the study. the interviews were audio recorded using a digital recorder. the recordings were transcribed verbatim. main outcome measures: challenges and barriers clinical pharmacists experience in interdisciplinary teams in hospital setting. results: the main findings are that the pharmacists' role is little known to other health care professionals, particularly at hospitals with short tradition for clinical pharmacy services. clinical pharmacists have great motivation from being able to influence drug treatment for patients. from the perspective of the participating pharmacists they succeed in interdisciplinary cooperation when their professional knowledge solves the patients' drug-related problems. communicating recommendations to physicians with professional credibility has great importance for the intervention to be implemented. using the theoretical framework of communicating tensions, we argue that the pharmacists in our study use indirect communication to prevent physicians defensiveness to recommendations. lack of education in interprofessional cooperation and communication is apparent in this study. the participants also stated that there should be some form of quality assurance or education requirements before one can work as a clinical pharmacist. conclusion: training in communication for graduates and interprofessional collaboration during the undergraduate pharmacy education, can possibly help pharmacists with integration in interdisciplinary teams. increased attention to teamwork from the hospital leadership is essential for the implementation of interprofessional collaboration in a larger context. please specify your abstract type: descriptive abstract (for projects) background and objective: antifungal therapy in the icu, particularly therapy targeting resistant aspergillosis, mucormycosis and systemic candida, is often of lifesaving importance. posaconazole and voriconazole are the antifungal agents of choice. our aim was to compile a tool that can be used at the icu to address aspergillosis, mucormycosis and systemic candida in an optimal manner. design: female patient, age + , liver transplant, crp [ mg/ l, creatinine [ lmol/l. abdominal x-ray imaging revealed four large abscesses and laboratory analyses confirmed mucormycosis. posaconazole intravenous ( mg one times daily) and liposomal amphotericin b ( mg/kg/day) were initiated. the inflammatory markers remained unchanged days following initiation of therapy with no change in size or number of abscesses and the patient developed sepsis. amphotericin b dose was increased to mg/ kg/day. after week the inflammatory parameters and size of abscesses began to fall. the dosage form of posaconazole was switched from intravenous to mixture. the dose remained the same and within h the crp rose to mg/l. results: pharmacist intervention revealed a missing loading dose of intravenous posaconazole as well as incorrect dosage of the per oral form due to bioavailability variation. posaconazole mixture dose was increased to mg two times daily. through serum concentration analysis of posaconazole was suggested prior to the dose increase. the serum concentration was . mg/l (range [ . - . ) . through serum concentration days later was . mg/l. both crp and abscess size were on the decline. a dosage and tdm pocket card for posaconazole therapy of mucormycosis, aspergillosis and candida was compiled. conclusion: optimal systemic fungal infection therapy is essential, especially in the critically ill. of special importance is tdm and correct dose adjustment when dosage-form changes occur. please specify your abstract type: research abstract background and objective: potentially inappropriate prescriptions and omission of prescription, respectively ip and op, are common issues in the pharmacotherapy, especially in vulnerable population, such as elderly and children. there are many available tools detecting ip and op for geriatrics, however, similar tools are less common in paediatrics. therefore, a first target tool for paediatric population: popi «paediatrics: omission of prescriptions and inappropriate prescriptions» was created and was validated by delphi method in . we aim to evaluate inter-rater reliability between health care professionals, who apply popi. our study also assessed their satisfaction and the accessibility of this tool. setting and method: twenty cases with or without ip or op were selected. these cases were identified in a previous retrospective ip-op prevalence study on . patients. these patients were admitted to the emergency department of a university mother and child hospital, between october and march . one doctor and one pharmacist, who participated in the creation of popi tool, identified ip and op in cases and composed ''standard answers''. these cases were then reviewed independently by eleven clinicians (including generalists, paediatricians, pharmacists, residents, general practitioners), who did not experience this tool before. inter-evaluator agreement was calculated by using the agreement kappa test. the satisfaction of users was also evaluated. main outcome measures: inter-evaluator agreement, the median time of use and the satisfaction of users. results: a high level of agreement of ip and op detection was recorded (ip: k median = . ; op: k median = . ). the easy use of popi was approved by % evaluators. the median time of use was min s per case (quartiles : . - . ) . as a result, there were % of clinicians satisfied with the provided popi and they would like to apply this tool in their daily practice. conclusion: popi demonstrated a good interrater reliability and is easy to use. this strong validation by many specialists prove popi is a reliable tool. it can be applied daily at work in paediatric section by doctors and pharmacists. other multicentre and prospective study should be conducted to evaluate economical and clinical impacts of popi. please specify your abstract type: descriptive abstract (for projects) background and objective: drug dosing during cvvh is challenging due to changes in pharmacokinetic parameters brought about by the patients' deterioration in health and factors associated with the physical process of filtration. this is of particular significance in the icu. in addition, there is the issue of the patients' diuresis or lack of such. this will affect the total clearance (cl total ) of the drug. the dose of antibiotics must therefore be calculated individually taking into account all of the above as well as changes of filtration parameters. our aim was to illustrate how such dosage calculations can be undertaken. design: a -year-old male patient, weight kg, diagnosed with stenotrophomonas maltophilia infection. the trimethoprim/sulfamethoxazole dose was . mg trimethoprim/kg/day every h as specified for anuric patients on cvvh. patient was initially anuric for days after which diuresis was started. the dose was recalculated. results: creatinine clearance (crcl) related to cvvh during the anuric period was calculated accounting for ultrafiltration rate, sieving coefficient, blood-flow, haematocrit concentration and pre-dilution. the value was ml/min. following diuresis on day , remaining kidney function was assessed by measuring urine and serum creatinine. the value for crcl renal ( ml/min) was added to the extracorporeal clearance, and gave a total clearance of ml/min. this warranted dose adjustment of trimethoprim/sulfamethoxazole since this drug requires normal dosage at crcl [ ml/min. conclusion: during cvvh, the presence or absence of diuresis must be taken into consideration when dosing antibiotics. in anuric patients, the cvvh-machine set up constitutes crcl total , but in patients with diuresis, the remaining crcl renal should be added. please specify your abstract type: descriptive abstract (for projects) background and objective: the aim of the study is; to evaluate patients' home (prescribed and non-prescribed) and hospital medication during hospital admission by computing medication regimen complexity index and investigating possible drug-drug interactions. design: patients (aged and older) who applied internal service during months ( days/a week) were included to the study. patients' medical profile were obtained from patients' file. their home medication and hospital medication were calculated with medication regimen complexity index ( ) and checked drug interactions with micromedex drug interaction program. results: a total of from of patients who applied to the internal service (male . %, female . ; the mean age of patients was . ± . .) were included to this study during months. of them, . % had low education level (\ education years), . % had and more chronic diseases of them, % hospitalized last months before this hospital admission. the most prevalent diagnoses documented at admission were kidney disease ( . %), cardiovascular disease ( . %) and cancer ( . %). the mean of patients' home medication number was . ± . and the mean of their mrci scores was . ± . . % in patients hospitalized in the last months. at least one possible drug-drug interactions were found in . % of patients at home medication and in . % of patients at hospital medication, respectively. the mean number of possible drug-drug interactions at patients' home medications was . ± . , while the mean number of possible drug-drug interactions at patients' hospital medications was . ± . . of them, . % had polypharmacy at home medication. the frequency of possible drug-drug interactions and the score of medication complexity index was found high among patients' hospital medications when compared with their home medications. conclusion: the potential role of pharmacist including medication reconciliation and medication review could improve rationale drug use during hospital admission. coronavirus. experts' local committee has approved to use oral ribavirin for the treatment of these respiratory viral infections. we aimed to assess the effectiveness and safety of oral ribavirin as main treatment in respiratory viral infections. setting and method: from may to october , we performed a retrospective monocentric study including patients who received oral ribavirin for non-hcv infections. main outcome measures: viremia negativation was used to determine the response rate to oral ribavirin. specific toxicities (anaemia, cytopenia, liver dysfunction) and renal function were assessed biologically. results: thirty-five immunocompromised patients (f/m: / , age: ) were included. underlying conditions were lung transplant (n = ), heart transplant (n = ), pulmonary fibrosis (n = ) and acute myeloblastic leukaemia (n = ). the median duration between transplantation and infection was . years ( . - . ). nine patients were exclusively infected by rsv, by hpiv ( hpiv- ; hpiv- ; hpiv- ; hpiv- ; non-identified hpiv), by hmpv and by coronavirus. there were six co-infections: rsv/ hpiv- , rsv/coronavirus, hpiv- /hpiv- and hpiv- or /coronavirus ( patients). all the patients were admitted in pulmonary division, except for the patient with heart transplant who was in cardiac intensive care unit. the administered dose was mg tid or mg tid if there was renal insufficiency ( patients). the median duration of the treatment was days . four patients prematurely discontinued the treatment due to severe toxicity or therapeutic change; three didn't respond to the treatment (no data for the last one). four patients were re-treated despite having a virological response to the first cure. one patient treated for a hpiv- /coronavirus coinfection had an hpiv- relapse days after ribavirin discontinuation. concerning the three other patients, they received a second cure to treat a new infection (coronavirus, hpiv- and hmpv, in opposition to hpiv- twice and hpiv- respectively). virological response rate was % ( / for rsv, / for hpiv, / for coronavirus and / for hmpv). two non-negative viremia patients (rsv and hpiv- /coronavirus) received intravenous ribavirin after oral ribavirin therapy. no patient died from viral infection. twelve patients presented specific toxicity: one hepatic cytolysis and cholestasis, eight haemoglobin decrease, two pancytopenia and one mucositis. conclusion: despite the poor number of patient, our study shows that oral ribavirin seems to be efficient to treat hpiv, hmpv and coronavirus in immunocompromised adults. we observed known side effects that could generally be managed. oral ribavirin may thus represent a therapeutic strategy in several respiratory viral infections. please specify your abstract type: descriptive abstract (for projects) background and objective: reconciliation of medicine lists is important to ensure correct medical treatment of patients both in hospital and other healthcare levels. while reconciliation upon admission is part of the normal routine at surgical ward b, molde hospital, there has been less focus on reconciliation at discharge. as such, this study aimed to ensure reconciliation and correct transfer of medical information at discharge. design: medicine lists of all patients discharged from surgical ward b, molde hospital between week and in (n = ) were investigated. the forms were gathered and counted based on the tasks signed for to ensure completed reconciliation and sufficient information given to the patient. the count was performed every - weeks, and the forms in each count was pooled together as one point of measure. the quality of medicine lists in discharge lists was evaluated based on the norwegian patient safety program criteria. medicine lists in discharge lists from week to (n = ) were pooled together and compared to medicine lists in weeks - (n = ). results: the results of reconciliation was divided into the subsections of surgical ward b, and represent the number of completed tasks as signed for in the reconciliation form. the surgical subsection showed a significant increase in patients with pre-checked medicine lists and reconciled medicine lists over the measured time period. similar results were not found in the orthopaedic subsection. as for the quality of medicine lists in the discharge lists, significant improvement was seen in all set criteria, with the exception of ''source'' in the surgical subsection. in the orthopaedic subsection however, no significant improvement was seen in any of the criteria other than ''indication for use''. conclusion: the implementation of reconciling medicine lists at discharge was successful. however, both subsections need to work further to ensure continuation and improvement of the process. furthermore we found varying results in the writing of medicine lists depending on subsection. still, regardless of the individual results of the two subsections there is big room for improvement to ensure that sufficient medical information is included in the discharge papers. please specify your abstract type: descriptive abstract (for projects) background and objective: from july clinical pharmacists began conducting medication histories and reviews (pharmacist notes) at the emergency surgical ward (esw), north zealand hospital (nzh). inclusion criteria are acute patients using c drugs or c risk drug (antidiabetics, anticoagulants, antipsychotics, benzodiazepines, opioids and digoxin). the aim of the service is to identify drugrelated problems and secure correct medication reconciliation between the medicine the patient is admitted with and the medicine in the electronic medication system (ems) in the hospital. the service ensures that the patients' medication follows across healthcare sectors. the objective is to determine if the discrepancies between the medicine the patient is admitted with and the medicine in ems (documented in the pharmacist notes) are used by the physicians. in addition to determine if the pharmacist interventions increase the physicians' acceptance rate of the discrepancies. design: data were collected at the esw at nzh (capacity of beds). data consist of pharmacist notes conducted from august to may . pharmacist notes were compared to the patient record and ems to identify if the pharmacist notes were considered by the physicians. in order to increase physicians' acceptance rate of the discrepancies suggested in the pharmacist notes, interventions were made according to the model for improvement. throughout the period, the focus was on oral delivery of the pharmacist notes. in december the pharmacist optimized the clinical relevance of the discrepancies, by creating and testing a list of products (including vitamins, herbal drugs, glucosamine etc.) which the pharmacist should not intervene on. in december the pharmacist also started to follow up on the pharmacist notes not considered by a physician the previous day to ensure that the physician considered the discrepancies. results: there were identified discrepancies between the patients' actual medication at admission and ems at the hospital in patient records ( . discrepancies per patient). in total discrepancies were accepted by the physicians ( . discrepancies per patient). the physicians' acceptance rate was based on the acceptance of one or more of the discrepancies in the pharmacist note. baseline data were collected from august to november , where out of pharmacist notes were accepted by the physicians resulting in an acceptance rate of . %. from december to may the interventions made by the pharmacist contributed to an increase in acceptance rate to . % ( out of notes accepted). if the pharmacist notes were not delivered orally to the treating physician the acceptance rate was % ( out of notes accepted). conclusion: the pharmacist interventions contributed to an increase in the physicians' acceptance rate of discrepancies from . to . %. a result indicating that the pharmacist notes contributes to an increase the quality of the medication process across sectors. hp-pc : how the centralization of medicines manufacturing enable to generalize the pharmaceutical validation? samantha oses * , soizic vandierdonck, vincent servant, dominique breilh please specify your abstract type: research abstract background and objective: the centralization of the reconstitution of injectable anti-infective drugs enhance to decrease costs and several risks. this minimization of the risks operates at several levels such as i) reduction of the staff exposure and external contamination of preparations during the reconstitution phase (with controlled atmosphere areas, isolators, etc.), ii) improvement in the quality of the management of infective diseases thanks to a pharmaceutical validation systematically performed after the prescription and before the reconstitution phase. the main objective of the study was to describe and quantify pharmaceutical validation on injectable anti-infective drugs prescriptions restored in a pharmaceutical reconstitution unit. setting and method: an observational descriptive study was carried out on each prescription with at least one injectable anti-infective drug that has to be reconstituted before administration. the process was as follows: -prescription by the physician on an electronic prescription software, -pharmaceutical validation and if necessary pharmaceutical intervention (pi) made by phone call, -reconstitution at the pharmacy, -administration to the patient. the pharmaceutical validation methodology followed the french society of clinical pharmacy (sfpc) guidelines ''prescriptions screening and analyses level'' published in the good practices of clinical pharmacy and one resident and one pharmacist were devoted to the activity every day. main outcome measures: the pharmaceutical validation was quantified by the number of pi by patient, which were categorized according to the sfpc guidelines. results: during months, a total of pi were collected. they concerned patients with an average of . pi per patient. among them, . % ( ) concerned paediatric population. antibiotics were involved in . % ( ) then followed by . % ( ) cases ( . % ( ) biological assessment issues, . % ( ) absence of therapeutic drug monitoring (tdm) and . % ( ) the drug hasn't been adapted to the weight), dosage adjustments in . % ( ), information missing concerning the treatment indication in . % ( ) and miscellaneous pi in . % ( ) such as wrong clinical service on the prescription, etc. approval rate of physicians was . % ( ). conclusion: this study has shown that even if prescriptions were secured by electronic prescription software, the pharmaceutical validation remains essential. in that case, the centralization of the reconstitution of injectable anti-infective drugs enabled to generalize this activity on all prescriptions of the hospital. however, the pharmaceutical validation was focused only on anti-infective drugs, that was not fully efficient and must be extended to the whole prescription. it is a priority to develop a comprehensive and exhaustive validation on every medical prescription; however, this activity is highly time consuming and needs larger and more trained staff. hp-pc : the start/stopp criteria as a helping tool to the pharmacists' medication review in the acute admissions unit of the regional hospital in horsens hans pedersen * please specify your abstract type: research abstract background and objective: polypharmacy occurs often increasing the need for patients' medications to be reviewed. the start/ stopp criteria help detects potentially inappropriate prescriptions in older people. in this study we aimed to measure and categorize the different start/stopp criteria found in medication reviews in the acute admissions unit of horsens and the acceptance rate. setting and method: patients admitted to the acute admissions unit were selected based on their age and the number of prescriptions in a period of months. patients years or more which received six or more drugs were included in the study. only patients who later were transferred to another medicine ward were included in the study. the pharmaceutical medicine review was performed by a clinical pharmacist using minimum two different sources; the electronic medical record and medication-lists. the guideline of pharmaceutical medicine review in the hospital pharmacy central denmark region was used as the standard-guideline. in addition, thestart/stopp criteria version was used. main outcome measures: the number of start/stopp criteria found in medication reviews. the different start/stopp criteria were scored equally with one point each. results: patients, males and females, out of , were included. the mean age was years and the patients received in average prescribed drugs. at admission the average number of stopp criteria were . ± . and . ± . for the start criteria. in average, % of the purposed stopp criteria were accepted by the physicians. the most frequently accepted stopp criteria were in the category of drugs that predictably increases the risk of falls in older people. the benzodiazepines where the most common drugs to be discontinued. in the start category, % of the suggested start criteria were accepted, which included: calcium and vitamin d supplement, beta- agonist and bisphosphonate. conclusion: the present study demonstrates that it was possible to integrate the start/stopp criteria as a helping tool in the medication reviews in the acute admissions unit of horsens. the start/stopp criteria were found within the different categories, however only a minor part of the registered start/stopp criteria were accepted by the physician. please specify your abstract type: research abstract background and objective: the objective of this work is to assess prescribing practices of somatostatin analogues in a surgery department, and to analyse the conformity of switching from immediateacting octreotide to the long-acting release (lar) form, in accordance to laboratories' guidelines. setting and method: retrospective observational study. a focus was realized on patients admitted in a digestive surgery unit between january and december , . the patients' medical records were reviewed for clinical features, diagnosis workup and treatment strategies. main outcome measures: medical records for patients with diagnosis of gastro-entero-pancreatic or endocrine tumors who had received injections of lar octreotide during hospitalization were reviewed and the economic impact of prescriptions errors has been evaluated. results: of the evaluated patients, ( %) were hospitalized in surgery digestive unit; mean age at first administration of octreotide was years and % were male. the male and female ratio was . : . reasons for hospitalization were: digestive system neoplasms ( %), fistula ( %), intestinal obstructions ( %) and other pathologies ( %). of the patients treated with octreotide, ( %) received a lar form. only four patients received doses in accordance with guidelines: one at mg/month lar form and three at mg/month lar form, after having respectively been treated by intravenous octreotide at and mcg/day during - days. medical prescriptions of the remaining patients did not comply: all patients received mg/month after an intravenous treatment of mcg/day, instead of mg/month. from a financial perspective, these misuses have led to an additional cost of . euros for the hospital, excluding tax ( mg: . €/unit and mg: €/unit). conclusion: despite the publication of octreotide release form proper use recommendation in our hospital, % of patients of digestive unit are not right treated. a new guideline will be written added by doses of long-acting release and economic data. this work will be transmitted to specialists by clinical pharmacists. hp-pc : pharmaceutical process for intrathecal analgesia in clinical oncology practice vivien pigeon * , guillaume binson, claire grignon, antoine dupuis please specify your abstract type: descriptive abstract (for projects) background and objective: in some cases, patients with cancer pain remain painful despite the use of high dose of intravenous opioids and intrathecal analgesia becomes the ultima recourse to manage acute pain. until , intrathecal syringes were prepared by nurses in the unit care which involve a risk for patients. therefore, the aim of this work is to describe the set-up of the prescription and preparation process with the potential benefits for the safety. design: multidisciplinary concertation took place between pharmacists, physicians and surgical teams and several points were discussed to secure the process: • identification of patients with high level of infection risk; • identification of critical points of the pharmaceutical process; • validation of quality control and drug stability studies regarding drug compounding involving morphine, ropivacaine, baclofen and clonidine, alone or in admixture. results: multidisciplinary concertation lead us to define the most important points to set up the pharmaceutical process for intrathecal analgesia: • chosen patients are cancer patients; • implementation of a prescription software to secure the prescription step; • production of syringes by the pharmacy department implying several criteria: • preparation in controlled atmosphere area; • training of pharmacy technicians; • implementation of quality control and drug stability studies at °c in syringe over h and at °c in pumps over month; • microbiological control and bacterial endotoxin level. the implementation of a pharmaceutical process for intrathecal analgesia gave us the opportunity to reorganize the care of cancer patients tolerant to high dose of opioids. in this process, the pharmacy department plays a major role leading to decrease the risk of infections and errors of dosing. ingrid plessala *, , xavier deviot , thomas sidibe , zohra mostefaï , michèle minvielle , marta wyrtwal , roselyne gervais pharmacy, geriatrics, saint-denis hospital centre, saint-denis, france please specify your abstract type: descriptive abstract (for projects) background and objective: proton pump inhibitors (ppis) are indicated in gastro-oesophageal reflux and peptic ulcer disease. they are widely prescribed, often in off-label indications. the objective of this work was to reassess ppis prescriptions in collaboration with geriatricians. proton pump inhibitors (ppis) are indicated in gastro-oesophageal reflux and peptic ulcer disease. they are widely prescribed, often in off-label indications. the objective of this work was to reassess ppis prescriptions in collaboration with geriatricians. design: prospective study in three geriatric wards. the study included ppis treated patients from these three geriatric wards. dose, indication of the ppi, age, gender and duration of treatment have been recorded for each patient. the relevance of each ppi treatment has been reassessed by a geriatrician, a pharmacist and a junior pharmacist, regarding the indication and the patient's clinical condition. following this re-evaluation, three situations arose: • to maintain ppi at the same dose ( mg or mg) • to maintain ppi but half dose (from mg to mg) • to stop ppi corrective actions have been recorded in patients' files to allow their traceability. results: patients were included in the study. % of ppis prescriptions were off-label, % had no indication mentioned in patient's file and % were conform to the marketing authorization. % of patients have been on ppis medication longer than months, which is the recommended treatment' duration in france, % longer than a year and % longer than years. in collaboration with the geriatricians, ppi prescriptions were maintained for % of patients. we reduced the dose in % of cases. finally, we decided to stop a third of the ppis prescriptions. conclusion: ppis prescriptions are often longer than recommended. this can lead to side effects for patients. in france, lack of new recommendations since may explain this frequent misuse of ppis. there is also a reserve from doctors to stop these treatments, especially with fragile patients. in our case, the relevance of each ppi treatment was re-evaluated in three geriatric wards and we succeeded in shortening and stopping ppis medications in half of the situations. to assess the impact of this action on our geriatricians, a new review of ppis prescriptions relevance is programmed in . ppis prescriptions are often longer than recommended. this can lead to side effects for patients. in france, lack of new recommendations since may explain this frequent misuse of ppis. there is also a reserve from doctors to stop these treatments, especially with fragile patients. in our case, the relevance of each ppi treatment was re-evaluated in three geriatric wards and we succeeded in shortening and stopping ppis medications in half of the situations. to assess the impact of this action on our geriatricians, a new review of ppis prescriptions relevance is programmed in . hp-pc : oral anticoagulants and heparin for children: standardized protocols for prescription, dispensation and administration alexandra liauzu , marie-françoise hurtaud-roux , ronan bonnefoy , caroline farnoux , philippe sachs , theresa kwon , olivier bourdon , sophie ajzenfisz , sonia prot-labarthe *, pharmacy, hématologie clinique, ap-hp hôpital robert-debré, cardiologie, néonatologie, ap-hp hôpital robert debré, réanimation pédiatrique, ap-hp hôpital robert-debré, néphrologie, pharmacy, ap-hp hôpital robert debré, coordonnateur de la gestion des risques associés aux soins/ responsable du système de management de la qualité de la prise en charge médicamenteuse, ap-hp hôpital robert-debré, paris, france please specify your abstract type: research abstract background and objective: high-alert medications (ham) are medications that are associated with a high risk of serious harm if used improperly. we already identified paediatric ham used in our institution to identify safety measures for their use. anticoagulants and heparin were part of these high-alert medications. we aim to write standard protocol of use for low weight heparin and oral anticoagulant used in our mother-child teaching hospital. our secondary objectives were to decrease medication errors, anti-xa and inr unexplained variability and to help nurses to administer the drugs (standard dilution, oral solution available) setting and method: we carried out a literature search on pubmed Ò , on websites of several learned or professional societies and agencies. the results of the literature search were compiled on written protocol and presented to our institute drug safety-steering committee composed of four doctors, two head nurses, two pharmacists, and one risk manager. main outcome measures: not applicable. results: the protocols concerned enoxaparin, tinzaparin, warfarin but we chose to also include protamine. the most difficult issue was to have standardized dilution and protocol for all ages and weight: from premature to adolescents and all units of care (from cardiology to intensive care unit, nephrology and neonatology). we took into account the administration errors we had in our hospital and the preexisting protocol to avoid any drastic error-prone change. the final version of these protocols will be presented on the final communication with web link to upload them. conclusion: for now we did note evaluate the impact of these protocols but a before/after analysis of error reports and users evaluation will be done. however, these protocols can help all health professionals working in paediatric units for benchmarking. hp-pc : does a hospital formulary system impact timely medication administration and quality of inpatient care? anne-valérie putallaz *, , vera jordan-von gunten , pierre-auguste petignat , pierre turini , johnny beney division of pharmacy, institut central des hôpitaux, division of internal medicine, medical coordinator for quality of care and patient safety, hôpital du valais, sion, switzerland please specify your abstract type: research abstract background and objective: the prevalence of drug omissions is often underestimated but their impact can be clinically relevant. we hypothesized that delays in the administration of non-formulary/nonstored drugs could impair the quality of care. the aims of this study were: °to determine the time between the prescription and the administration of the first prescribed dose and, if applicable, to calculate how many doses were omitted. °to analyse the clinical relevance of the identified delays. setting and method: three months retrospective study of electronic records of patients hospitalized on the internal medicine wards of a network of hospitals supplied by a centralized pharmacy. this pharmacy is located in one of the sites; other sites are - km apart. main outcome measures: . for the main hospital site and the three distant sites: • median time between the prescription and the administration of the first prescribed dose • mean number of omitted doses for formulary and non-formulary/ non-stored drugs. . categorization of patient's harm caused by the delays of timecritical drugs, according to the ncc-merp taxonomy of medication errors. results: ' prescriptions were analysed. calculated delays for non-stored/non-formulary drugs were longer than for formulary drugs. however, the median time to administration is less than h for both formulary and non-stored/non-formulary drugs; and more than % of formulary drugs and around % of non-stored/non-formulary drugs were administered within h following their prescription. there was no significant difference in the mean number of omitted doses or in the delays between the site where the centralized pharmacy is located and the other sites, except for one of them. a delay representing . or more omitted doses was found for ( . %) prescriptions. among them, only were considered potentially clinically relevant. none of them caused severe harm to the patients involved. conclusion: in our setting, non-stored/non-formulary drugs take more time to be delivered than formulary drugs, but more than % of formulary drugs and around % of non-stored/non-formulary drugs are administered within h following their prescription. none of the patients who experienced delays underwent severe harm. our study showed that delays also occur for formulary drugs but no systematic cause of omission was identified; further studies should focus on all dose omissions during hospitalization. penelope randuineau *, , roger jeremy , lauriane cornuault , anne lecoeur , franck lemercier , isabelle javerliat , thomas tritz service de pharmacie à usage intérieur, service de chirurgie vasculaire, hôpital ambroise paré, boulogne-billancourt, france please specify your abstract type: descriptive abstract (for projects) background and objective: a french national survey of inpatient adverse events reveals that nearly half of adverse drug events (ade) are preventable. medication errors behind these ade occur mainly during the transition steps of care pathway. in this context, medication reconciliation process has been implemented in our vascular surgery department. the objective of this study is to identify unintentional discrepancies (uid) and assess their potential clinical impact design: a pharmacy resident or a pharmacy student reconciliated patients: aged older than or with at least five chronic treatments at admission or suffering from chronic diseases. patients were considered reconcilable if at least two reliable sources on usual patient's treatment were available. these many sources of data (patient interview, prescription or interview of general practitioner, reference dispensary, drug box …) were compared to the admission prescription during the first h of hospitalization to detect and correct uid. based on gravity scale promoted by the french high authority of health, two pharmacists (a resident and a senior) and a vascular surgeon reviewed every uid in order to define their potential clinical impact. the uids were considered minor if it leads to no consequence for the patient, clinically significant if it leads to essential monitoring, major if it could cause temporary clinical consequences, and critical if it could result in permanent clinical consequences or the involvement of the prognosis. results: between february th and may st , a total of patients have been reconciled. patients were excluded due to a lack of reliable sources. mean age was . years old (± . ) and sex ratio m/f was . . % of the reconciliated patients' admissions were scheduled. the mean number of medication was . (± . ). patients ( %) had at least one uid and the mean uids per patient was . (± . ). the most common types of uids were omission ( %), incorrect dose ( %) and incorrect administration frequency ( %). more than % of these uid presented a potential clinical impact: an adverse effect (high blood pressure, hyperglycaemia) was observed for nine patients and lead to therapeutic optimization and monitoring; uid were considered to have potential clinically significant impact ( %), a potential major impact ( %) and a potential critical impact. conclusion: these results appear consistent with those reported in literature. vascular surgeons have appreciated the approach and would like systematic medication reconciliation before surgery. as a major part of admissions were scheduled, we would like to establish the reconciliation before the patient's hospitalization every time it's possible. this new organization should facilitate the care pathway before surgery and decrease preventable postoperative adverse events. hp-pc : delirium in elderly patients: successful use of melatonin gaëlle jouin , aurélie reiter-schatz , pierre bentzinger , fatem-zohra laalou , bénédicte gourieux *, pharmacy-sterilization, orthopedic's intensive care unit, university hospital of strasbourg, strasbourg, france please specify your abstract type: descriptive abstract (for projects) background and objective: postoperative delirium happens to about one-third of elderly patients and is a major cause of morbidity and mortality. it is reported that haloperidol, an antipsychotic, has been the agent of choice for managing delirium. however, it induces cerebrovascular adverse effects and greater mortality. the hyperactive type of delirium is known to be associated with a low melatonin level and the loss of a normal melatonin secretion rhythm. the postoperative administration of melatonin to elderly could decrease the symptoms of delirium. the purpose of this study was to evaluate melatonin effectiveness in a cohort of patients suffering from postoperative delirium. design: a retrospective study of melatonin prescriptions has been conducted over a months period. medical background, type of surgery, symptoms of delirium, use of antipsychotics and benzodiazepines have been studied in all patients who received melatonin in an orthopaedic surgery unit. length of hospital stay, time between delirium and melatonin administration and the effect of melatonin had been evaluated. results: a total of patients were included: average age was . years ( - ), sex ratio m/f = . twelve patients ( %) were hospitalized because of an infection (prosthesis or osteoarticular). in % of cases (n = ), the prescription of melatonin was started when the patients were hospitalized in our intensive care unit. nine patients ( %) were under chronic treatments like benzodiazepines or antipsychotics. the average length of hospital stay was days ( - ). melatonin was started on an average of days after surgery , and administered at the dose of mg xr in the evening, during an average of days ( - ). cognitive impairments requiring a prescription of melatonin were: confusion ( %, n = ), agitation ( %, n = ), daytime sleepiness ( %, n = ), temporal-spatial disorientation ( %, n = ), nocturnal awakening ( %, n = ), hallucination ( %, n = ), difficult falling asleep ( %, n = ). the average time to recover from confusion was days, agitation days, daytime sleepiness days, temporal-spatial disorientation days, nocturnal awakening days, hallucination days and falling asleep days. melatonin treatment helped stopping benzodiazepines treatment in six patients ( %). conclusion: after administration of melatonin, delirium symptoms were improved for all patients and benzodiazepines treatment stopped for six patients. earlier prescription of melatonin could regulate sleep-wake cycle and reduce the duration and incidence of delirium. please specify your abstract type: research abstract background and objective: denosumab (xgeva Ò ), a fully human monoclonal antibody targeting rankl, which inhibits bone resorption, is indicated to prevent skeletal complications in patients with solid tumors and bone metastases. about % of patients develop hypocalcaemia, a common adverse event that may induce spasms, muscle cramps, paraesthesia, prolonged qt interval, tetany, convulsions… we report the management of ionic supplementation and physicochemical incompatibilities in a case of hypocalcaemia due to denosumab. setting and method: the clinical case was analysed with the pharmacovigilance regional center. main outcome measures: a year old patient, with nodal and bone metastasis in prostate cancer, was treated with denosumab (stopped with the last injection months before, on the th of march). he went to emergency on the th of may with asthenia, anorexia, nausea, diarrhoea, qt prolongation. biological results showed hypocalcaemia (corrected calcaemic = . mmol/l) and hypophosphatemia (phosphorus \ . mmol/l). concomitant calcium and phosphorus intravenous supplementation started with loading doses ( g of calcium and . g of phosphorus) and then a week of following daily intakes: phosphorus ( g iv and . g oral); calcium ( g iv and . g oral). however, low-serum corrected calcium and phosphorus levels persist at . mmol/l and . mmol/l. results: incompatibility between phosphorus and calcium by formation of soluble or not-soluble complexes is described in literature. in our case, calcium and phosphorus were mixed in a same infusion. after a week of supplementation, calcium infusion is continued with increased dose ( g/day) and phosphorus infusion is stopped. phosphorus oral supplementation remains stable ( . g per day); calcium oral supplementation is increased ( . g per day). h between intakes is applied to avoid digestive complexation. h later, corrected calcium levels are normalized at . mmol/l and phosphorus levels are still low. therefore, as hypocalcaemia due to denosumab induced a secondary hyperparathyroidism and thus hypophosphatemia; phosphorus levels are expected to increase subsequently. conclusion: this case report shows that recurrent hypocalcaemia with denosumab is possible few months after administration. supplementation with large amount of calcium is needed and administration methods may impact the effectiveness of supplementation. indeed, it seems that the incompatibility between phosphorus and calcium did not allow an effective supplementation. gunnhild langdal *, , , ida rudberg , lone holst , anne-lise sagen major , central norway hospital pharmacy trust, Å lesund, centre for pharmacy, university of bergen, bergen, møre og romsdal health trust, Å lesund, norway please specify your abstract type: descriptive abstract (for projects) background and objective: drug interactions (dis) can cause side effects and lack of therapeutic effect. the objective of this study was to describe the prevalence of dis at the medical department of Å lesund hospital, and to investigate how dis were managed by clinical pharmacists and physicians. design: at the medical department, Å lesund hospital, clinical pharmacists serve seven out of ten wards, from which patients were included during a five weeks period. the clinical pharmacists selected patients for screening for potential dis (www.interaksjoner.no) as int j clin pharm ( ) : - usual (= pharmacist group). detected dis were classified according to a predetermined classification system, and it was registered whether the physician implemented suggested changes in prescription. for patients not selected by clinical pharmacists (= non-pharmacist group), a pharmacy student performed the search for dis. results: in total patients were admitted. on average, each patient had . dis, and . % of the admitted patients had at least one di. the prevalence of dis was significantly higher among the patients in the pharmacist group compared to the patients in the non-pharmacist group (median@@@ vs. , respectively, p \ . ). the groups differed significantly regarding number of drugs used, age, duration of hospital stay and number of warfarin users. . % of the dis detected in the pharmacist group were discussed with the physician. the remaining . % were considered not necessary to discuss for various reasons e.g. because they were considered not clinically relevant ( %) or already adjusted for in clinical practice ( %). for dis the clinical pharmacist suggested a change in prescription, and of these suggestions ( %) were implemented by the physician. conclusion: just over half of the patients were selected by the clinical pharmacist for screening of dis, and the pharmacist seemingly made a reasonable priority of patients with many drugs, old age, a long hospital stay and users of warfarin. only of dis was discussed with physicians. this indicated that pharmacists do a considerable work in assessing the relevance of dis before discussing with the physicians. it also seemed that changes in prescription suggested by the clinical pharmacist were reasonable. hp-pc : securing the paediatric use of oral chemotherapy: a proactive risk assessment samia mouffak *, , linda an , anne fratta , anne auvrignon , , nadia marquis , karine morand pharmacy, risk management committee, hematology, armand trousseau hospital -aphp, paris, france please specify your abstract type: descriptive abstract (for projects) background and objective: oral chemotherapy is an important part of the therapeutic strategy in childhood cancer or haematological malignancy. it also represents an emerging risk area in oncology practice. several medications errors involving oral chemotherapy were reported in children of our onco-haematology department, fortunately without clinical consequences. nevertheless, the potential severity of such errors led us to implement a failure analysis of the paediatric oncology care pathway in order to identify and prevent potential risks, and secure the paediatric use of oral chemotherapy. design: we conducted a failure modes, effects and criticality analysis (fmeca) which is a proactive risk assessment approach. first, process maps were detailed for each step of the oncology care pathway. it was performed by a multi-disciplinary group composed of physicians, coordinating nurse, hospital pharmacists and pharmacy resident. then, for each step of the medication-use process, the team identified the failure modes, their main causes and effects. finally, participants rated the expected severity, frequency and detectability for each failure mode, assigning a score on a five-point scale. a risk priority number (rpn) was then calculated by multiplying those three indexes. the risks getting a high rpn were categorized as critical risks and have been the object of safety improvements. results: failure modes were identified, including critical risk failure modes. critical failures were related to hospital discharge prescriptions and were about the dispensation of oral chemotherapy by pharmacy assistants. most failures were due to prescriptions heterogeneity, lack of clinical information reported on prescriptions, and lack of training of pharmacy assistants in reading oral chemotherapy prescriptions and in mistake detection. two improvement strategies were implemented. first, physicians' awareness led to the harmonisation of practices and to the standardisation of discharge prescriptions. then, to enhance pharmacy assistants' abilities, an educational program on oral chemotherapy dispensation was planned. conclusion: the implementation of a fmeca has highlighted the most critical risks of oral chemotherapy medication-use process. the awareness of all caregivers and the targeted changes in our practices allowed us to improve the safety of the paediatric oncology care pathway. please specify your abstract type: descriptive abstract (for projects) background and objective: the purpose of this study was to investigate if medication reconciliation and medication review, by using the integrated medicines management (imm) model, were suitable to assure the quality of patients' medical treatment at a gastrointestinal surgical ward. furthermore, to analyse frequency, type, handling and clinical relevance of medication discrepancies (mds) and other medication related problems (mrps). design: patients, above years of age, from two departments at a gastrointestinal surgical ward at a norwegian university hospital were included consecutively. medication reconciliation was performed at admission by a clinical pharmacist. the resulting medication histories were compared with the medications documented in the medical records. mds were detected and categorized. thereafter the clinical pharmacist identified mrps by reviewing the medical records systematically and categorized the revealed mrps. mds and mrps were presented for the physician with proposed solutions. the physician's actions to manage the mrps were registered. later a multidisciplinary team assessed the clinical significance of mds and mrps in a subset of patients. results: a total of patients were included. overall, mds and mrps were identified. at least one md was revealed in % of the included patients, whereas at least one mrp was identified in % of the patients. the most frequent type of mds was omission, whereas mrps most often were related to medications that were considered unnecessary. totally, % of the mds and mrps were discussed with the treating physician. the physicians followed the pharmacist's input in % of the discussed md-cases and % of the mrp-issues. longterm consequences of mds and mrps were considered more serious than short-term consequences for the patients. conclusion: medication reconciliation and medication review revealed, solved and prevented a large number of mds and mrps in this study. the results emphasize that pharmacist involvement, by using the multidisciplinary imm-model, contributed to more correct medical records and furthermore to quality assurance of the patients' medical treatment at a gastrointestinal ward. hp-pc : prevention and management of drug interactions in oncology day-hospital: results from a months study involving drug assessment and pharmaceutical report to oncologist pauline-saraï zeller *, , chloé hugard , céline mongaret , , juliette vella-boucaud , antonin maréchal , olivier bouché , dominique hettler , florian slimano , pharmacy, oncology day hospital, university hospital reims, clinical pharmacy, faculty of pharmacy, reims, france please specify your abstract type: research abstract background and objective: quality during transitions of care is a major concern in drug safety for patients. traditional hospitalization allows to reconciliation medication but there is not possible for dayhospitalization (patient's hospitalization short time and no outpatient medication prescribe by oncologists). however, lack of communication between health professionals may expose patients to drug-drug interactions (ddi). while ddi between oral antineoplastic and other drugs are well known, there is a lack of knowledge about ddi between parenteral antineoplastic (ak) and other drugs. in this pilot study, we aim to investigate prevalence and characteristics of ddi between ak and other drugs in real life and to propose a pharmaceutical report model to enhance patient's drugs safety. setting and method: during months, all new oncologic patients (thoracic and digestive) receiving chemotherapy in day-hospital have been recruited by clinical pharmacist. first it was conducted a patient-clinical pharmacist interview and carried out the best possible medication history (bpmh) by contacting at least three different sources of drug information. then, the bpmh has been confronted with oncologic treatment (including concomitant medications such as like antiemetic) with support at least with two different database of ddi analysis. finally pharmaceutical recommendations in order to manage potential relevant ddi were reviewed with oncologists then reported and inserted in personal health record (phr). main outcome measures: prevalence and description of potential clinically relevant ddi in an ambulatory oncology population. results: from november, to april, n = oncologic patients were included with following characteristics: mean age of . , sex ratio : , majority of oncologic thoracic localization ( %). number of oncologic concomitant medications per patient was . ± . (mean ± standard deviation). patients present an average of . ± . comorbidities (excluding cancer) and . ± . linked medications per patient. pharmaceutical analysis revealed potential clinically relevant ddi ( . ± . per patient): % of them concern antiemetics (ondansetron and aprepitant): pharmaceutical interventions were formulated (including recommendations to adapt chronic treatment) and % of them involved biological monitoring (for renal function, inr, potassemie or magnesemie). conclusion: our pilot study confirms high prevalence of ddi between oncologic and non-oncologic drugs. clinical pharmacy services with bpmh performing and pharmaceutical recommendation appears to be useful to enhance patient' drug safety in oncology dayhospital. we currently are deploying our study in order to convey a pharmaceutical letter to general practitioner and community pharmacist. hp-pc : loading dose of anti-infectives: elaboration of a tool helping pharmaceutical analysis julie soyer *, , cécile sanchez , guillaume beraud , nicolas venisse , pauline lazaro , antoine dupuis pharmacy, infectiology, pharmacokinetics, university of poitiers, poitiers, france please specify your abstract type: descriptive abstract (for projects) background and objective: the recent data on vancomycin and ceftazidime confirm that continuous infusion is the best way of administration of these antibiotics. moreover a loading dose before the administration is required for the antibiotics to prevent from the infratherapeutic period at the start of infusion and limiting the risk of resistance emergence. long half-life antibiotics and antifungals also require a loading dose to be effective. the aim of this study is to analyse the prescriptions of anti-infective requiring a loading dose in order to develop a tool to help pharmaceutical analysis. design: a prospective observational study was carried out during days in units. initially, pharmacists, residents and students were trained (role of the loading dose, drugs concerned). then, all patients with anti-infective requiring loading dose were included. some data were collected: weight patient, creatinine clearance, loading dose or not, dose, administration mode, monitoring of steady state concentrations (vancomycin and ceftazidime) and dose adjustment. the results were analysed and compared to bibliographic research before discussion during a multi-disciplinary meeting (pharmacists, infection control specialist and pharmacokinetic specialist). finally, a list of relevant pharmacist interventions was selected. results: out of the patients, were enrolled for prescription of anti-infective requiring loading dose. twenty-six prescriptions including vancomycin, ceftazidime, the others fluconazole, caspofungine, voriconazole and posaconazole. concerning vancomycin, the loading dose was prescribed in % of case, monitoring of steady state concentrations was performed in % of case and dose adjustment after first dosage was required in % of case. selected pharmacist interventions were: • to favour continuous infusion (excepted paediatric) • to keep loading dose at full dose even in patient with renal failure • to monitor steady state concentrations after the first h in patient with renal failure or obesity • to adapt dosage when the target concentration is not reached concerning ceftazidime, the interventions were: • to recommend continuous infusion: g/ h after loading dose of mg/kg • to monitor steady state concentrations in patient with renal failure a total of interventions (dosage, adaption of posology at the monitoring, patients with renal failure, obese, paediatric patient, administration…) were identified by the group of experts. conclusion: this study allowed creating a recap data sheet for students and hospital pharmacists. the selected interventions will allow the harmonization of practices. these recommendations have been validated by the commission of the anti-infective. finally, this study shows that the pharmacist has a key role in the management of antiinfective requiring loading dose. hp-pc : assessment of potentially inappropriate medications in orthogeriatric patients using the rasp list the detection of inappropriate prescribing. the objective of this study was to investigate if the rasp list (rationalization of home medication by an adjusted stopp list in older patients), an explicit screening method adapted to the belgian context, can be used to reduce the number of potentially inappropriate medications (pims) in orthogeriatric patients. setting and method: single-centre, interventional study conducted at the orthogeriatric department of the uz brussel, a -bed university hospital. the rasp list was first applied by a last year pharmacy student to the admission medication of orthogeriatric patients hospitalised in october . after potential adaptations to the medication by a liaising geriatrician, the rasp list was additionally applied by the same pharmacy student to the discharge medication of these patients. main outcome measures: detection and reduction of the number of pims. results: in total, orthogeriatric patients, from whom an informed consent was obtained, participated in this study. on admission, a total of pims were detected in this population. at discharge, the number of pims decreased to . the median number of pims per patient decreased from (on admission) to (at discharge). this difference was statistically significant (p \ . ; wilcoxon signed rank test). drugs of atc class n (nervous system) were responsible for the highest number of pims. conclusion: pims can be detected and reduced in the hospital using the rasp list. a structured and collaborative medication review between (student) pharmacists and physicians appears a good approach to reduce the number of potentially inadequate drugs. nevertheless, more research is necessary to substantiate this further as well as to assess the clinical impact of the findings. hp-pc : impact of implementing ward based dispensaries across a hospital site on both service delivery and patient care michelle sullivan, paul wright, christopher watson, malcolm smith, sotiris antoniou * please specify your abstract type: descriptive abstract (for projects) background and objective: waiting for medication at discharge is often quoted as a key factor for delaying patients leaving hospital. feedback from service users (patients and healthcare professionals) was for a more patient facing pharmacy service. this led to a phased installation of remote dispensaries on wards within the hospital to supply medicines. this new and innovative service enabled the supply function to be fully co-ordinated on the ward. this model was initially implemented on wards, which coupled with one-stop dispensing meant % of discharges require nothing to be supplied at the point of validation, % of discharge prescriptions meeting key performance indicator of being dispensed and ready within h with average turnaround time of min for a discharge prescription and a reduction in missed doses- . % in september to . % in march . this success prompted further installation of remote dispensaries in all clinical areas on site. design: implementation included; purchasing hardware, pharmacy labellers, locating appropriate computer terminals and stock cupboards. the main pharmacy labelling and stock control system was fully integrated at ward level, enabling the automatic reordering of replacement stock. identification of items and quantities to stock for remote dispensaries was also needed prior to role out. there was a need to scope staffing requirements including the redeploying of roles from a main inpatient pharmacy to patient facing areas. results: over items are supplied at ward level each month via satellite pharmacies for all wards, equating to more than % of the total dispensing workload for the site allowing for pharmacy staff to be redistributed from dispensary to the ward. this offered the benefit of being more patient facing and supporting other initiatives such as patient counselling and medicines reconciliation. the project has impacted the pharmacists as it has enabled them to focus on clinical aspects of service delivery, including attendance of ward rounds as well as supporting a ward team approach with the pharmacy technician. results of missed dose audit from june shows across the site % ( ) wards scored below the national . % target and ( %) wards had no unintentional missed doses. conclusion: ward based dispensing has led to pharmacists and pharmacy technicians being % ward based. as a constant presence on the ward, the team offer consistency within the pharmacy service for patients, nursing and medical staff. impact of pre-discharge planning has been beneficial to nurses, patients and work flow of the pharmacy teams. ward based dispensing has improved supply at discharge as well as promoting a more patient facing pharmacy service that has seen the pharmacy team instilled as integral to service delivery at ward level. kutay demirkan * , nursel surmelioglu, aygin bayraktar-ekincioglu clinical pharmacy, hacettepe university, ankara, turkey please specify your abstract type: research abstract background and objective: hacettepe university hospitals clinical pharmacy unit was established in april . this unit runs its services by clinical pharmacy postgraduate students under the supervision of two qualified clinical pharmacists as part-time and oncall basis, in adults, paediatrics and oncology hospitals. the aim of this study was to identify drug related problems and describe its management strategies in inpatient and outpatient settings by pharmacists in clinical pharmacy postgraduate education program. setting and method: during a total of months study period (period i: february-july , and period ii: november-february ), clinical pharmacy postgraduate students followed patients for - times in a week in different services in hospitals (internal medicine, internal medicine intensive care, infectious diseases, neurology intensive care, paediatric bone marrow transplant/haematology unit, paediatric intensive care, geriatrics and nutrition units) and drug related problems were identified and pharmacists' recommendations were listed. main outcome measures: determination and evaluation of drug related problems by pharmacist in hospital. results: a total of recommendations was provided for patients. those recommendations were classified as alteration or discontinuation of drug treatment ( . %), dose adjustment ( . %), change in drug administration time ( . %), inadequate treatment ( . %), healthcare staff training/consulting ( . %), patient education ( . %) and error/deficit in therapeutic drug monitoring ( . %). a majority of recommendations (n = ) were related with alteration or discontinuation of drug treatment provided mainly in departments of internal medicine (n = , geriatrics (n = ), neurology intensive care (n = ) and infectious diseases service (n = ). the following main reason for pharmacist's recommendation was related with dose adjustment (n = ) which were provided in departments of internal intensive care (n = ), infectious diseases service (n = ), neurology (n = ) and internal medicine (n = ). conclusion: clinical pharmacy practices are being carried out effectively in many services, particularly in internal medicine services, internal medicine intensive care unit and infectious diseases services. a collaborative and bed-side education in postgraduate programs in clinical pharmacy help to increase the knowledge and skills of students in real life circumstances and also maintain safe and effective drug therapy by an involvement of clinical pharmacists in hospital services. hp-pc : development of a tool to help pharmaceutical analysis in patients with hepatic failure barbara troussier *, , eric gautier , astrid bacle , florian charier , christine silvain , pauline lazaro pharmacy, gastroenterology, hepatology and gastroenterology, university hospital of poitiers, france, poitiers, france please specify your abstract type: descriptive abstract (for projects) background and objective: hepatic impairment can cause significant changes in the pharmacokinetics of many medicines. however hospital pharmacists can be helpless in performing pharmaceutical analysis behind the lack of precise guidelines. we need a strategy to first detect accurately patients with hepatic impairment, then lead us in dose adjustments. the objectives of this project were to develop a helping tool for hospital pharmacists in the pharmaceutical analysis of patients with hepatic failure's prescription and to select relevant pharmacist interventions. design: we first planned an investigation of patients with hepatic failure's management, with multidisciplinary experts groups. the study was conducted during one week in post-surgical, gastro-enterology, endocrinology, cardiology, pulmonology, geriatric departments and reanimation care units. a flowchart based on hepatic's biomarkers helped us including patients. criteria used to assess hepatic impairment could be: a stage c child-pugh score, prothrombin score inferior to %, bilirubin superior to micrograms per millilitres of blood without haemolysis, aspartate and alanin aminotransferases superior to three times the high normal value, and presence of a vitamin k antagonist interfering with those results. after a review of each included patient's prescription, we checked the major pharmacokinetic elimination pathway of each prescribed molecule (biliary or renal) and if hepatic biotransformation was expected. we also checked if the molecule could cause hepatic side effects. results: out of patients, patients were included for liver failure ( . %) and for a cholestasis ( . %) mainly in reanimation care units ( . %) and gastro-enterology ( %). among the lines of prescribed medicines, the main pharmacological classes encountered were cardiology, ( %) pain ( %), psychiatry ( %), haemostasis ( %) and antibiotics ( %). at the end of the investigation, the expert group decided on the relevant pharmacist interventions. these were based on dose adjustment of anti-infectious, psychotropic drugs, painkillers, oral anti-diabetics, anti-coagulants and corticosteroids. alternatives are proposed for each class. conclusion: to conduct a better pharmaceutical analysis, steps are necessary. first, any liver failure or cirrhosis must be detected thanks to the patient's biological results and medical record. then the patient's prescription can be analysed in order to highlight drugs that need a dose adjustment in a context of hepatic impairment. finally, the physicist and the pharmacist discuss about dose adjustments or alternatives if presence of contraindication with the drugs prescribed. soon the designed tool will be available to all pharmacists to harmonize clinical pharmacy practices. please specify your abstract type: research abstract background and objective: data regarding adherence rates to oral chemotherapy in lymphoma patients is limited. the aim was to assess pharmacist intervention on adherence to oral chemotherapy in patients suffering from hodgkin's (hl) and non-hodgkin's lymphoma (nhl). setting and method: following ethics approval, hl and nhl patients attending chemotherapy sessions at the medical investigations and treatment (mitu) at mater dei hospital accepted to participate. a questionnaire was compiled to evaluate adherence to oral chemotherapy and to assess pharmacist intervention. the questionnaire was divided into sections (a-c). the same questionnaire was used for both the first interview (t = ) and after weeks (t = ). an additional section (d) was incorporated at t = to evaluate pharmacist intervention. section a consisted of questions regarding patient management of lymphoma. section b incorporated the morisky -item medication adherence scale (mmas- ) to evaluate adherence to oral chemotherapy. a total mmas- score of zero indicates high adherence, a score between and indicates medium adherence and a score between and indicates low adherence. section c consisted of additional questions regarding medication adherence. between t = and t = , pharmacist intervention involved providing each patient with an information leaflet which was developed in this study, an individualised treatment chart and verbal advice. ibm Ò spss version and the wilcoxon signed-rank test were used to assess changes in medication adherence between t = and t = . main outcome measures: evaluation of pharmacist intervention on adherence to oral chemotherapy in patients suffering from hl and nhl. results: out of the patients with hl at t = , 'never' missed a dose, missed a dose 'once in a while' and 'sometimes' missed a dose. for the patients with nhl at t = , 'never' missed a dose, missed a dose 'once in a while' and 'sometimes' missed a dose. the reason for missing a dose was forgetfulness. all nhl and hl patients indicated the haematologist as their source of information about the management of lymphoma. of the nhl patients, scored low adherence and scored medium adherence at t = and after weeks (t = ) all nhl patients who participated scored medium adherence. of the hl patients, scored low adherence and scored medium adherence in the first interview (t = ) and after weeks (t = ) all hl patients who participated scored medium adherence. there was a statistically significant increase (p \ . ) in the number of patients who scored medium medication adherence between t = and t = for both nhl and hl patients. conclusion: this study shows how pharmacist intervention and extended professional services could be implemented in the clinical setting to impact on the management of hl and nhl patients. please specify your abstract type: descriptive abstract (for projects) background and objective: in may , an activity of medication reconciliation was implemented in the gastroenterology service to carry on the optimization of the medication care of patients due to the recent computerization of their prescriptions. design: this project, worked in collaboration with the gastroenterology service has been introduced in two medical committees. this activity gathers pharmacy students, the pharmacist, senior and junior doctors. reconciled patients are selected according to several criteria (advanced age, poly pathological, poly-medicated and those for whom a drug background is difficult to retrieve for the medical team). a minimum of information sources is used for the collection of the drug background. all information are synthetized on a paper, validated by the pharmacist and discussed again with the prescriber. results: on a -month period, patients were reconciled with on average age of . the reconciliation is executed on average . days after the entry in the service. . % of reconciliations are retroactive. the main sources of information used for the collection of the drug background are: in . % of the cases an oral interview with the patient and/or the family; in % of the cases the prescriptions, the hometown pharmacist ( . %) and a medical letter ( . %). . drugs are on average on the hospital prescription, and . % ( / ) of the patients are concerned with at least one non intentional divergence (nid). on average there are . nid/patient and . intentional divergences (id)/patient. the main types of nid are omissions ( . %), drug dose errors ( . %) and errors in administration frequency ( . %). after the detection of nid, the proposed modifications to the prescribers are accepted in more than % of the cases ( / ). the average time of a reconciliation is min. exchanges on the id and nid are made with the junior doctors in . % of the cases. conclusion: some nid are occurring for . % of the reconciled patients. it is therefore necessary to extend this new activity to reconciliation in other services in order to increase the interception of eventual medication mistakes and allow their correction. please specify your abstract type: research abstract background and objective: diuretic therapy is routinely used in the management of congestive heart failure (chf).,compliance with clinical practice guidelines is reported to result in improved outcomes for patients with chf such as reduced exacerbations. the aim was to assess the effect of pharmacist intervention on adherence to diuretic treatment in a hospital and community pharmacy scenario. setting and method: the study was undertaken at karin grech hospital (kgh), a geriatric and rehabilitation hospital, and in one community pharmacy. inclusion criteria for patients recruited from kgh were age over years, suffering from chf and on bumetanide therapy. the validated -item morisky medication adherence scale (mmas- ) was administered to patients on admission (t = ), repeated after two weeks hospital stay (t = ) and again one-month post-discharge (t = ). a total mmas- score of zero indicates high adherence, a score between and indicates medium adherence and a score between and indicates low adherence. in the community setting patients on diuretic therapy were chosen by convenience sampling. the same adherence scale was administered prior to pharmacist intervention (t = ) and one-month after pharmacist intervention (t = ). pharmacist intervention in the community setting involved dissemination of an informative leaflet regarding chf and diuretic therapy developed for the purpose of this study. main outcome measures: impact of pharmacist intervention on adherence to diuretic therapy in chf patients. results: a total of patients were recruited from the hospital setting, of whom were female and were male with a mean age of years (range - years). on admission (t = ), patients scored high adherence, scored medium adherence and scored low adherence to bumetanide therapy. following weeks at the hospital (t = ), the number of patients scoring high adherence increased from to and the number of patients scoring low adherence decreased from to . one-month post-discharge (t = ), patients scoring high adherence decreased from to and patients scoring low adherence increased from to (p \ . ). a total of patients were recruited from the community pharmacy, of whom were female and were male, with a mean age of years (range - years). after pharmacist intervention (t = ), the number of patients scoring high adherence increased from to , while the patients scoring low adherence decreased from to (p [ . ). conclusion: pharmacist intervention in the hospital setting improved adherence to bumetanide therapy. in the community pharmacy setting, there was a slight improvement in the compliance. pharmacist monitoring and patient support is important post-discharge to ensure patient compliance to therapy. conclusion: surveillance of aeds may be followed by combination of data from adverse drug reaction databases and drug utilisation data from prescription databases. focus on reporting adverse reactions is important for pharmacists and clinicians, especially for newly approved drugs. awareness of increased exposure of aeds to new groups of patients followed by data regarding safety aspects is important and contributes to improved pharmacovigilance. please specify your abstract type: research abstract background and objective: the medication review of polymedicated patients is a priority shared among all healthcare professionals. a multidisciplinary approach of these patients is necessary to achieve the best results for their treatment ( ) . the objective was to analyse the rate of acceptance of the recommendations made by the primary care pharmacist (pcp) to the general practitioner (gp) regarding the treatment of polymedicated patients. setting and method: setting: a primary health care centre ( , population). method: a review of the medical records of polymedicated patients (c chronic drugs for c months). the patients' data were collected from january to june from their clinical records. statistical descriptive analysis of data was performed. main outcome measures: drug related problems (drp) for each patient: interactions, contraindications, inadequate dosages, nonindicated drugs, omission of a necessary drug, duplications, medication with low therapeutic effect, and inappropriate medication for patients c years old. treatment alternatives proposed to gp's by pcp were also measured. results: patients were included in the study (average age: . ± . , % women). out of the patients, interventions were laid out to reduce the risks of drp's and to improve the efficiency of treatments. % of patients presented some drp or some intervention to improve the efficiency of their treatment, this mean an average . interventions for patient. the prevalence of intervention proposals were: non-indicated drugs ( %), interventions for improve the efficiency of treatments ( %), interactions ( %), inappropriate medication for patients c years old ( %), contraindicated drugs ( %), duplications ( %), medication with low therapeutic effect ( %), inadequate dosages ( %) and omission of a necessary drug ( %). % of these intervention proposals were accepted by the gp: % of the accepted proposals were carried out and from the remaining , . % led to a prescription from a specialist physician. in % of the cases, the patient did not accept the changes. . % were not carried out due to other issues. the main drug related problem was the prescription of non-indicated drugs and the most involved drug was omeprazole. conclusion: acceptance by gp's to changes proposed by primary care pharmacists was high. a significant number of changes was not accomplished due to the negative response by some patients and led prescriptions from a specialist physician. the gp greatly values the multidisciplinary aid in approaching the complexity of polymedicated patients. background and objective: case-reports provided evidence that influenza infections, particularly severe episodes, may exert neuronal damage in the cns and thereby increase the risk of depression. it was the aim of this study to analyse the association between influenza infections and the risk of developing incident depression. setting and method: we conducted a case-control analysis using the large uk-based primary care database clinical practice research datalink (cprd). this database contains anonymous longitudinal data from primary care. at present, it contains over million person-years of data from some million active patients. the study encompassed , patients below the age of years with an incident major depression diagnosis between and , and we matched each case to one control patient on age, sex, general practice, number of medical encounters, and years of history in the cprd prior to the index date. main outcome measures: major depression diagnosis was identified by read-codes based on icd- codes (f ), with a minimum of three prescriptions for antidepressant drugs recorded after the diagnosis. we calculated relative risk estimates of developing depression in association with previous influenza infections, stratified by the number, timing and severity of such events, and we adjusted for a variety of comorbidities, smoking status, alcohol intake, body mass index, use of oral corticosteroids, and benzodiazepines. results: patients with a previous influenza infection had an increased risk of developing depression (or . , % ci . - . ) compared to patients with no history of influenza infections. a recent influenza infection recorded within - days prior to the index date yielded an adjusted or of . ( % ci . - . ), and an increasing number of previous influenza infections was associated with increasing odds ratios (c recorded influenza infections, adjusted or . , % ci . - . ). we did not see any differences in the relative depression risk associated with influenza with regard to a previous influenza vaccination. conclusion: this study suggests that influenza infections are associated with a moderately increased risk of developing depression. please specify your abstract type: research abstract background and objective: warfarin is known for its interactions with many drugs. elderly patients are particularly sensitive to warfarin interactions. to evaluate the incidence of potential drug interactions when prescribing new drugs to elderly patients on warfarin, a prospective observational study was conducted. setting and method: patients on warfarin older than years were included and monitored for months in community pharmacies in croatia. data regarding new prescribed drugs was obtained from pharmacy records at the moment of dispensing or by patient selfreporting. the potential interacting drugs were identified using the lexicomp Ò lexi-interact online software. only the clinically significant (levels c, d, x of clinical significance as classified by lexicomp Ò lexi-interact online) interactions were included in this analysis. main outcome measures: number of new proscribed drugs, level of interaction with warfarin, mechanism of interactions. results: we included elderly patients with an average age of years. in the follow-up period, new drugs were prescribed to patients ( . %). there were prescriptions of new drugs and ( . %) of those were drugs with a clinically significant interaction with warfarin. there were prescriptions of drugs with level c of interaction ( . %), and ( . %) with level d. there were no drug interactions of level x. in the group with level c the most prescribed drugs were antibiotics with prescriptions: amoxicillin/clavulanate %, clindamycin %, ciprofloxacin %, norfloxacin %, azithromycin %, cefuroxime %, clarithromycin %, doxycycline %. the remaining prescriptions included tramadol with paracetamol %, rosuvastatin %, simvastatin %, fluvastatin %, levothyroxine % and torasemide %. the dominant mechanism of the potential interactions was pharmacokinetic. in the group with level d the most prescribed drugs were nonsteroidal anti-inflammatory drugs with prescriptions-diclofenac %, ibuprofen %, indomethacin %. among other drugs, prescriptions were antibiotic sulfamethoxazole with trimethoprim %, fenofibrate %, miconazole %, and fluconazole %. the dominant mechanism of the potential interactions was pharmacodynamic. conclusion: pharmacists should actively monitor prescribing of new drugs to elderly patients on warfarin in order to reduce the risk of clinically significant drug interactions. please specify your abstract type: research abstract background and objective: explicit criteria of potentially inappropriate medications in the elderly (pims) have been published in the usa, canada, australia and many eu countries. there is a lack of studies describing prevalence of pim use in central and eastern europe. the aim of the eu cost action initiative wg b ( wg b ( - is to evaluate the registration rates and use of pims in central and eastern europe compared to other eu countries participating in this initiative. this abstract describes preliminary findings on different registration rates of pims in different eu countries. setting and method: researchers/members of the eu cost action initiative from the czech republic, serbia, hungary, spain, turkey and portugal were asked to fill in evaluation tables for the list of pims in the period - / . items available in these evaluation tables related to: registration of individual pims on the pharmaceutical market, registered doses, drug forms, availability of pims on prescription or as otc drugs, prescription limits and the most frequently used brand names. data were evaluated using comparative descriptive statistics. main outcome measures: overall prevalence of registered pims in different countries, cross-country differences in availability of individual pims. results: of pims . % were registered in at least participating country. for the czech republic ( . %), turkey ( . %), spain ( . %) and hungary ( . %) overall prevalence rates of registered pims were found to be similar. however, these prevalence rates substantially differed in serbia (low prevalence- . %) and portugal (high prevalence- . %). substantial differences were found also in the lists of individual pims registered in different countries. these lists were similar in spain and portugal compared to the czech republic, hungary, serbia and to turkey. conclusion: although overall prevalence rates of registered pims were similar in the majority of evaluated countries (except serbia and portugal), availability of individual pims was substantially different. our pilot results confirmed that there are substantial geographical/ regional differences in europe in the lists of pims available (in spain and portugal compared to central and eastern europe and compared to turkey). please specify your abstract type: research abstract background and objective: inappropriate prescribing is a common circumstance found in polymedicated patients. screening tools for identifying potentially inappropriate prescription (pip) and pharmacist interventions for evaluating them have been developed to decrease this ( ) . the aim of this study was to evaluate the effectiveness of a pharmacist provided intervention to reduce pips in polymedicated patients. setting and method: the design was a quasi-experimental study focusing on a single group before and after intervention. the study took place from july to december of at three primary care centres ( , population). polymedicated patients were those using c chronic drugs for c months. main outcome measures: reduction in the rate of pip per polymedicated patient (number of pips found divided by the total number of polymedicated patients) before and after intervention, and the influence of the following variables: type of pip (inappropriate medication for patients c years old, medication with low therapeutic effect, duplication of benzodiazepines (bzd) or angiotensinconverting enzyme (ace) inhibitors, combination of anticoagulant and antiplatelet, combination of non-steroidal anti-inflammatory drug (nsaid) with a diuretic and ace inhibitor, nsaid in cardiovascular disease, chronic antipsychotic in dementia, chronic bzd, or chronic nsaid), gender and age of patients with at least one pip, and the main prescribed drugs involved in the pips based on atc classification system of world health organization. results: there were and polymedicated patients before and after intervention, respectively. . % (n = , before) and . % (n = , after) of the total patients had at least one pip. the number of pips was reduced from to , while the rate of pip per polymedicated patient decreased from . to . , achieving the limit established by the regional health authority. . % (before) and . % (after) of patients had more than one pip at the same time, up to pips per patient. before and after intervention, more than half of patients with at least one pip were c years old, and approximately out of were c years old. also before and after intervention, out of patients with chronic nsaid and with bzd duplication were women. out of patients with combination of anticoagulant and antiplatelet were men. the main pips before and after intervention were, respectively: chronic prescription of bzd ( . vs. . % of the total pip), medications with low therapeutic effect ( . vs. . %) and inappropriate medication for patients c years old ( . vs. . %). the main atc group involved in the total of pips was drugs for the nervous system, and the five most prescribed drugs were all bzd (lorazepam being the first). conclusion: pharmacist provided intervention was able to reduce pip in polymedicated patients. gender, age and atc classification of drugs involved were factors in the pips. please specify your abstract type: research abstract background and objective: up to % of women are exposed to selective serotonin reuptake inhibitors (ssris) during pregnancy. information on their effect on birthweight and gestational age remains conflicting. the aim of this sibling-controlled prospective cohort study is to address shared genetic and family-level confounding to investigate the effects of prenatal ssri exposure and maternal depression on birthweight and gestational age. setting and method: we used the norwegian mother and child cohort study (moba) and the medical birth registry of norway (mbrn). our study population consisted of siblings; were prenatally exposed to ssris and were unexposed to any antidepressant medication. random and fixed effects analysis with propensity score adjustment was used to evaluate the effects on birthweight and gestational age. main outcome measures: birth weight. gestational age. results: ssri exposure during two or more trimesters was associated with a decrease in birthweight of g [ % confidence interval (ci) to ] and a decrease in gestational length of . days ( % ci . to . ). neither maternal ssri use in one trimester, lifetime history of major depression nor depressive symptoms during pregnancy were associated with these pregnancy outcomes. conclusion: prenatal exposure to ssris during two or more trimesters may decrease birthweight and gestational length. our results indicate that neither maternal depression nor shared genetics and family environment fully explain this association. please specify your abstract type: research abstract background and objective: the drugs burden index (dbi) is a tool to evaluate the burden of medications with anticholinergic and sedative effects and this exposure has been associated with poorer physical and cognitive function in older people. objectives were; to determine the cumulative burden of anticholinergic and sedative medicines in older adults with intellectual disability (id) using the dbi, to examine the relationship between dbi score with demographics and comorbidity. setting and method: data from wave of the intellectual disability supplement to the irish longitudinal study on ageing (ids-tilda), a nationally representative study of ageing people with id in ireland. dbi scores were calculated for all participants with available medication data (n = ). bivariate associations between dbi and demographic and clinical characteristics were examined with a significance level of . main outcome measures: dbi scores of participants categorised into low ( ), medium ( - ) and high (c ). dbi score categories were related to demographics, cognitive effects and to a modified functional comorbidity index (fci), which is associated with physical function in older adults. results: of participants, . % ( ) had dbi exposure; . % were exposed to any anticholinergic medication, . % to any sedative medication; mean number of dbi medications . (± . ), mean dbi score: . (± . ). ( . %) participants had dbi score , ( . %) - , and ( . %) c . antiepileptics accounted for the greatest contribution to cumulative score ( . %), antipsychotics ( %) and antidepressants ( %). there was no significant association between higher dbi score and sleep difficulties (p = . ). there was a significant age gradient associated with higher dbi score (p = . ) and significant association between higher scores and increased comorbidity scores; mean fci of . in those with dbi c , . in dbi - and . in those with dbi . conclusion: cumulative exposure to sedative and anticholinergic medicines was high in older adults with id. higher dbi scores were associated with higher comorbidity and associated poorer physical function. optimising use of medications with anticholinergic and sedative effects through medicines review by pharmacists as part of multidisciplinary teams using a tool such as the drug burden index may reduce functional decline and improve quality of life among older adults with id. please specify your abstract type: research abstract background and objective: poor adherence to pharmacotherapy may have considerable consequences for the patients' health and for healthcare costs to society. there was observed that diabetes patients have higher risk of later health complications development. it is necessary to be adherent to non-and pharmacological recommendations as well, to improve the clinical outcomes and decrease the cardiovascular risk (cvr). the aim of this study was to evaluate the medication adherence and cvr in group of patients with diabetes, and to find an association between them. setting and method: the methods were based on a questionnaire survey using a modified -item morisky score and score charts ( ). medication adherence and cvr were evaluated in the whole group (n = , males and females, range - years) as well as in subgroups according to age, gender, (no-/ex-) smoking, level of education, residence, number of used medicines, exercises, compliance to the diabetic diet, and total cholesterol levels. the survey was realized in three ambulatory diabetic centres in slovakia. the study has been approved by ethics committee of university hospital bratislava -ruzinov. all participants signed an informed consent. main outcome measures: the results of medication adherence were evaluated as follows: points = full adherence, - points = partial adherence and - = non-adherence. the cvr (estimating -year cardiovascular attack risk) was evaluated according to score charts using data from questionnaire and medical records-gender, age, smoking, total cholesterol levels and blood pressure. the results showed a partial medication adherence in the study group in average ( . ± . ). the average value of cvr in the study group was . %. the highest average medication adherence has been observed in males b years ( . ), with elementary education ( . ), in ex-smokers ( . ), in patients with regular physical activity-at least times a week ( . ), in patients non-adherent to the diabetic diet ( . ), in patients using medications ( . ), and in patients with satisfactory ( . - . mmol/l) total cholesterol levels ( . ). the lowest cvr has been observed in females b years ( . %), in no-smokers ( . %), with elementary education ( . %), in patients with irregular physical activity ( . %), in patients adherent to the diabetic diet ( . ) , in patients using medications ( . %) and in patients with satisfactory ( . - . mmol/l) total cholesterol levels ( . ) . on the other hand, the highest cvr has been observed in males [ years ( . %), smokers ( . %), secondary educated patients ( . %), without any physical activity ( . %), in patients partially adherent to diabetic diet ( . %), using medications ( . %) and, surprisingly, in patients with satisfactory (\ . mmol/l) total cholesterol levels ( . %). conclusion: our survey has showed that medication adherence in our study group has been decreased and cvr has been increased. cvr and adherence to pharmacotherapy in the study group did not correlate with each other. the medication adherence, cvr and their relationships are specific in every patient. please specify your abstract type: research abstract background and objective: studies show that quality of life (qol) of patients with diabetes mellitus can influence medication adherence, satisfactorily improving clinical outcomes and reducing the morbidity and mortality rates and disease progression. this applies even upside down-medication adherence could significant contribute to improving patient qol. the aim of this study was to evaluate the medication adherence in group of patients with diabetes, to evaluate their qol and find a correlation between them. setting and method: the methodology was based on a questionnaire survey using a modified -item morisky score and questionnaire eq- d- l, including visual analogue scale (vas). medication adherence and qol were evaluated in the whole group (n = ) as well as in subgroups according to age, gender, level of education, monthly income, number of used medicines and type antidiabetic treatment. the survey was realized in three ambulatory diabetic centres in slovakia. the study has been approved by ethics committee of university hospital bratislava-ruzinov. main outcome measures: the results of medication adherence were evaluated as follows: points = full adherence, - points = partial adherence and - = non-adherence. the qol in levels of dimensions results were evaluated as follows: the lowest qol in every dimension = point, the highest = points. the highest vas evaluation has been points and every patient should mark number on the scale - to indicate his/her health on current day. results: the results showed a partial medication adherence in the whole group in average ( . ± . ). the average value of the qol in the study group was . and vas . . the highest medication adherence has been observed in males ( . ± . ), patients \ years old ( . ± . ), with primary education ( . ± . ), with monthly income over € ( . ± . ) and in patients using medications ( . ± . ). the highest qol and vas (qol; vas) has been observed in males ( . ; . ), patients \ years old ( . ; . ), university educated ( . ; . ) , with monthly income over € ( . ; . ) . qol has been highest in patients using medications ( . ), vas has been highest in patients using medication ( . ). we have observed the highest level of medication adherence in patients treated with combined therapy-with oral antidiabetic agents and insulin ( . ), the lowest in patients treated with only insulin therapy ( . ). highest qol was recorded in patients treated with oral antidiabetic agents ( . ), and the lowest qol in patients with insulin therapy ( . ). the highest vas has been observed in patients using only oral antidiabetic agents ( . ), the lowest in patients using combined therapy ( . ). conclusion: survey has showed that medication adherence and qol in our study group has been decreased. qol and adherence to pharmacotherapy in the study group did not correlate with each other. the medication adherence, qol and their relationships are specific in every patient. the role of health care professionals should be in education and counselling with patients to improve qol and medication adherence as well. please specify your abstract type: research abstract background and objective: to assess the appropriateness of antibiotic prescriptions used for urinary tract infections (uti) in the elderly. setting and method: we included patients aged years and older, hospitalized in the geriatric department and for whom a urine culture was performed between march and may . a prescription was qualified as inappropriate: when the antibiotic prescribed was not the narrowest compared to the culture result, or when there was a contra-indication, or when the treatment duration was shorter or longer than recommended. prescriptions were consistent with the guidelines when they were identical to those adopted by the french society for infectious diseases in december . main outcome measures: appropriateness of antibiotic prescription (type and duration) results: elderly patients were included (women: . % (n = ), mean age: . years). % of antibiotic choices were appropriate and % of treatment durations were consistent to the guidelines. urinary clinical signs were mentioned in the medical files for . % of the cases (n = ). patients received an empirical antibiotherapy ( . %). . % (n = ) of urine cultures were positive with bacteria, escherichia coli being the most prevalent (n = ). the urine culture results led to a change in antibiotics for . % of the cases. for cystitis, . % of the antibiotics chosen were appropriate (n = ). the main reasons of non-conformity were the lack of deescalation (to amoxicillin or pivmecillinam), and the prescription of ciprofloxacin when the bacteria was in vitro resistant to other fluoroquinolones. the average duration of effective antibiotherapy for cystitis was . days (appropriateness: . % (n = )). for pyelonephritis, . % of the antibiotics chosen were appropriate (n = ). the average duration of effective antibiotic treatment was . days (appropriateness: . % (n = )). . % of the patients had a transurethral catheterization (n = ). another infection was diagnosed for . % of the patients (n = ). conclusion: according to these results, it appears important to reemphasize to the prescribers the guidelines around the uti diagnosis and treatment in order to improve the prescriptions appropriateness in elderly patients. it is particularly necessary to promote the de-escalation of antibiotherapy (with pivmecillinam for example which has recently become available in our hospital) and to insist about the recommended durations of treatment. please specify your abstract type: research abstract background and objective: to measure the use of potentially inappropriate medications (pim) in the general elderly population several criteria lists exist, e.g., beers criteria. last year, a set of explicit criteria for assessing pharmacologically inappropriate medication use in nursing homes was developed; the norwegian general practice-nursing home criteria (norgep-nh). the aim of this study was to investigate the prevalence of pims in nursing home patients using this new assessment tool. furthermore, we studied possible associations between the use of pims and factors like gender, age, geographical area and the number of drugs used. setting and method: cross-sectional study comprising nursing home patients from two geographical different regions in norway; tromsø city (n = ) and lofoten islands (n = ). data was collected from november to january . pims were identified by norgep-nh. we used logistic and poisson regression to examine possible associations between the use of pims and factors like gender, age, geographical area and the number of drugs used. main outcome measures: number of pims per patient, and odds ratios (or) and marginal effects for associations. results: nursing home patient used a mean (sd) of . ( . ) drugs; . ( . ) regularly and . ( . ) as needed. at least % of patients used one pim. concomitant use of three or more psychotropic drugs was the criterion most commonly identified ( %), followed by the use of antidepressant ( %) and hypnotics ( %). an increasing number of regularly used drugs increased the odds of having pims (or: . ), as well as it lead to . more pims per extra drug used. on average, patients c years had . fewer pims than patients \ years. no statistical significant associations were seen between having pims and gender, nor geographical area and the use of as-needed medication. yet, statistical significant differences were identified in some criteria. conclusion: this is the first study that explicit uses norgep-nh. our results confirm that nursing home patients often use potentially inappropriate medications. this is an area where further work is necessary, not to measure the prevalence of pim, but to develop interventions in order to prevent pims from being used. pe : use of pharmacy dispensing data to measure adherence and identify nonadherence with oral hypoglycaemic agents please specify your abstract type: research abstract background and objective: a framework for calculation of adherence for oral hypoglycaemic agents (ohas) based on data from health-insurance claims is available. pharmacy dispensing data aid identification of nonadherent patients in pharmacy practices. however, use of these data for calculation of oha adherence requires additional methodological categories. we examined the impact of different methodological choices on estimation of oha adherence using pharmacy dispensing data. setting and method: a framework for adherence calculation for pharmacy dispensing data was developed from health-insurance claims. a basic scenario was developed from methodological categories. consequences of choices for different parameters within these categories on the scores of the three adherence measures were calculated from dispensing data. main outcome measures: for oha use between july and july , three adherence measures were calculated: ( ) average medication availability (ama); ( ) mean rate of adherent patients with an ama c % (mrap ); ( ) please specify your abstract type: research abstract background and objective: ulcerative colitis (uc) is a chronic inflammatory disease usually affecting young adults and impacting on patient's quality of life. although many biological agents (bas) have been approved for the treatment of moderate-to-severe uc in patients who have responded inadequately to conventional therapy, the selection of bas is controversial due to the lack of head-to-head trials. indirect economic comparisons of these costly drugs are available from national healthcare perspectives that are not the italian ones. therefore, the objective is to evaluate cost-utility of bas for the treatment of refractory moderate-to-severe uc both in italy and in the lombardy region. setting and method: a markov model (considering transition states: remission, clinical response, relapse) was constructed using the software r . . markovchain-package to evaluate incremental cost-utility ratios (icur) of adalimumab, infliximab, infliximab biosimilar, golimumab and vedolizumab treatments of patients over a ten-year time horizon from the perspective of the italian (n) and lombardy region (r) healthcare system. clinical parameters were derived from clinical trials. costs (which have been actualised- . %) were obtained from the national database and regional public tender. utility was expressed as qaly (quality adjusted life years). main outcome measures: icur. results: costs per treatment were different from a n and r perspective (adalimumab - %; infliximab - . %; infliximab biosimilar - . %; golimumab - . %; vedolizumab - %). direct healthcare costs (treatment cost, visits, lab tests, hospital admissions) were calculated over years of treatment per patient: adalimumab (n: € , . , r: € , . , - . %), infliximab (n: € , . , r: € , . , - %), infliximab biosimilar (n: € , . , r: € , . , - . %), golimumab (n: € , . , r: € , . , - . %), vedolizumab (n: € , . , r: € , . , - . %) with associated qaly respectively of . , . , . , . , . . from a n perspective, infliximab biosimilar was dominating compared to all other treatments. the icur of vedolizumab/infliximab biosimilar was € . for years (willingness to pay (wtp) € . /qaly). from a r perspective, adalimumab was dominating compared to all other treatments. the icur of vedolizumab/adalimumab was € , . for years (wtp € , . /qaly). conclusion: national and regional cua produced different results. as regional price discounts can occur, local analyses are needed to estimate the economic impact of therapies to ensure optimal choice. please specify your abstract type: research abstract background and objective: automated dispensing systems (ads) have been implemented to reduce overall medication errors related to picking, preparation and administration of drugs. costs of drug storage between ads and classic dispensing system (cds) had not been yet performed in france. our objective was to assess economic impact of ads compared to cds. setting and method: retrospective quasi experimental study was conducted in university hospitals in , one with ads ( beds, ads) and one with cds ( beds, cds ( ) for ads and ( ) for cds (p \ . ). mean number of costly drug per system was for ads and for cds. the global stock value in the wards was , € in ads and , € in cds representing respectively . and . % of total pharmacy stock value. conclusion: our data demonstrate that despite the same storage capacity, ads allow the storage of more expensive drugs such as innovative drugs fully reimbursed up to national reimbursement prices, due to the lower risk of pilferage. this preliminary study was focused mainly on stock value. subsequently, another study is conducted to evaluate cost of these two drug storage systems, satisfaction of pharmaceutical technicians and nurses and time allowed for systems reloading. please specify your abstract type: descriptive abstract (for projects) background and objective: in france, pharmacists are not entitled to substitute an original biological drug with its biosimilar, due to specific issues of efficiency, safety, and patient monitoring. our hospital referenced a biosimilar of infliximab on january . according to the french medication safety national agency's recommendations, it has been decided that naïve patients would be treated with biosimilars, and changes between specialties would be proscribed. the objective is to compare prescribing practices between infliximab and its biosimilar, year after its introduction. design: a database tracking patients treated with infliximab was set up. data comparing prescribing practices of biosimilar and reference treatment were analysed between june and may . regional and national infliximab consumption between january and february were used to compare the practices of our hospital with other hospitals. the past and future savings were estimated from repayments data of the regional health agency. results: infliximab was administered to patients, of which ( %) were naive. patients were treated with biosimilar (i.e. . % of all patients), of which were naive. in the end, nearly % of naive patients actually received the biosimilar and . % of patients treated with infliximab switched specialties during treatment. in % of cases, biosimilar prescriptions were consistent with the recommendations (vs. % for infliximab). in % of cases the off-label prescriptions of the biosimilar were explained in the patient record (vs. % for infliximab). in february , the share of biosimilars was % in france, % at regional level and % locally. in year, infliximab and its biosimilar's consumption in our hospital have increased by % in quantity and only % in expenditure (+€ m expenditure). negotiating a lower purchase price and costs has enabled the hospital to save € , (vs. € , during the previous year). because of the decline of refund rates, the gains would have been zero without using the biosimilar but € , if it had been prescribed to every naive patient. conclusion: current data from the literature on security and effectiveness of infliximab biosimilars are very reassuring and the french medication safety national agency doesn't exclude the possibility of changing specialties during treatment. in our hospital, there is room to improve the efficiency of treatment with infliximab. feedback on prescribing practices will be given to prescribers and a campaign to widespread prescriptions of biosimilars will be made. the arrival of biosimilars on the market is a real economic opportunity for hospitals, which are increasingly financially constrained in particular by the arrival of therapeutic innovations which are more and more expensive. setting and method: the study used health claims data on prescription ppis from st january to st july obtained from the health insurance institute of slovenia. to assess medicine use and costs before and after trp implementation data were aggregated into four periods: jan-dec , pre-baseline period; jan-dec , baseline period; jan-sept , transition period between announcement and introduction of trp; oct to jul , period after trp enforcement. main outcome measures: medicine costs; defined daily doses (ddds) dispensed per inhabitants per day; market share; herfindahl-hirschaman index (hhi); number of active substance switches; number of exceptions when medicine is fully reimbursed since physicians may choose option ''not to switch medicine'' when adverse consequences are predicted. results: average monthly cost of ppis declined from € , , in pre-baseline period to € , in period after trp introduction although the consumption increased from . to . ddds/ inhabitants/day. cost of ppis decreased the most in baseline period ( %), however trp induced . % cost reduction compared to the transition period. the reference pantoprazole was market leader already in the transition period, but its use increased significantly after trp introduction and represented % of total ppis consumption. manufacturers' market shares were constant before trp, whereas trp caused decrease of the largest market share for %. still, this resulted in the minor market concentration change; hhi was on average . before and . after trp introduction. further, at least one active substance switch was detected in approx. and % of patients before and after trp introduction, respectively. similarly, the proportion of exceptions when medicine was fully reimbursed increased from . % in transition period to . % in period after trp introduction. conclusion: enforcement of trp for ppi contributed to approx. € m annual cost savings. from the payer's perspective the new policy was proven to be effective in reducing pharmaceutical expenditure; however trp also affected physician prescribing pattern and use of ppis. pec : blood coagulation factor: improvements of the supply chain samantha oses * , serri traore, sonia caroline sorli, lea damery, philippe cestac, sylvie pomies, julien tourel please specify your abstract type: descriptive abstract (for projects) background and objective: most of the antihemophilic factor (ahf) must be held by a teaching hospital to face serious bleeding events. to ensure better availability, offsite-stocks at critical points are required (emergency unit, intensive care unit, etc.). however, this management system increases the risk of economic loss and alteration of the quality due to expired products. in this context, we carried out an optimization of the supply and management system of the ahf. to identify critical points of the supply and management system and to implement improvement solutions. design: a multidisciplinary working group belonging to a regional management centre of haemophilia was set up. two lines of improvement were discussed: i) optimization of stocks ii) optimization of the supply system. results: the optimization of stocks has led to the modification of the threshold of the lowest stock (ls) for ahf out of . in % of cases, this stock modification has exceeded %. the overall cost of ls has been reduced by . % ( , €) for the general stock at the central hospital pharmacy (hp) and by . % for offsite-stocks ( , €). the ahf mainly involved in this reduction was fvii mg ( , €), then followed by the strengths of mg and mg ( , € for each). in order to improve the ahf management, several propositions have been implemented: ( ) developing an online, easily accessible and monthly updated spreadsheet that displayed several accurate data such as the shortest expiry date and the storage location. this operative tool is shared between all pharmacists involved in ahf management in order to facilitate a stock rotation and decrease economic losses, ( ) regular reminders to physicians and health care staff concerning the guidelines for inventory management and the importance of checking the drug expiry date, ( ) presentation of the financial results and raising awareness on ahf costs to the medical consultant[ppip ] and ( ) optimizing stock distribution based on consumption on the different hospital sites for better patient care management (pcm). conclusion: this optimization of stocks and improvement of the supply chain have led to a direct cost saving of , €. however, a more accurate assessment has to be performed to quantify the direct and indirect impact on pcm and cost saving. this work has been done in a context of a sharing operative network at a regional level. the aim of such project is to share, to optimize and to improve practices, knowledge, human and medical health resources at a widespread level to enhance the security and quality of health services and to promote cost and time saving. please specify your abstract type: descriptive abstract (for projects) background and objective: the overall pharmaceuticals consumption in hospitals is rising, which has led to an increasing expenditure, challenging health care professionals and threatening patients safety. clinical trials in hospitals have increased over the past few years and currently play an important role, giving access to new investigational medicinal products and also avoiding costs with standard treatments. the objective of this study is to evaluate the savings of centro hospitalar do porto, a central university hospital with beds and currently clinical trials, with patients included in clinical trials between january and may . design: retrospective observational study over months. all the clinical trials ongoing between january and may were analysed and the data was collected based on: pathology and doses established; number of treatments per patient and the medium prices of standard treatments that patients would be receiving if they were not in the clinical trial. results: there were clinical trials ongoing between january and may , but only were selected to be included in this study. the total number of patients included was . the clinical trials selected for this study were conducted in medical specialties: in dermatology, in immunology clinical unit, in hemato oncology, in gastroenterology, in ophtalmology and in neurology. during these months, with all ongoing clinical trials, centro hospitalar do porto was able to save, in medical products, more than million euros. conclusion: during the period of time established, of the clinical trials ongoing, were not selected due to: not including patients or not having an alternative treatment. hospitals and patients can benefit from clinical trials not only financially but also by preserving resources and medication. on centro hospitalar do porto, the pharmacists specialized in clinical trials, as members of the study team, are more and more required to perform specific tasks, their contribution has been increasing over the years and also have become more aware of all the advantages from participating in clinical trials. these savings can be used to provide a better assistance and contribute, in general, to a higher quality health care. please specify your abstract type: descriptive abstract (for projects) background and objective: several studies show a misuse of opioid maintenance treatment (omt) in detention. in fact, buprenorphine (bup) when it's misused, could present the same effects as heroine. in order to reduce misuses, the pharmacist decided to switch all the patients under bup to buprenorphine/naloxone (bup/nlx). bup/ nlx prevents patients from misusing by a withdrawal syndrome when it's issued by another route of administration than sublingual route. in france, bup/nlx is more expensive than bup which may explain why this therapeutic strategy is not often observed. the purpose of this study is to evaluate the extra cost after switching patients from bup to bup/nlx in order to decide if this choice could be maintained. design: to identify our population, we used the administration reports drugs written by nurses. please specify your abstract type: research abstract background and objective: haemophilia b is an x linked genetic disorder characterized by spontaneous or prolonged haemorrhages due to factor ix (fix) deficiency . within the next few years, new treatments are willing to hit the market. among them are recombinant extended half-life products that will reduce by half the number of injections and will potentially improve the patient quality of life. the aim of the study is to describe the development of haemophilia treatments market between and and to forecast the potential impact of these new therapies on the haemophilia market. setting and method: national and french hospitals of paris (aphp) consumption data of fix between and have been studied. new therapies in development or soon to be marketed have been identified. potential benefits and interest in the therapeutic care of these new products were discussed with haemophilia's medical experts. main outcome measures: quantity (ui) and value (euros) of fix aphp and national consumption. results: in , recombinant (rfix) and plasma-derived factors (pfix) were on the french market. the ap-hp's purchases of these factors represent almost million ui and million euros, which comprise % of national fix expenditures. in france and aphp, ambulatory care is a major part of the use of these treatments with nearly % of the fix purchases in . french rfix consumptions are higher than pfix consumptions ( % against %). in the ap-hp hospitals, rfix even account for % of consumptions against % for pfix. both national and ap-hp rfix purchases have steadily increased between and . the added competition arising from new treatments may lead to more competitive market procedures in hospitals and may reduce costs of haemophilia treatments. according to haemophilia doctor, long-acting (la) fix would offer obvious benefits like fewer infusions and presumably fewer bleeds. these treatments will mainly be used in a prophylactic wayin ambulatory care-than in a curative way (such as surgical use). conclusion: the therapeutic extent of these new treatments is still hard to define. the choice of treatment must remain consensual between physicians and patients. please specify your abstract type: descriptive abstract (for projects) background and objective: good practice about medicines imposes to health institutions a close monitoring of prescriptions, especially off-label prescriptions. patient care should take into account clinical profile, respect of guidelines and health expense control. we report here a case highlighting the significant role of the clinical pharmacist in care units to ensure medication good use in a castleman syndrome, a rare disease due to human herpesvirus (hhv- ) and associated with human immunodeficiency virus (hiv) infection. design: case report. results: our patient, a years old man (creatinine clearance rate (crcl): ml/min), was diagnosed with hiv infection in february (cd at ui/l), leading to introduce a therapy by emtricitabine-tenofovir, darunavir, and ritonavir. the evolution was hampered by repeated episodes of acute renal failure (arf; crcl: ml/min) and pancytopenia (hemoglobinemia at . g/dl, leucopoenia at . g/l, and thrombopenia at g/l). because of hhv blood pcr at copies/ml, transient crises with pancytopenia, arf, and hiv infection, a diagnostic of kaposi sarcoma herpesvirus (kics), an atypical castleman syndrome, was retained. given the lake of data in literature for this rare disease, a multidisciplinary team (medical specialists and clinical pharmacists) was gathered to choose an appropriate therapeutic strategy. treatment regimen consisted of: day , intravenous etoposide at mg; day , rituximab at mg/ m ; following one week later by rituximab day and oral etoposide at mg the day after. good communication between medical specialists and pharmacists enables the patient to get an optimal and personal treatment. relaying the information by clinical pharmacists in care units to pharmacists in charge of good practice facilitate the reimbursement. conclusion: clinical pharmacists in care unit help to optimize therapeutic strategies according to their experiences and scientific works. cooperation with physicians is improved, as well as prescriptions follow-up of off-label drugs, and health patients fully respected. quality and relevance of prescriptions are strengthened, with a better control of economic expenses. please specify your abstract type: research abstract background and objective: the maltese government launched the hpv vaccination scheme in and the national healthcare system (nhs) has since provided the cervarix Ò vaccine free of charge to girls aged . the aim of this study was to assess the cost of the administration of hpv vaccines in the healthcare system of malta. this study was based on the scheme provided by the nhs. the number of girls born per year was used to estimate the annual cost for vaccinating year old girls, based on the wholesale price and tender price respectively. the estimated yearly cost using the wholesale price was approximately € , while the average estimated cost based on the tender price was approximately € , . this signifies that cost savings based on the tender price compared to wholesale costs were of approximately € , . the cost for the cohort who completed the three dose schedule using the tender price on average was of € , per year. this result proved to be more than the anticipated cost. a reason for this could be that the number of girls aged increased possibly due to an influx of immigrants. including boys in the vaccination scheme would increase costs by an average of € , per year. conclusion: this study shows that procuring branded vaccines using the tendering process reduces expenditure for the government and the tax payer. wholesale prices were found to be more expensive than tender prices. this proves that the tendering system in malta is a potent system with many advantages for the tax paying public. the impact of the tendering process must therefore, be safeguarded. please specify your abstract type: research abstract background and objective: with the old age, presence of comorbidities, and overcrowding in mass gatherings such as the annual hajj pilgrimage in saudi arabia, there is a high risk of spreading infectious diseases among pilgrims and then within their country of origin. knowledge and application of hygiene principles in such an environment is therefore important to reduce the transmission of infectious diseases. up to date, there have been no studies to evaluate pilgrims' knowledge, attitude and practices toward mers-cov during the annual hajj pilgrimage in order to see whether there is a need for these aspects to be improved. setting and method: a cross-sectional survey study was conducted with a convenience sample of participants. participants were pilgrims, aged over , and able to speak arabic or english. a selfadministered structured questionnaire was distributed during hajj season in mecca. descriptive and multiple linear regression analysis were used in data analysis. main outcome measures: assessing pilgrims' knowledge, attitude and practices regarding mers-cov. results: two hundred and fifty-seven participants completed the study, % of whom were female, and the median (iqr) age was ( . - . ) years. pilgrims had moderately correct knowledge and accurate attitudes towards mers-cov with median scores of (iqr - ) and (iqr: - ) respectively. they were less educated about management ( %), hallmark symptoms ( %), high-risk individuals ( %) and source of coronavirus ( %). almost % of participants showed a negative attitude towards the use of protective measures such as avoiding food prepared under unsanitary conditions and contact with live animals. some participants ( %) were unable to comply with hygiene practices, particularly washing hands with soap and water or disinfectant after sneezing/coughing and wearing a face mask in crowded areas. educational level and employment status were significantly associated with knowledge whereas gender and age were significantly associated with attitude and practices respectively (p \ . ). the correlation between knowledge, attitude and practices was significant (correlation coefficient: . ; p \ . ). better knowledge was found to be a predictor for positive practice. conclusion: these findings aided in the assessment of the adequacy of current pilgrims' educational measures. they will also provide insight when designing future interventions to promote specific messages to improve knowledge, change attitude and improve practice regarding mers-cov. please specify your abstract type: research abstract background and objective: the prevalence of type diabetes significantly increased in the paediatric population, which is affected by obesity worldwide. today, type diabetes accounts for % of all cases of new-onset diabetes in adolescents. preventive health care particularly taking place at community pharmacies may involve risk assessment for the children and the adolescents, early referral for seeking relevant medical care and patient education on healthy lifestyle choices. the aim of the study is to conduct a type diabetes risk assessment program for the kids b years of age of whose parents visited the community pharmacies involved in the study and also to identify the behavioural parameters that might be associated with this risk. setting and method: the study was conducted in community pharmacies. all patients with kids aged b years who visited the study pharmacies during one-week period were informed about the study and invited to participate in the study. patients who gave their informed consent were included in the study. all data were provided by the parents. demographic data, height and weight of the kid, as well as data regarding the behavioural features (eating habits, exercising, time spent in front of a screen, etc.) of both the children and the parents were collected using standardized forms. type diabetes risk test consisted of questions and identified subjects at risk. the parent of the kid who was identified to have risk for type diabetes was referred to a physician for further examination. also, information regarding type diabetes and the importance of preventive measures such as converting to a healthy life-style was provided. main outcome measures: main outcome measures were the percentage of kids identified to be at risk of developing type diabetes and the behavioural parameters associated with type diabetes risk. results: the study involved subjects. of the subjects % were identified to be at risk of type diabetes. more girls than the boys had the risk ( vs. . %). those with type diabetes risk were older, taller, heavier and had higher body mass index. they were spending more time in front of a screen (tv, pc, tablet, smart phone); . % were spending more than h a day. although the kids' eating habits were similar for those with and without risk, the parents' of the kids with risk ate out more frequently, consumed rice, pasta and pastry more frequently. both the kids with risk and their parents exercised more regularly and frequently. conclusion: this study shows that pharmacist have a vital role in identifying children and adolescents at risk for type diabetes; thus at early management of this condition. identifying and addressing the behavioural parameters associated with the risk will be helpful in lifestyle modification interventions. please specify your abstract type: descriptive abstract (for projects) background and objective: analyse and promote the reporting of adverse drug events (ade), to improve the quality and safety of care to be able to control the risks. design: a software is available on the intranet website of the institution, to enable health professionals to report ade. the drug and medical devices commission (comedims) of the hospital, centralizes these statements and always makes a multidisciplinary and overall analysis of the event, using a collection sheet which is based on the pdca model (plan, do, check, act). it proposes the nursing and medical teams axes of improvement. results: in , only ade were reported and analysed by the comedims, including from the paediatric centre ( %), particularly sensitized to this issue. health professionals are divided as follows: healthcare executives ( %), nurses ( %), pharmacists ( %), residential students ( %), doctors ( %) and others ( %). the main impacted steps of the drug circuit are: administration ( %), prescription ( %) and the use or implementation of a sterile medical device ( %). identified causes include related following factors: operational tasks and procedures ( %), health professionals ( %), work environment ( %), organization and management ( %), drugs or associated medical devices ( %). the number of ade reports, taking into account the size of the institution, remains very low. in january , the comedims decided to broadcast a communication campaign to promote ade reporting, on the hospital website via the intranet. three months after the release, this document was viewed times, and the number of reports increased by % compared to the same period in . conclusion: in front of the low number of returns of adverse drug events, and relying on the charter of non-punishment, the come-dims wants to increase health professionals' awareness. in our hospital, where e-learning about drug-related iatrogenesis is already available, the communication campaign with poster and analysis of adverse events seems to be a useful complementary tool to enhance awareness of medication safety concerns. please specify your abstract type: research abstract background and objective: the migration of modern social networks to the internet has facilitated the transition of traditional pharmacy networks online. the ubiquitous nature of social media (some) combined with merging of personal and professional personas have led to organisations publishing guidance on online behaviour and responsible use of social media. the research to date on the use of social media as a support for professional practice in general is limited. as the pharmacy profession evolves to embrace the technologies which underpin core services and mainstream online daily social activities, it is important that research tracks and evaluates its use and impact within the profession. the objective of this research was to explore and describe how and why pharmacists interact with hosted networks on social media. setting and method: two one-hour online hosted micro-blogging twitter chats were held in december via the #weph network. topic guides were developed around 'exploring the use of twitter and wepharmacists' in line with the wenetwork guidelines (#wecommunities), informed by existing literature, discussion with the #weph moderator after review by an expert panel. all research was carried out in accordance with university governance processes and association of internet researchers guidelines. themes were inducted from analysing the textual content of the chats using the topic guide as a framework. the research was approved by the school of pharmacy and life sciences ethics committee. main outcome measures: tweets per chat results: each of the chats had over million impressions with participants representing international pharmacy practice. themes of e-professionalism and online privacy emerged as concerns; however, the benefits included using social media for education, networking, support mechanisms and career development. tweets highlighted personal experiences of 'trolling' (angry, offensive behaviour) and the effect on user interaction with social media. twitter was also recognised as a career development tool and, in particular, collaborative outcomes around mentorship networking early career pharmacists with more experienced colleagues. conclusion: results support the responsible use of social media as a force for inclusion, breaking down geographical barriers in support of pharmacy practice. further research is underway including a systematic review of guidance on the use of social media by registered healthcare professionals. please specify your abstract type: research abstract background and objective: it is estimated that half of the , persons with diabetes in norway have not been diagnosed. with early treatment, life expectancy can be increased and the incidence of longterm complications and health costs reduced. community pharmacies may be able to help uncover undiagnosed diabetes, but being diagnosed with diabetes can lead to strong emotional reactions, and how the diagnosis is given may influence the experience. the aim of this study was to explore how norwegian people living with type diabetes (t d) experienced being diagnosed, and what led up to the diagnosis. in addition, their attitudes towards a planned community pharmacy service to identify undiagnosed t d was investigated. setting and method: three focus group interviews with people with t d were conducted using a semi-structured interview guide. eleven participants were recruited through a course about type diabetes. the interviews were audio-taped and transcribed in modified verbatim form and analysed in accordance with malteruds principles of systematic text condensation. the study was approved by the norwegian data protection authority, and did not require approval from the regional committee for medical and health research ethics. main outcome measures: how people with t d describe their experiences of being diagnosed with t d, how the disease was revealed and reactions towards using community pharmacies to perform risk assessment for t d. results: none of the participants were diagnosed due to their own suspicion of having diabetes. some saw their doctor because of unspecific symptoms such as fatigue and thirst, and were thereafter diagnosed with t d. others were diagnosed through a routine checkup. negative reactions like shock, discontent and denial were commonly used to describe the experience of being diagnosed with t d, but some participants also expressed a more relaxed attitude, especially if they were familiar with the disease through family members. participants expressed a strong wish for more and better information following the diagnosis. ''it's a jungle out there'' was used to describe how difficult they felt it was to find trustworthy and understandable information. they described change of lifestyle, side effects from drug use, and stigma as challenges following the diagnosis. while in general the participants were positive to using community pharmacies to uncover undiagnosed diabetes as this could help reduce the number of people who were undiagnosed, some were sceptical. they questioned whether the pharmacy staff had the necessary competence of the for this type of service, and saw it as the doctor's responsibility. conclusion: more information and support when people are diagnosed with diabetes may lead to that the experience being diagnosed will be more adaptable and that the challenges living with diabetes are reduced. community pharmacies are important healthcare providers, and risk assessment of t d at the pharmacy can be valuable. however, the pharmacies may also be helpful to reduce the information gap. please specify your abstract type: research abstract background and objective: chemotherapy-induced nausea and vomiting (cinv) is a disruptive and unpleasant side effect in chemotherapy patients and is associated with decline in patients' quality of life and decrement in the adherence to effective chemotherapy regimens. setting and method: chemotherapy naive patients were included in this study. consistency with guidelines were assessed according to mascc/esmo . flie questionnaire was administered to patients before chemotherapy, and days after receiving chemotherapy to assess the difference in the quality of life due to chemotherapy administration. main outcome measures: patients were categorized into two groups as consistent with guidelines group (acute (gcga) and delayed (gcgd)) and inconsistent with guidelines group (acute (giga) and delayed (gigd)). flie score differences between the two groups were assessed. results: the median flie score for patients prior to chemotherapy was and a dramatic decline was noticed post chemotherapy (flie score ; p \ . ). the post-chemotherapy score were for nausea and for vomiting ( . , respectively). although the flie score differed significantly between gcgd and gigd (p \ . ), these differences were not significant in gcga and giga. conclusion: the significant drop in flie scores in the study ( pre-to post-chemotherapy) reflected substantial declination in patients' quality of life. the lower postchemotherapy flie score of nausea emphasized the negative impact of nausea, and to a lesser extent vomiting on the patients ability to complete normal daily activities such as enjoying meals and maintaining social activities. although there were no significant differences in flie scores between giga and gcga groups for acute cinv prevention, significant differences were noted between gigd and gcgd (p \ . ). the flie score was lower for gigd patients. this result implied guideline inconsistency associated with high incidence of nausea which negatively affect patient quality of life. as for the degree of compliance with gp, the results are expressed as percentage of compliance compared to the ideal of %. prescription criterion was fulfilled to %: all requirements of pntb were performed using standardized procedure. in what concerns validation, % of pntb prescriptions were validated by a pharmacist. the invalidated prescriptions were made outside opening hours of the pharmacy service, which is open monday to friday from : to : and on weekends and holidays from : to : . % of the dispensations were individualized and not pntb stocks were found in hospital wards. as for preparation, % were supplemented with micronutrients. pntb of kabiven peripheral administration ml are not supplemented in our centre. of the remaining prescriptions central administration, % were supplemented. in all cases, the addition of micronutrients was performed in laminar flow hood in pharmacy service and the corresponding galenic validation was performed. finally, in the process of administration, % of pntb identified with a complete label: name of the patient, medical record number, type of pntb, qualitative and quantitative composition, date of administration and infusion rate. conclusion: use practices of pntb of our centre are far from those recommended by the sefh standards. this initial evaluation will serve for improvement measures that increase the quality of prescribing and safe use of pntb, in order to minimize errors that can occur with the use of this therapeutic modality. please specify your abstract type: research abstract background and objective: methadone maintenance treatment was developed in malta in and is provided to patients by sedqa, the national agency against drug and alcohol abuse. methadone is the most frequently prescribed opioid in opioid substitution treatment and is dispensed through a centralised service through the substance misuse outpatients unit. in , patients were in opioid substitution treatment, of who were on methadone. in , the government introduced a take-home methadone program. the prescribing, purchasing and dispensing of methadone are regulated by subsidiary legislation . . the objectives were to determine whether community pharmacists in malta would be willing to dispense and supervise the consumption of methadone and to investigate the involvement of community pharmacies in the development of a regionalised methadone dispensing service. setting and method: the study was set in community pharmacies. a cross-sectional study, through the use of a questionnaire, was performed to quantitatively analyse whether pharmacists in malta would be willing to dispense methadone. the questionnaire consisted of questions divided into sections, with each section assessing a particular aspect of community pharmacists' attitudes towards methadone dispensing. community pharmacies were then chosen via a systematic sampling procedure. a hard copy of the questionnaire, addressed to the managing pharmacist, along with a cover letter, instructions on how the questionnaire was to be returned, and a prepaid self-addressed envelope was distributed via postage to community pharmacies. an online format of the questionnaire was also circulated to community pharmacists through the pharmacy council. data was analysed using spss version . main outcome measures: community pharmacist's attitudes towards methadone dispensing. results: a total of responses were obtained and a response rate of . % was achieved. eighteen percent of the pharmacists (n = ) who responded to the questionnaire worked in a community pharmacy located in the north of malta, % in the centre, % in the south, % in the southeast and % in gozo. thirty-two percent of community pharmacists were willing to dispense methadone to drug misusers. the number of community pharmacists who are willing to dispense methadone increased to % if they were provided with appropriate education and support. twenty-nine percent of community pharmacists were prepared to handle the duty of supervising the consumption of methadone while % had never learnt about methadone and its clinical application within opioid substitution treatment. conclusion: community pharmacists should be provided with education and training regarding methadone substitution treatment before embarking on a new regionalised methadone dispensing service within community pharmacies. this would allow more community pharmacists to become involved in a new dispensing methadone service. pt : evaluation of regorafenib in patients with colorectal cancer please specify your abstract type: research abstract background and objective: the colorectal cancer is the second more frequent cancer in europe and the third in the world. regorafenib is only approved in adult patients with metastatic colorectal cancer who are previously been treated with available therapies or are not considered suitable candidates to these treatments. regorafenib is an oral anti-tumor drug that blocks the kinases involved in the tumor angiogenesis (vegfr , - , - , tie ), the oncogenesis (kit, ret, raf- , braf, brafv e) and the tumor microenvironment (pdgfr, fgfr).in this study, we are reviewed the reports of the patients with colorectal cancer who are been treated with regorafenib in our hospital and analysed the information in order to evaluate the efficacy and safety of regorafenib. setting and method: descriptive and observational study about the use of regorafenib from april to the present day. the variables studied, obtained from the software applications archinet and diraya, were: sex, age, pathology, location of metastasis, posology and adverse effects of regorafenib, tumor markers (cea y ca . ) before and after the treatment with this drug and the mutational state of kras. main outcome measures: the tumor markers cea and ca . only decreased in the . % of the patients after the regorafenib treatment. results: regorafenib was taken by patients ( %men).the average age of these patients was . ± . years old. the patients took regorafenib to treat: metastatic and non-intervened gastrointestinal stromal tumors (gist) e-iv that progressed with the previous treatment of imatinib and sunitinib ( . % patients), intervened colon adenocarcinoma e-iv ( . % patients), sigma adenocarcinoma e-iv ( . % patients) and unresectable and non-intervened rectal adenocarcinoma e-iv ( . % patients).all patients presented metastasis in different locations on the body: liver ( . % patients), diaphragm ( . % patients), intestine ( . % patients) and lung ( . % patients).the % of the patients started the treatment with mg of regorafenib, administrated once a day for weeks followed by one week without this drug; while the . % of the patients started the treatment with mg. however, the . % had to decrease the initial dose and the . % of the total patients had to get off the treatment because of the development of side effects. the most frequent adverse effects were: hypertension associated with headache, hyperbilirubinemia, elevation of ast and alt, intense asthenia. the . % of the patients presents native kras. the native kras was presented in the % of the patients treated with regorafenib who had an appropriate development of the illness (decrease of cea and ca . ) conclusion: the decrease of cea in the . % of the patients and the high development of side effects reveal that regorafenib has low effectiveness and security in the control of the progression of colorectal cancer. in addition, it is supposed that this drug has better results in native kras patients. however, more studies are necessaries in order to demonstrate the effectiveness of regorafenib in this pathology. pt : evaluation of nintedanib in patients with non-small-cell lung carcinoma (nsclc) please specify your abstract type: research abstract background and objective: the nsclc means a high rate of mortality in developed countries. patients diagnosed with nsclc who debut with advanced or metastatic disease have a median survival of months. one of the innovative drugs approved to improve survival in nsclc is nintedanib: an inhibitor of multiple tyrosine kinases, which can be found in some receptors on the surface of cells involves in the growth and spread of cancer cells (''pdgfr'', ''fgfr'' and ''vegfr''). nintedanib is not yet marketed in spain. hospital pharmacists are responsible for applying this treatment as ''expanded drug'', only after the elaboration of an exhaustive report. in this study, we have reviewed all the reports and classified the information in order to present our clinical practice. the objective of this study is to evaluate the effectiveness and safety of nintedanib in patients with nsclc treated in a tertiary hospital. setting and method: descriptive observational study of the use of nintedanib from november to september . sex, age, body mass index (bmi), pathology, smoking habits, line of treatment, posology and adverse reactions of the treatment with nintedanib and tumor markers (cea an ca . ) before and later the treatment with nintedanib were collected from medical history through archinet informatic application. main outcome measures: the tumor marker cea decreased in % of the patients and ca . no decreased in any patient after nintedanib treatment. results: nintedanib was used in patients ( % men and % smoker).the average age of these patients was years old. the average bmi was kg/m ( - ).all patients received nintedanib together with docetaxel for metastatic nsclc with adenocarcinoma histology and with non-mutated egfr and alk in third line treatments. posology: all patients started the treatment with nintedanib mg/ h from day to day every weeks; but patients had to reduce the initial dose to mg/ h ( patient) and mg/ h ( patient) because of some adverse reactions. the side effects were: asthenia, diarrhoea, alteration of transaminases, muscle pain and cramps, weight loss and mucositis. conclusion: the decrease of cea in % of the patients reveals that nintedanib is effective in controlling nsclc progression which involves an increase of the survival and the quality of life of these patients. however, more studies are required to demonstrate the efficacy of nintedanib in this illness. please specify your abstract type: research abstract background and objective: patients with sore throat symptoms often seek fast, meaningful relief when presenting to their local pharmacy. flurbiprofen is a non-steroidal anti-inflammatory drug, which has been developed as a spray and lozenge to provide targeted relief for the main underlying process responsible for the symptoms of sore throats, inflammation. to study the relief provided by flurbiprofen . mg delivered as a spray or lozenge, we conducted a multicentre, randomised, double-blind, double-dummy, parallel group, activecontrolled, single-dose, non-inferiority study. setting and method: adult patients with acute sore throat were randomly assigned to take one dose of either flurbiprofen . mg spray plus a placebo lozenge, or flurbiprofen . mg lozenge plus placebo spray at sites across russia. main outcome measures: patients rated sore throat relief using the sore throat relief rating scale (strrs; a -point scale, = no relief, = slight relief, = mild relief, = moderate relief, = considerable relief, = almost complete relief, = complete relief) at timed intervals throughout h starting from min post completion of first dosing ( min after administration of the spray, and min after the lozenge had fully dissolved). adverse events (aes) were recorded over h post-dose. results: patients were assessed (n = for spray, n = for lozenge). [ % of patients in either treatment group experienced some relief (a score of [ on the strrs) at min post-dose, which increased to % of patients by h. - % of patients reported 'at least moderate relief', which is a well-recognised measure of a clinically meaningful effect at min post-dose, which increased to - % of patients by h. over the h post-dose, a total of drugrelated aes were reported by patients across both treatments and no severe adverse events were reported. conclusion: flurbiprofen . mg delivered as a lozenge or spray provides fast, clinically meaningful relief from sore throat. pt : analising antiangiogenics prescription in an ophtalmology service after a protocol implementation silvia cornejo-uixeda * , ivan de la vega-zamorano, celia aparicio-rubio, olga carrascosa-piquer, manuel prieto-castello, agustin sanchez-alcaraz pharmacy, hospital universitario de la ribera, alzira, spain please specify your abstract type: descriptive abstract (for projects) background and objective: after some years using antiangiogenics in our hospital, we observed a large variety of use. considering the high cost of these treatments, we proposed ophthalmology service to develop a protocol of use, attending efficiency criteria. in this paper, we analyse the protocol implementation repercussion. design: a protocol of use was designed with the main of unify criteria and to use the most efficient treatment depending on the specific situation on each patient. once it was implemented, we compared two periods, the period after the implementation (january-may ) and the period before of it (january-may ). the protocol designed is the following: the cost for each injection and patient was the following: aflibercept €, bevacizumab €, ranibizumab €. results: in the period, patients were treated with antiangiogenics. ( %) with aflibercept, ( %) with bevacizumab and ( %) with ranibizumab. in the period, patients were treated, ( %) with aflibercept, ( %) with bevacizumab and ( %) with ranibizumab. the consumption of aflibercept decreased a %, bevacizumab consumption increased % an ranibizumab increased a %.we also observed, some patients had more than one diagnostic at the same time. once the protocol was implemented, the percentage of use was the following: % . please specify your abstract type: research abstract background and objective: drug prescribing is the most common medical intervention in the elderly. however, elderly patients are more sensitive to the drug's effects due to pharmacokinetic and pharmacodynamic changes associated with aging. chronic diseases and co-morbidities often require the use of a large number of medications. therefore, when prescribing drugs for the elderly, the choice of suitable drugs, dosage and duration of treatment should be carefully considered as well as clinically significant drug interactions. inappropriate prescribing is often associated with an increased risk of adverse drug reactions, increased morbidity and mortality, and health care costs. the aim of this study was to determine the incidence of potentially inappropriate medications (pim) prescriptions in the elderly (c years) using the original protocol developed by mimica matanovic and vlahovic-palcevski. setting and method: we enrolled patients hospitalized in clinic of internal medicine. data about patients' medications was collected during patient interview taken by the pharmacists on hospital admission. pharmacotherapy was analysed using the original protocol developed by mimica matanovic and vlahovic-palcevski in order to detect pims. main outcome measures: number and type of potentially inappropriate medications, potential clinically significant interactions. results: the average age of patients was years (range - ), and the average number of drugs per respondent was . (range - ). a total of patients ( . %) were taking at least one pim. the most common pim were long-acting benzodiazepines, central antihypertensive moxonidine and non-steroidal anti-inflammatory drugs (nsaids) in patients with hypertension. in the study population, patients ( . %) have taken at least one combination of drugs that could result in a clinically significant interaction. the most common combinations included application of nsaids and antihypertensive drugs or diuretics, concomitant use of multiple medications with effects on the central nervous system and drug combinations that can cause hyperkalaemia. conclusion: this study revealed the high prevalence of inappropriate prescribing. clinical application of this protocol could be an effective method for improving and optimizing drug prescription with the aim to reduce the number of side effects and the morbidity and mortality associated with the drug use in the elderly. please specify your abstract type: research abstract background and objective: to reduce adverse effects of conventional amphotericin b formulation (deoxycholate or d-amb) it can be infused in intralipid Ò (a fat parenteral nutrition), or lipid-based formulations can be used (i.e. amphotericin b lipid complex (ablc), amphotericin b colloidal dispersion (adcd) and liposomal amphotericin b (l-amb)). studies evaluating safety profiles present conflicting results. the aim of our study was to gather evidence on nephrotoxicity rates of d-amb versus lipid-based formulations in immunosuppressed patients susceptible to invasive fungal infection. setting and method: a systematic review, including randomized controlled trials (rcts) that compared the use of d-amb and amphotericin b lipid-based was performed. a search was conducted in pubmed, scopus, web of science and scielo. results were synthetized and meta-analysis was performed using software review manager . . main outcome measures: nephrotoxicity rates. results: eighteen rcts were identified (n = participants). the result from the meta-analysis favours the treatment with the lipidbased amphotericin b formulations (or: . ( . , . ) and presents a low heterogeneity (i = %). about % of patients from lipid-based treatment group presented an increase in serum creatinine of one to two times, which corresponds to stage one or two of acute renal failure (arf). and % presented an increase of tree times in serum creatinine achieving a stage three in arf (severe) which will require dialysis. while in group treated with conventional formulation int j clin pharm ( ) all of these patients, except one whose treatment adherence was inadequate, were cirrhotic ( / ), liver transplanted ( / ) and/ or presented hepatocellular carcinoma ( / ). / patients were coinfected with hiv. / patients ( %) were genotype . the total genotype patients treated with daas (svr /relapsed) were , which means that . % ( / ) of all genotype patients has had a relapse. / patients ( %) were treated with ledispavir/sofosbuvir ( . % of a total of patients (svr /relapsed) treated with this option). % of patients who suffered a relapse were treated with daas sofosbuvir, simeprevir, daclatasvir, previously to the introduction of the newest antivirals (dasabuvir + ombitasvir/ paritaprevir/ritonavir, ledispavir/sofosbuvir), which represents . % of the total of patients treated with the older option. conclusion: relapses rate was . %, slightly lower than reported in other studies. according to the references, these results show that genotype is the one presenting more relapses. all the patients presented a deteriorated performance status, except for one whose treatment adherence was inadequate. patients treated before april , when the newest daas where introduced, showed more relapses. more studies have to be developed in the near future since other daas will appear, the treatment options will be amplified and the number of relapses is expected to decrease. please specify your abstract type: research abstract background and objective: the inappropriate use of antibiotics remains a major issue since it causes bacterial resistance, longer hospital stay and increased mortality. antibiotic prescriptions must be monitored: the clinical pharmacist has a key role in ensuring patient safety and quality of pharmaceutical care. therefore, an antimicrobial stewardship program has been implemented as part of a national project of the italian society of hospital pharmacy (sifo). the objective is to describe the results obtained at the hospital. setting and method: a multidisciplinary antimicrobial management team has been implemented including clinical pharmacists, microbiologists and infectious disease specialists. the pharmacist examines drug charts on a daily basis in the department of medicine and supports clinicians to improve the appropriate use of antibiotics. data from time-points were extracted from medical records and collected in an excel database: t (november -january ) and t (february -april ). main outcome measures: type of infection, antibiotic consumption data, type of isolated pathogens, patient allergies, clostridium difficile infection assessment and adverse drug reactions (adr). results: records were analysed (t -t ), of which contained at least one antibiotic prescription. the most frequent infections were urinary tract ( %), respiratory ( %) and gastro-intestinal ( %). antibiotic therapy was started in . % of cases due to aspecific increase of c-reactive protein (crp). ddds were calculated for each treatment and were grouped by type of infection and setting (empiric vs targeted): ceftriaxone, meropenem and metronidazole were the most widely used antibiotics for empiric therapy. at t , an increase in the use of piperacillin-tazobactam instead of meropenem was observed. the ddd of ceftriaxone for targeted therapies decreased significantly, while an increase was observed for carbapenems, levofloxacin, glycopeptides and, in case of mdr bacteria, tigecycline. three allergies to antibiotics were reported in medical history. there were clostridium difficile infections ( relapses), confirmed by antibiogram. a total of adrs were identified: of these were related to antibiotics. conclusion: antimicrobial stewardship is a fundamental step to optimise antibiotic management, ensure patient safety and improve quality of care. the results obtained so far demonstrate the added value of a multidisciplinary team in controlling bacteria resistance and in the improving the use of antibiotics. please specify your abstract type: descriptive abstract (for projects) background and objective: the aim of this study was to analyse effectiveness and safety of pirfenidone, an anti-inflammatory and antifibrotic agent used for treatment of idiopathic pulmonary fibrosis. design: a retrospective, descriptive, observational study including all patients treated with pirfenidone at the hospital between march and june ( month) was carried out. to identify patients and collect data the outpatient medication dispensation software farhos Ò and the electronic medical record software hcis Ò were used. statistical analysis was carried out using microsoft excel Ò . demographic (age and sex), clinical (forced vital capacity (fvc), diffusing co capacity (dlco) and six-minute walk test (wt m)) and therapeutic (dosage and adverse reactions) variables were collected. results: throughout the study period, a total of patients ( males) started treatment with pirfenidone, with a median age of . years ( - ). during this period patients were excluded for lack of monitoring. the median fvc, dlco, wt m values prior to pirfenidone therapy, were % ( [ %), . % ( [ %) and m ( - m) respectively. all patients met the inclusion criteria of capacity trial according to fvc and wt m; however of them didn't meet the dlco criteria (at least %).'' all patients were monitored every months. the median in fvc percentage change at the end of the study was - % (- % to + %). patients ( %) showed an improvement on fvc during treatment with a median change of %. in the other eight patients fvc value decreased with a median of - %. only one patient would be candidate to discontinue treatment due to a lack of efficacy, according to discontinuation criteria established at the hospital (absolute decrease of c % in fvc during first year of treatment). dlco percentage was measured in patients, with a median change of % (- % to + %). dlco decreased in patients. wt m was monitored in patients, with a median change of - . m (- m to + m). adverse effects related to pirfenidone were gastrointestinal disorders ( / ), increase of hepatic ggt ( / ), and dermatologic toxicity ( / ). six patients ( %) required a dose reduction because of gastrointestinal adverse effects. five patients ( %) discontinued treatment with pirfenidone due to hepatotoxicity ( ), gastrointestinal ( ) and dermatologic effects ( ). one patient died. conclusion: half of the patients improved fvc during the period of the study. the other half, showed a decrease in fvc value which was similar to the median obtained in capacity trial. gastrointestinal disorders were the most frequent adverse effects and cause of discontinuing treatment. treatment monitoring is important to achieve therapeutic benefit and control the adverse effects. the national centre for epilepsy, oslo university hospital, oslo, norway please specify your abstract type: research abstract background and objective: systematic medication reviews in interdisciplinary teams can help to identify potential and actual drugrelated problems (drp). the centre for development of institutional and home care services in oslo, norway, conducted medication reviews for polypharmacy patients with mental disabilities in - , based on a lack of knowledge about drug-related problems in this patient group. the objective was to examine prescribing patterns, frequencies and types of drp in patients with mental disabilities. setting and method: the forms for medication reviews were developed by the national patient safety campaign in norway. the nurse/social educator recruited eligible patients, observed them, and ordered test if needed. the clinical pharmacist (jwa) reviewed the medications to identify drps. the interdisciplinary case conference took place at the different general practitioners' offices being responsible for the individual patients. the general practitioner, the nurse/social educator and the pharmacist were present, and in some cases, also patients took part. main outcome measures: an independent researcher (aqm) collected and analysed the data based on the drp-forms containing information on the prescribed medicines, strength, dose, indication, a description of drp and suggested interventions to resolve them. results: overall, patients with mental disabilities, aged - years, consented to have a medication review. they used on int j clin pharm ( ) : - average medicines (range - ). the team identified drp in of the patients (average . , range - ). overall, % of all drp were resolved. for one-third of the medicines, an action was taken to improve the prescribing. the most commonly medicines were analgesics ( %), antiepileptics ( %) and anxiolytics ( %). the most frequent drps were unnecessary drug choice ( %), side effects ( %) and too low dose ( %). drps were most common in antipsychotics ( %), antidepressants ( %) and anxiolytics ( %). conclusion: patients with intellectual disabilities take more medicines and have many drps compared to other patient groups. they are also more prone to taking combinations of cns-active medicines and therefore more at risk of side effects and drug interactions. pt : protocol feasibility and patient findings when using a dry extract of zingiber officinale roscoe (ginger extract gr ) during pregnancy please specify your abstract type: research abstract background and objective: there is limited information about the use of dry extracts of ginger root. the objectives of this study are ( ) to evaluate the feasibility of a pilot study with a food supplement among pregnant women ( ) to learn what the patient findings are when using the dry extract of ginger during pregnancy. this abstract deals with the intermediate evaluation of a study conceived to investigate the safety of the ginger extract gr during pregnancy. setting and method: a prospective, interventional and real life pilot study with pregnant women between and weeks of gestation and having symptoms of nausea and vomiting or digestive complaints. the included patients can use the ginger extract gr for digestive comfort during pregnancy when needed. during the use, the score of digestive discomfort is noted and the researcher reports adverse events. main outcome measures: ( ) number of included patients as an indicator of feasibility: including a number of patients was taken as a target ( ) analysis (qualitative and quantitative) of the patient diaries, more particularly patient behaviour, wellbeing and impressions. results: within twelve weeks, patients were included with an average age of . years and a median age of ( - ) years. patients used gr : patients were dissatisfied, patients had a neutral opinion and patients were satisfied to very satisfied. one miscarriage occurred at a gestational age of almost weeks (only tablets of gr were used, with no relevant medical history in preceding pregnancies). two patients were hospitalized, of which with hyperemesis gravidarum. one patient complained about heartburn and one patient experienced a bad taste and heartburn. three patients have indicated that they experienced more nausea after taking the tablets. patients experienced no adverse events. the remaining patients were not yet evaluated. of the included patients, six patients decided not to use the product: because their gastrointestinal complaints were not serious enough, because problems of swallowing (using ginger gums instead). one patient was afraid for the negative consequences for her unborn child. the last of the nonusers indicated that she had no confidence in the product. conclusion: conducting a pilot study with the ginger extract gr in case of pregnancy is feasible. the majority of the evaluated patients were satisfied. signing the consent form does not guarantee the intake of the product. pregnant women remain very cautious in the use of unknown products during their pregnancy, even though it concerns a food supplement and not a drug. the severity of symptoms does not give a good indication whether or not and how often the product will be used. please specify your abstract type: descriptive abstract (for projects) background and objective: to analyse effectiveness and safety of ibrutinib, an oral inhibitor of bruton tyrosine kinase, in patients with mantle cell lymphoma (mcl) who have received at least one prior therapy. design: a descriptive observational study was carried out. all patients with relapsed or refractory mcl who started treatment with mg of daily ibrutinib between september and june were included. patients were identified and followed through electronic medical record. demographic and baseline clinical characteristics of patients were collected: age, sex, ecog (eastern cooperative oncology group scale), number and type of prior regimens, simplified mipi status (mantle-cell lymphoma international prognostic index), and disease stage (relapsed or refractory). progression free survival (pfs) and response to treatment were recorded to evaluate effectiveness. adverse effects related to ibrutinib and possible interactions with concomitant medication were documented to measure safety. statistical analysis of the data was carried out using microsoft excel Ò and spss Ò . results: throughout the period of study a total of patients ( males and female) with a mean age of . ± . years started treatment with ibrutinib. the median of previous treatments were ( ) ( ) ( ) ( ) ( ) including first-line treatment with high dose chemotherapy ( %), steam-cell transplantation ( %), rituximab ( %), bortezomib ( %) and lenalidomide ( %). the median ecog value prior to ibrutinib therapy was (range - ). the mipi status was intermediate risk in patients and high risk in , the disease stage was relapsed in % of the patients. partial response was reported in patients. the mean pfs estimated at the end of the study period was months ( % . - . ). adverse effects related to ibrutinib were: fatigue ( %), diarrhoea ( %) leucocytosis ( %) and infections ( %), including upper respiratory and urinary tract infections, sinusitis and pneumonia. one possible interaction between ibrutinib and everolimus was found in a liver transplant patient. close monitoring of everolimus plasmatic levels was recommended. conclusion: the mean pfs estimated in our study was similar to the median obtained in the pivotal phase ii trial. infections were the most frequent adverse effects. concomitant medication to ibrutinib should be checked, as ibrutibib is metabolised by cyp a and interactions may be frequently present. pharmacy, hiv unit, germans trias i pujol hospital, badalona, spain please specify your abstract type: descriptive abstract (for projects) background and objective: dolutegravir (dtg) is one of the preferred options for initial antiretroviral therapy (art) due to its high efficacy, good tolerability and low potential for drug-drug interactions. nevertheless, an unexpectedly high rate of dtg discontinuation (up to %) due to adverse events in the clinical practice has been recently reported. therefore, we aimed at assessing the dtg discontinuation rate and reasons for discontinuation in our hospital. design: single-centre, retrospective study from september to june of patients cohort with art both naive and pretreated. patients who had started dtg-based art containing regimen were identified and the reasons for the discontinuations were analysed. data were collected using the primary care service program and the electronic prescription program. results: out of patients attended by pharmacy department in our hospital, patients ( males, mean age years (range - )) had started a dtg-based art. out of them, patients were art naive and art-experienced. at the moment of starting dtg, mean cd cells were cell/mm (range - ) and hiv- rna load in plasma was detectable in patients. treatment discontinuation was reported in / patients ( . %) with a median treatment time of days (range - ). / patients ( . %) were naïve and / patients ( . %) pre-treated. most of the patients ( ) were in single tablet regimens (str) containing dtg in combination with abacavir and lamivudine, whereas the rest were in combination with other antiretroviral drugs. the main reason for treatment discontinuation was toxicity in / patients ( . %). the rest of the patients discontinued due to other motives (clinical trial inclusion ( / ), treated in another hospital ( / ), exitus ( / ) and others ( / ). reasons for the discontinuation were classified in different side effects: / ( . %) related to central nervous system (cns) (insomnia, psychiatric disorders such as anxiety, nightmares and depression), / ( . %) gastrointestinal effects, / ( . %) headaches, / ( . %) musculoskeletal effects, / ( . %) fatigue, / ( . %) allergy and / ( . %) for other reasons. some patients reported various toxicities at once. conclusion: more than % of patients treated with dtg discontinued by toxicity reasons. it is important to note that half of these patients had cns adverse effects. please specify your abstract type: research abstract background and objective: hcv therapy has been revolutionised recently by the approval of antiviral agents direct-acting (daa) facilitating the treatment of patients coinfected with hiv/hcv. however, potential drug interactions and overlapping toxicities of both treatments represent the major challenges in adapting therapy. to analyse the prescription profile of direct acting antivirals (aad) in patients coinfected with hiv/hcv. setting and method: retrospective observational study from january to january in a specialty hospital. the data were collected from the hospital program of clinical stories, archinet Ò , and the outpatient program farmatools Ò . the results were analysed using the statistical program r-commander. main outcome measures: inclusion criteria: adult patients coinfected with hiv/hcv with undetectable viral load. the following variables were collected: age, gender, hcv genotype, degree of fibrosis, patient type (naïve or pre-treated), baseline cd count, cd levels end of treatment, sustained viral response (svr) and hcv treatment. results: patients, of whom were men, mean age years were included. patients received daclatasvir and sofosbuvir for hcv, patients had genotype a and b respectively, patients genotype and patient genotype . patients had fibrosis f f , . of the patients they had not received previous treatment (naïve) and had failed to treatment. hiv treatment was modified in patients, patients achieved svr. the cv was undetectable to hiv treatment change for all patients. cd levels increased in all patients at the end of treatment for hcv with a median of cells/ul and at the beginning and end respectively. patients received ombitasvir/paritaprevir/ritonavir and dasabuvir, who had a genotype a. these two patients had received previous treatment and had a f and f fibrosis. none of them was modified hiv treatment and only one got svr. cv remained undetectable and cd slightly increased after the treatment. patients received ledipasvir and sofosbuvir, patients had genotype a, patients genotype b and patient genotype . patients had f fibrosis and had f . patients had received previous treatment (naïve). the hiv treatment was modified only in one of the patients, patients achieved svr. cv increase in patients after the treatment while cd followed the trend of increasing. conclusion: the aad that caused fewer changes in the hiv treatment were ombitasvir/paritaprevir/ritonavir and dasabuvir followed by ledipasvir/sofosbuvir. sofosbuvir and daclatasvir present a greater number of interactions with hiv drugs so they behaved to a major change. more patients are needed to assess more accurately the aad leading to a minor modification. please specify your abstract type: research abstract background and objective: the simplification strategies reduce the amount of tablets and the toxicity in order to facilitate adherence in patients with virological suppression. the strategy more studied is monotherapy with a ritonavir-boosted protease inhibitors (pi/r). to analyse the effectiveness of monotherapy with pi/r in pre-treated patients infected with hiv. setting and method: retrospective observational study. selected hiv patients treated with pi/r monotherapy at any time of pharmacotherapeutic history to / / , with at least one clinical and analytical control months before the beginning. data were collected from the medical record archinet Ò and outpatient farmatools Ò program. variables included were age, sex, duration of monotherapy, virological failure, treatment failure, cd % during monotherapy. main outcome measures: inclusion criteria: virological suppression for year prior to the start of monotherapy, no previous ip virological failure, high cd count ([ cell/ml) and a high level of drug adherence. the effectiveness is defined as the percentage of patients without virological failure ( consecutive plasma viral load (vl) [ copies/ml) and without treatment failure (any event causing retirement monotherapy). results: patients with monotherapy, which represent % of patients with antiretroviral therapy (art) at our institution were identified. were excluded ( co-infected with hepatitis virus, with insufficient data and no had more than months included), including patients in the analysis, with a mean age of years and % were men. the median of time monotherapy treatment was . years ( . days), ( . %) patients received darunavir/r and ( . %) lopinavir/r. the effectiveness of monotherapy treatment during the follow up period was % with undetectable pvl at follow-up. the median of cd % over the treatment time was cell/ml ( %). conclusion: the effectiveness of treatment with ip/r monotherapy in our hospital obtained good results. according with our results treatment adherence plays a very important role. this is a current and valid strategy that brings benefits to the patient and to the healthcare system. please specify your abstract type: research abstract background and objective: the access to investigational drugs for patients who are not included in a clinical trial and without authorized therapeutic alternatives is known as compassionate use. the incorporation of the evidence-based medicine in the area of oncohaematology has implied that an important part of clinic therapy validated by evidence that could not be controlled from an administrative point of view. this is due to the continuous and progressive development of investigation and information on cancer treatment and the delay of the administration regulation. the use of drugs in this way is regulated by royal decree / ( / ). the objective of the study is to describe the use of cancer drugs through compassionate use in the last years in a specialty hospital. setting and method: descriptive retrospective study on a specialty hospital. all the applications for a compassionate use drugs were analysed from january until october . the data were obtained from medical records programme diraya Ò and from an excel database of medicines in compassionate use of the pharmacy service. main outcome measures: the following variables were registered: • number of patient clinic history • authorized medicine • authorization date • applicant service results: we recorded requests of cancer drugs in compassionate use during the years of study. oncology was the service that recorded more authorizations with %, followed with gynaecology with . % and finally endocrinology and haematology with . %. drugs of the requests were approved ( %) and unauthorized ( %) in the years of study. the year in which more applications were received was ( . %) and the least requests were received in ( . %), being the year where all requests were authorized. in fewer applications were authorized, %. in the years , and were authorized . , and . %, respectively. a total of different active drugs were received during the study, the most requested bevacizumab ( %) for grade iii oligoastrocytoma, ovarian cancer (monotherapy), metastatic gall bladder cancer and metastatic platinum-resistant ovarian cancer, everolimus ( %) for indications of neuroendocrine carcinoid tumour and metastatic breast cancer, nab-paclitaxel ( %) for invasive lobular carcinoma indications of high-grade and metastatic pancreatic cancer, ipilimumab ( %) for the indication of metastatic melanoma, and regorafenib for indications of colorectal cancer and metastatic gist i pre-treat with imatinib ( %). the solicitude of drugs through compassionate use needs effective commissions of pharmacy and therapeutics, along with the medical management to establish an agile and faster requesting circuit and the consequent use monitoring. please specify your abstract type: research abstract background and objective: to describe the standard procedure for the elaboration and control of a magistral formula (mf) to assess their effectiveness in two patients with cutaneous metastases of malignant melanoma refractory to other treatment. setting and method: medication for compassionate use was requested for two patients of and years with histopathologic diagnosis of cutaneous metastases of malignant melanoma in the left thigh and left heel in which the lack of response to first-line treatments made to be valued to start with adesleukina intralesional therapy. the first week was infiltrated mu ( ml) in lesions less than cm, mu ( ml) in the larger lesions and repeating each week until complete remission of the lesions. in the nd patient we proceed in the same way but the second week was infiltrated mu ( ml). the following week, infiltrated mml, in metastases and we turn to weekly infiltrations. the response was assessed by clinical disappearance of the lesions treated. complete response (cr) is defined as a clinical disappearance of lesions and partial response (pr) greater than % reduction of the lesion diameter. main outcome measures: we performed a literature search (pubmed, trissel, spc) for all studies published to determine the standard procedure for preparing and monitoring the mf (processing, preservation, stability, dose and indication). results: the standard procedure of preparation and quality control was carried out following the rules established in rd / . it was made in a vertical laminar flow cabinet. the aldesleukin vial was reconstituted with . ml api ( mu/ml) and then diluted with . ml of a solution of . % albumin, % glucose as stabilizer, to avoid aggregate formation, preparing ml syringes ( mu/ml). it was obtained a homogeneous and clear solution without precipitate or opalescence appearance. stable days in a refrigerator ( - °c), protected from light. initially patients had approximately a total of injuries. after months of treatment it was obtained a cr of most lesions in the first patient and rp of the second patient injuries. treatment was well tolerated. the side effects presented were only a flu-like syndrome in the second patient. conclusion: intralesional administration aldeslukina has been effective in treating malignant melanoma skin metastases in our patients, allowing the extension of its use in patients with the same involvement refractory to other primary treatments. the results are similar to those of the publications consulted. please specify your abstract type: research abstract background and objective: chronic infection with hepatitis virus c (hcv) affects about million people worldwide and is a leading cause of liver cirrhosis and hepatocellular carcinoma. the new direct acting antivirals against hcv have revolutionized the treatment of this disease. due to the high cost of these drugs it is necessary to assess their use in clinical practice. to evaluate the effectiveness of daclatasvir in combination with sofosbuvir in patients with hcv monoinfected in a specialty hospital. setting and method: retrospective observational study of patients who began treatment with the combination of daclatasvir and sofosbuvir from january to january in a specialty hospital. the data were collected from the hospital program of clinical stories archinet Ò and the outpatient program farmatools Ò . the results were analysed using the statistical program r-commander. main outcome measures: the sustained virologic response (svr) was considered the primary endpoint of the study. as secondary variables were analysed: sex, duration of treatment, naïve patients or pre-treated, degree of fibrosis, hcv genotype, concomitant use with ribavirin, viral load (vl) before treatment and medical service. results: there were included patients of whom were men. baseline characteristics were: patients with genotype , genotype b, with genotype a and genotype . the degree of fibrosis in the study was patients with f , f and to f . among the patients infected with hcv genotype , had not received prior treatment (naïve) and had failed therapy. the duration of the treatment was weeks to patients and weeks for patients. only patients receiving ribavirin of these had genotype and genotype b. from ribavirin patients it was greater the number of patients in whom the treatment duration was weeks ( patients versus with p-value = . ). the digestive service attended to patients while patients were followed by infectious. the median cv was , , iu/ml. svr was achieved in . % of patients with hcv genotype in . % with genotype b and % with genotype and a. after weeks of treatment % of patients achieved svr and % after weeks. only one patient died during treatment. the results are similar to those obtained in clinical trials. svr has not been influenced by hcv subtype, duration of treatment, degree of fibrosis, pre-treatment or by concomitant use of ribavirin. further studies are needed to evaluate the efficacy of this treatment. please specify your abstract type: research abstract background and objective: the safety and efficacy of medications can vary significantly between patients as a result of genetic variability. as genomic screening technologies become more widely available, pharmacists are ideally suited to utilize this tool to optimize medication management. the objective of this study is to evaluate the feasibility of implementing personalized medication services into community pharmacy practice and to assess the number of drug therapy problems identified as a result of pharmacogenomic screening. setting and method: the study was designed as open-label, nonrandomized, and observational. two community pharmacies in toronto, ontario offered pharmacogenomic screening as part of their professional services program. prior to initiation, participating pharmacists received structured, comprehensive training in pharmacogenetics. pharmacists then facilitated voluntary subject enrolment among patients who they believed would benefit from screening and met inclusion criteria. eligible patients received a simple buccal swab followed by dna analysis using pillcheck Ò . pillcheck Ò is a genotyping assay that translates genomic data and generates a personalized, evidence-based, report that provides insight into patients' inherited drug metabolic profile. upon receiving the report, pharmacists invited patients back to the clinic for interpretation of the results. clinically significant drug therapy problems were identified and recommendations for medication optimization were forwarded to the primary care physician. main outcome measures: number of clinically significant drug therapy problems identified by pharmacists as a result of pharmacogenomic testing. results: patients were enrolled in the study. average age was . years and patients were taking a mean of . chronic medications. pharmacists cited the most common reasons for testing as ineffective therapy ( . %), to address an adverse reaction ( . %), and to guide initiation of therapy ( . %). an average of . drug therapy problems were identified per patient. pharmacist recommendations included change in therapy ( . %), dose adjustment ( . %), discontinuation of a drug ( . %), and increased monitoring ( . %). generally, physician feedback was positive but did reveal an opportunity for a broader understanding of the technology. conclusion: these results highlight the readiness of community pharmacists to adopt pharmacogenetic screening into practice and their ability to leverage this novel technology to positively impact medication management. community pharmacists are ideally suited to both offer personalized medication services and interpret genomic results. please specify your abstract type: descriptive abstract (for projects) background and objective: visual impairment is a common geriatric syndrome and glaucoma/miotic eye drops treatment is a frequent therapeutic option. pharmacist's role in medication reconciliation is an effective process for reducing medication errors and supporting safe medication use. we observed that mentioned medication reconciliation was occasionally not performed during hospital stay and could be cause of delirium because of visual impairment. the aim of this study was to evaluate the influence of omission errors of eye drops treatment on incidence of acute confusional state. design: we conducted an observational, descriptive and retrospective study in an orthogeriatric unit with an average of patients with hip fractures per year ( % surgically treated). data collection was performed from june to march . reconciling medications at admission was performed by implementing the tools and resources of the canadian patient safety institute (cpsi). we extracted from our electronic database (filemaker pro Ò ): • demographic patient data (age and gender). • name and posology of the glaucoma/miotic eye drops treatment. • medication reconciliation performed and identification of professional in charge (pharmacist, geriatrician or orthopaedic surgeon) registration during hospital stay. • protocolar management of delirium with tiapride occasional intramuscular administration performed if necessary was also registered to establish the incidence of acute confusional state. results: thirty-two patients ( women and men) were included, median age year-old . in patients, eye drops reconciliation treatment was performed by the pharmacist in of the patients, the geriatrician in cases and the orthopaedic surgeon in . in patients, the mentioned medication reconciliation was not performed (pharmacist absentism). considering the patients on eye drops treatment during hospital stay, ( . %) of them suffer from acute confusional state. on the other hand, among the patients without medication reconciliation, delirium was registered in cases ( . %). concerning ocular topic treatment, . ± . active principles per patient were observed, being the most frequent timolol ( . %), brinzolamide ( . %) and latanoprost ( . %). conclusion: we consider of paramount importance the pharmacist evaluation availability at an orthogeriatric unit, minimizing the impact of acute confusional state during hospital stay by medication reconciliation. please specify your abstract type: descriptive abstract (for projects) background and objective: to report the therapeutic management of haemorrhagic rectocolitis onset in a lung-transplanted patient with mycophenolate-induced diarrhoea. design: case report. results: a -year-old-man lung transplant patient for alpha -antitrypsin deficiency in receiving mycophenolate mofetil, tacrolimus and corticosteroid developed chronic diarrhoea worsened by sigmoid and cecal necrosis in , and treated successfully by sigmoidectomy. severe diarrhoea attributed to mycophenolate mofetil reappeared in april , which motivated a switch to mycophenolate sodium. the absence of clinical improvement in june led to stop mycophenolate sodium and introduce azathioprine at mg/day (absence of mutation for the thiopurine methyl transferase gene). one month later, the patient presented melena, diarrhoea, bloating, nausea, and knee pain, attributed to azathioprine. this latter was stopped and mycophenolate mofetil was rechallenged associated with symptomatic treatment (i.e., diosmectite and loperamide). in january , a colonoscopy, performed in a context of profuse chronic diarrhoea with mucus during months, highlighted haemorrhagic rectocolitis. therefore, the patient initiated sulfazalasine therapy with no clinical improvement, and then high doses of oral corticosteroids. because high-dose of oral corticosteroids was not recommended as a long-term treatment, mercaptopurine was proposed as a new therapeutic option. mercaptopurine has no indication as an immunosuppressive treatment in solid organ post-transplant supportive care. however, as the active metabolite of azathioprin, an immunosuppressive drug widely used in transplantation, mercaptopurine has immunosuppressive functions towards t-lymphocytes. after multiprofessional collaboration between gastroenterology, pneumology and pharmacy specialists, it was decided to stop mycophenolate mofetil and introduce mecaptopurine at . mg/kg/day, as immunosuppressant for haemorrhagic rectocolitis as well as lung transplantation. this unusual lung transplant immunosuppressive therapy, associated with tacrolimus, improved digestive disorders and patient's quality of life. currently, mercaptopurine is biologically and clinically well tolerated. the dosage of blood residual concentrations of purinethol metabolites ( -thioguanine and -methylmercaptopurine) is going to be performed. conclusion: immunosuppressive therapy in solid organ transplantation is a real challenge for patients who have comorbidity onset. despite a lack of data in the literature, a multidisciplinary collaboration based on comprehensive pharmacology skills is essential to choose the best therapeutic option in this type of patients. please specify your abstract type: descriptive abstract (for projects) background and objective: the use of complementary medicines (cm) in oncology is the subject of broad but still controversial interest. a large part of patients with cancer uses cm, including complementary drugs, during their treatment period. indeed, according to different studies, this proportion ranges from to %. importantly, the risk of interaction between cm and anti-cancer drugs is not negligible; hence we need to identify these cm to ensure the security of our patients and the success of their treatment. design: to achieve this purpose, a monocentric retrospective analysis was conducted with collection of data by pharmacy students during medication reconciliation of hospitalized patients from january to june . collected data are patients' characteristics, prevalence of cm use and potential cm-anticancer drug interactions. results: patients were included in the study ( men- women); median age was [ - years]. a total of . % (n = ) were using a least one cm, most frequently homeopathy ( %, n = ) or phytotherapy ( %, n = ); some patients were using a combination of two cm ( %, n = ). cm are mainly used by women in comparison to men ( . % versus . % and p = . , chi square test). for phytotherapy, at least different herbs were described by patients and among them the most frequently used were mistletoe (viscum album), propolis and fireweed (epilobium angustifolium). data analysis showed that % (n = ) of patients were at risk of potential cm-anticancer drug interaction. moreover this risk was increased to % if we considered only patients taking phytotherapy. interactions included pharmacokinetic ( %, n = ), such as altered hepatic metabolism, and pharmacodynamics ones ( %, n = ). conclusion: in conclusion, our work clearly demonstrates that the use of cm by patients is associated with high risk of relevant drug interaction with their anti-cancer treatment. even if further investigations are necessary to clarify the clinical impact of these interactions, the use of cm must be considered during prescribing process. please specify your abstract type: research abstract background and objective: since their reimbursement, the direct oral anticoagulants (doacs) are increasingly used for stroke prevention in atrial fibrillation (af). the objective of this study was to identify the proportion of real life patients with af eligible for doac therapy, based on the inclusion and exclusion criteria used in the clinical studies and based on the officially approved indications as mentioned in the summary of product characteristics (smpc). setting and method: data for this retrospective cross-sectional study was extracted from the uz brussel stroke registry, containing anonymized data of patients with a suspected stroke. characteristics of patients with documented af were compared with the patient characteristics in clinical trials and the approved indications in the smpc. main outcome measures: proportion of real life patients with af eligible for doac therapy. results: data of patients with af was analysed. based on the selection criteria of the clinical trials, significantly less patients were eligible for treatment with rivaroxaban compared to dabigatran etexilate ( . % versus . %; p = . ), but not compared to apixaban ( . %; p = . ). based on the indications and contraindications in the smpc, significantly fewer patients were eligible for apixaban compared to dabigatran etexilate and rivaroxaban ( . % for apixaban, . % for dabigatran etexilate and . % for rivaroxaban; p \ . and p \ . , respectively). significantly more patients were eligible for doac therapy based on the indications and contraindications in the smpc compared to the inclusion and exclusion criteria of the clinical trials ( . % versus . %; p \ . for dabigatran; . % versus . %; p \ . for rivaroxaban and . % versus . %; p \ . for apixaban). conclusion: when taking into account the selection criteria from the pivotal clinical trials with doacs for stroke prevention in af, less than half of real life patients are eligible for therapy with one of the doacs. however, the indications mentioned in the smpcs of these drugs are less strict. please specify your abstract type: research abstract background and objective: idiopathic pulmonary fibrosis (ipf) is a disease in which tissue deep in the lungs becomes thick and stiff, or scarred, over time. the formation of scar tissue is called fibrosis. pirfenidone is an anti-fibrotic and anti-inflammatory agent, thus offers a new hope for treating progressive fibrotic diseases. int j clin pharm ( ) : - our objective is to set a description of idiopathic pulmonary fibrosis patients treated with pirfenidone, as well as the adverse reactions observed. setting and method: descriptive study in which all patients have received pirfenidone. the data were obtained through the dispensing program of outpatient (farmatools) and review of medical records of the hospital database (archinet) and clinical station (diraya). main outcome measures: we have extracted from each patient baseline data, comorbidities, dose received, reported adverse reactions and data about haematology and biochemistry. results: we have a total amount of patients treated with pirfenidone, all diagnosed with idiopathic pulmonary fibrosis, including women and men. the age of patients is between and years, with an average of . years. all patients are ex-smokers and one of them is also ex-alcoholic. concerning concomitant pathologies, patients have diabetes mellitus, have arterial hypertension, and one of them has ischemic heart disease. another has upper gastrointestinal bleeding prior, among others chronic pathologies. pirfenidone dose received was the usual dose in of the patients: days - mg every h, days - mg every h and a maintenance dose of mg every h. in one patient due to its low imc the dose received was smaller ( - days mg every h, days - mg every h and maintenance dose of mg every h). in relation with the adverse effects, digestive discomfort were observed in of the patients, causing the interruption of the treatment in one of them (with prior gastrointestinal bleeding). in the other patient it was relieved by lowering the dose received. also, one patient has experienced photosensitivity. alterations in transaminase levels were observed in patients but that didn't force to discontinue the treatment. no alterations were observed in the blood count. conclusion: treatment with pirfenidone is being generally well tolerated by patients. it has improved their life-quality and reached the objective data of a slowdown in disease progression. currently, the number of patients is no enough to give conclusive information in relation to the drug effectiveness. please specify your abstract type: research abstract background and objective: to describe the total amount of patients treated with a magistral formula of sodium cromoglycate mg without excipients: indications, concomitant therapy and the response to therapy. setting and method: we run a descriptive study in which we included the totality of patients in treatment with a magistral formula of sodium cromoglycate mg without excipients in a tertiary hospital. the data were obtained through paracelso (development of magistral formulas program), as well as with farmatools (dispensation program of outpatient) and the review of medical records from the hospital database (archinet), and diraya clinical station. main outcome measures: from each patient we extracted data relative to sex, age, diagnosis, time in treatment with the formula, dose received, response to therapy, concomitant antihistamines treatments and adverse effects. results: a total of patients in treatment with a magistral formula of sodium cromoglycate mg without excipients were reviewed: women and men with a mean age of . years old (range - years). regarding the indication of the prescription, patients have been diagnosed of indolent systemic mastocytosis and the remaining were diagnosed of mast cell activation syndrome. in all cases, the diagnosis was established by examination of the bone marrow in the mastocytosis studies institute of castilla la mancha (spain). on average, patients took the treatment . months, with a range between months and months. the dose received was mg every h in patients, having to be increased to mg times daily in a case with poor response to the therapy. in the remaining patients, the treatment response has been optimal. in relation to the concomitant anti-allergic treatment received, patients took fexofenadine daily during the study. no cases of adverse effects related to the therapy received have been reported. conclusion: both indolent systemic mastocytosis and mast cell activation syndrome are considered rare diseases, and we should indicate that in spain there are no commercial medicines available of sodium cromoglycate without excipients for its treatment. the treatment with this magistral formula of sodium cromoglycate mg without excipients has been effective and well tolerated in all patients, improving the symptoms associated with their condition as well as their quality of life, and also, assuming a solution to the lack of marketing of the drug currently in spain. please specify your abstract type: research abstract background and objective: to analyse the prescription profile, safety and effectiveness of new therapies available for the treatment of hcv genotype b in a tertiary hospital. setting and method: we run a retrospective observational study in which we included a total amount of patients infected with hcv genotype b treated with the new therapies against hcv from february to december in a tertiary hospital. the data were obtained through the outpatient dispensing program farmatools and the review of the medical records from the hospital database, archinet and prescription hepatitis c portal of the andalusian health service. main outcome measures: from each patient the following information was collected: sex, age, viral genotype (gen.), naive/nonnaive, hiv coinfection, presence of cirrhosis, degree of hepatic fibrosis measured by fibroscan, treatment prescribed and duration, adverse effects, sustained viral response (svr) and the service that made the prescription. results: a totality of patients with hcv gen. b were reviewed which . % of them were men with a mean age of . years (range - years). of the patients were naive and only of them were hiv co-infected, there were a . % of cirrhotic patients. regarding the degree of hepatic fibrosis, patients had grade f , f grade patients, patients grade f and f grade patients. the most commonly therapy prescribed was lepidasvir + sofosbuvir in patients ( without ribavirin and with ribavirin ) using a treatment schedule of weeks in of them. the treatment was discontinued in one case because of the adverse effects, achieving svr in the remaining patients. the combo treatment with paritaprevir/ombitasvir/r + dasabuvir was prescribed in times ( without ribavirin and with ribavirin) choosing only in one of them for a treatment period of weeks. there were no treatment discontinuations and svr was achieved in all patients treated in this way. patients received simeprevir + sofosbuvir for weeks ( without ribavirin and with ribavirin), one patient of the left the treatment due to adverse effects. svr was found in the remaining patients who completed treatment. sofosbuvir + daclatasvir was prescribed to patients, associating ribavirin in only one case. a treatment duration of weeks was used in patients and weeks in the remaining two. one patient failed rvs without any incidences of adverse effects in any case. interferon + ribavirin sofosbuvir + was prescribed to patients in -week regimen which was well tolerated achieving svr. digestivo service treated the % of the total amount of patients. conclusion: new therapies for hcv have been used in all the treated patients and the older drugs have been relegated. about the effectiveness, svr was achieved in . % of patients. regarding the safety, only patients have discontinued the treatment due to adverse effects representing less than % dropout rate of the therapy. please specify your abstract type: descriptive abstract (for projects) background and objective: thanks to pharmacogenetics we can identify and predict different responses to the same drug among different individuals. during these last years we have noted a big increase of dosing guidelines and advices about the use of several drugs due to the influence of different polymorphisms. the aim of this study is to describe and evaluate the use of pharmacogenetics in our hospital from april , when we started our first research about pharmacogenetics, to the actual time, using these information in our daily clinical practice; and indeed quantify the number of different tests and the number of different clinical advices done because of pharmacogenetic information, by different healthcare specialty areas and drugs. design: we reviewed all the pharmacogenetic test requests in our hospital from april to april , noting which health specialty and for which drug was asked the test. polymorphisms were genotyped using taqman Ò genotyping assays technology by independent laboratories to confirm the results. results: from april we were asked for pharmacogenetic tests from different healthcare specialty areas: rheumatology ( . %), infectious diseases ( . %), oncology ( . %), cardiology ( . %), vascular surgery ( . %), neurology ( . %), ophthalmology ( . %); this information was asked about different drugs: clopidogrel ( . %), trastuzumab ( . %), ranibizumab ( . %), azathioprine ( . %) and tocilizumab ( . %). from all the genotypes, ( . %) were done after using the drug (study phase) and ( . %) were done previous to the use of the drug in daily clinical practice to make a ''clinical recommendation''; from these recommendations affected to the prescription of clopidogrel. conclusion: during the last years we could implement the use of pharmacogenetics in the daily clinical practice in our hospital in different healthcare areas affecting drugs and we started research studies previous to its use on the clinical practice for other three different drugs. please specify your abstract type: descriptive abstract (for projects) background and objective: the drug burden index (dbi) is a tool used to quantify the anticholinergic and sedative burden of medication on an individual. it has been independently associated with poor physical and cognitive performance in community-dwelling older people. objectives were: to create an inventory of medications used in ireland with clinically significant anticholinergic and/or sedative activity and to decide upon the minimum daily dose (mdd) for each medication. design: medications with potential anticholinergic and/or sedative burden were identified by literature review and examination of the summary of product characteristics (smpc) for all medications registered in ireland. each medicine was classified as anticholinergic or sedative. drugs with both anticholinergic and sedative properties were classified as primarily anticholinergic. the mdd, a key component of the dbi score calculation, was selected by reference to the irish smpc. other options which were also considered for this value include the defined daily dose (ddd) of a medication, as available from the world health organisation (who), and the mdd as outlined in the british national formulary (bnf). mdds were decided upon regardless of indication as the lowest effective therapeutic dose as specified in the smpc for the medication. the final list of medicines and mdds to be included in the inventory was then defined by consensus of three pharmacists. results: in total, medicines with potential anticholinergic and/or sedative activity were considered for inclusion. a final list of medications was identified by consensus ( anticholinergic, sedative). of these, ( %) were agents which act primarily on the nervous system. the three main therapeutic groups contributing to the inventory of dbi medications were antipsychotics ( medications), antidepressants ( medications) and antiepileptics ( medications). conclusion: creation of an inventory of medications with anticholinergic and/or sedative properties, in combination with the individual mdds, was achieved. this is a useful resource for use in analysis of drug burden in an older population. it could help in both identifying patients who would benefit from medication review as well as analysing population medication data. please specify your abstract type: research abstract background and objective: vancomycin is an antibiotic widely used to treat infections such as bacteraemia, infective endocarditis, osteomyelitis, meningitis and pneumonia. nowadays, optimal trough concentration is stablished between and mg/l to avoid development of resistance or - mg/l to improve penetration in complicated infections. some articles have been published explaining the methodology to calculate an expected trough level in steady state. our aim was to compare the trough serum value estimated by the mathematical method with a two-compartimental bayesian forecasting model. setting and method: observational retrospective study carried out in a tertiary hospital from january to december . non obese adult patients with creatinine clearance (crcl) \ ml/min and who have achieved steady state level were included. vancomycin serum values were measured using a chemiluminescence's immunoassay (cmia) and bayesian analysis was performed with abbottbase pksystem Ò (pks Ò ). the statistical analysis was made with medcalc software Ò . bland-altman plot and passing-bablok regression were used to compare both methods. main outcome measures: sex, age, weight, dose, creatinine, and size were collected from clinical history. serum trough values (cminr) were collected from cmia. trough values were estimated using two methods: mathematical method (cminf) and bayesian calculations (cminb). results: patients were included, with a mean age of (± . ) years. % were male and % female. they received a median dose per h of ( - ) mg. the mean of cminr was . mg/l ( % ci . - . ), cminb . mg/l ( % ci . - . ), cminf . ( % ci . - . ) . correlation coefficients (r) comparing both methods were significantly different: r between cminf and cminr was . ( % ci . - . ), while r between cminb and cminr was higher: . ( % . - . ). bland-alman plot analysis showed both methods cannot be used interchangeably. the regression equations estimated by passing-bablok regression were y = - . + . x and y = - . + . x. conclusion: bayesian method has demonstrated better correlation with real measures than mathematical method. most part of our patients could be underestimated or overestimated using mathematical methods which could cause toxicity or lack of efficacy, so this method is unsuitable for clinical use. bayesian estimation remains the best option for optimal dosing of vancomycin. please specify your abstract type: research abstract background and objective: combination therapy with digoxin and acenocoumarol is common in patients with atrial fibrillation (af). getting optimal concentrations of digoxin leads an appropriate response; taking into account its narrow therapeutic range and all the factors which can affect to its pharmacokinetics. interaction between them has been studied, even though its mechanism is not clear yet. patients who are taking both drugs need higher doses of digoxin; because they get lower concentrations by using the same dosage. the objective of this study was to analyse digoxin concentrations in patients treated with this combination compared to expected concentrations according to population parameters. setting and method: retrospective observational study from december to march performed by pharmacokinetic unit. patients included had chronic treatment with acenocoumarol and digoxin, which determination were realized in the steady state before the next dosage. patients with toxics concentrations of digoxin, or who were suspected nonadherence, were excluded. the plasma digoxin concentrations were determined through the autoanalyzer architect c- Ò (petinia). dosage adjustment was realized by the program abbot pharmacokinetics system (pks). a comparative between the real measured concentrations in patients and estimated concentrations were realized based on population parameters. finally, in order to get optimal concentrations, some dosage changes were proposed based on pharmacokinetic monitoring. data collected: population characteristics (gender, age, weight, and height), analytical data (potassium, urea, creatinine and clearance). main outcome measures: digoxin serum concentrations (optimal range . - ng/ml). results: data from patients, . % women with a mean (sd) age of . ( . ) years were included in the study. at baseline, potassium, urea, creatinine and clearance mean (sd) was . ( . ) mmol/l; . ( . ) mg/dl; . ( . ) mg/dl; . ( . ) ml/min. . % of the patients had lower concentrations than expected according to population parameters. finally, digoxin dosage was increased in . % of patients, it was maintained in . %, and it was decreased in . %. conclusion: digoxin concentrations in patients with af in combination therapy of digoxin and acenocoumarol are lower than would be expected in most cases. it is important monitoring digoxinaemia to achieve optimal concentrations and a good clinical response. further studies are needed to determine the relevance of this interaction in clinical practice. please specify your abstract type: research abstract background and objective: tocilizumab (tcz) is a humanized monoclonal antibody inhibitor of il- receptor, indicated in combination with methotrexate in the treatment of rheumatoid arthritis (ra) in patients with inadequate response or intolerance to prior therapy. interleukin is involved in the pathogenesis of rheumatoid arthritis via its broad effects on immune and inflammatory responses. previous studies have shown that c-allele at the - g[c (rs ) polymorphism is related with a bad response to tocilizumab (according to eular criteria). the aim of our study was to explore the potential role of il- genetic polymorphisms as a predictor of tocilizumab efficacy in rheumatoid arthritis (ra) patients and check this association depending on the genotype. setting and method: the il- (g[c) (rs ) genetic variant was genotyped using predesigned taqman Ò genotyping assays technology and analysed on a viia Ò real-time pcr system. main outcome measures: clinical response was evaluated at , , and months according to the eular criteria. patients were classified as ''responders'' (good and moderate response according to eular criteria) and ''non-responders''. the statistical analysis was performed using spss v. . results: we recruited patients with ra treated with tocilizumab, these were aged . ± . (mean ± sd), ( %) were women. the mean das at baseline was . ± . . of these patients, the il- g[c genetic polymorphism was significantly associated with ''responders'' at months after the baseline (cc vs non-cc p = . , or . , % ci . - . ) but not at (p = . ), (p = . ) and (p = . ) months. conclusion: the il- g[c may be useful as a genetic marker of tocilizumab efficacy at months. other polymorphisms, clinical parameters and other pharmacological treatment during the follow-up may be checked about their influence on the response to tocilizumab. tdmp : daptomycin pk/pd profile in neutropenic cancer patients with beta-lactam-resistant gram-positive infection nancy perrottet *, , frederic tissot , laurent decosterd , thierry buclin , guy prod'hom , christina orasch , oscar marchetti , farshid sadeghipour , , thierry calandra , véronique erard pharmacy service, infectious diseases service, laboratory and division of clinical pharmacology, service of biomedicine, institute of microbiology, lausanne university hospital, lausanne, school of pharmaceutical sciences, university of geneva, university of lausanne, geneva, switzerland please specify your abstract type: research abstract background and objective: the pharmacokinetics (pk) and pharmacodynamics (pd) of many antibiotics are modified in neutropenic patients and few data are available on daptomycin in this population. this prospective study aimed to assess the pk/pd profile of daptomycin in the treatment of neutropenic patients with beta-lactamresistant gram-positive cocci infections. setting and method: this substudy was performed in the context of a prospective pilot study on daptomycin versus vancomycin in adult hemato-oncological patients with febrile neutropenia and proven or suspected infection with methicillin-resistant staphylococci or betalactam-resistant enterococci. patients received daptomycin mg/ kg/day ( mg/kg/day for enterococci) for c days as a -min infusion. main outcome measures: pk analysis using a published non-linear mixed effect model with nonmem Ò , followed by comparison of parameters with values published for healthy subjects. pd analysis based on auc/mic (area under the concentration-time curve/minimal inhibitory concentration). according to eucast, an auc/mic ratio [ is required for bacteriostatic effect against staphylococci and [ for a two-log reduction in bacterial count. for e. faecium, an auc/mic ratio of . has been suggested for bacteriostasis and . for a -log bacterial count reduction. results: model-derived mean auc observed in patients was . ± . mg h/l, maximum concentration (cmax) ± mg/ l, minimal concentration (cmin) . ± . mg/l. clearance was . ± . l/h and volume of distribution at steady sate . ± . l, both values found higher than those reported in healthy subjects. all patients ( / ) with a staphylococcal infection achieved auc/mic values predictive of bacteriostatic effect on staphylococci, and out of values associated with two-log bacterial killing. of note, infection relapse occurred in the only patient with suboptimal daptomycin exposure (auc/mic of ). the pd targets were also reached in the two patients with e. faecium infection. an asymptomatic elevation of creatine phosphokinase was reported in two patients ( u/l and u/l) with cmin of . and . mg/l, respectively. conclusion: daptomycin pk profile in neutropenic cancer patients indicated higher total clearance and volume of distribution, along with lower total exposure, compared to healthy subjects. despite this, standard dosages allowed attainment of pd targets in / patients with a staphylococcal infection (two-log drop) and / with e. faecium infection ( -log drop) . please specify your abstract type: research abstract background and objective: individual clinical response to infliximab can be influenced by their pharmacokinetics and immunogenicity, so therapeutic monitoring of drug levels (tdm) can guide these biologic treatments. the objective was to analyse the suitability of serum infliximab trough levels (sitls) in patients with inflammatory bowel diseases (ibd) receiving dose schemes based only on clinical response. setting and method: prospective and descriptive study of patients with ibd treated with infliximab and under tdm. medical records were reviewed. dose schemes were established according to clinical guidelines ( mg/kg every weeks) and optimized based on an index of clinical response (mayo, pcr…). sitls (therapeutic range - mcg/ml) and anti-drug antibodies (ada) were measured in all of patients by elisa (promonitor Ò ). ada presence was considered as a therapeutic failure indicator. informed voluntary consent was obtained from all patients. main outcome measures: sitls and ada. results: a total of patients, with a median age of years (range ), were included in the analysis. infliximab standard dose according to clinical guidelines were administered to patients: . % showed sitls under the therapeutic range ( . % with ada). in eight patients with maintained good clinical response, dose decrease or interval elongation had been implemented: % of these patients showed sitls below the therapeutic range ( % with ada). it had been necessary to increase the dose or shorten the interval in patients due to inadequate clinical response: . % of these patients with sitls below the therapeutic range ( % with ada). conclusion: optimization based on clinical response of infliximab treatments in patients with ibd is not always an effective strategy, since it leads to a high percentage of patients with sitls below the therapeutic range and adas. tdm together with clinical response should guide the optimization of infliximab treatments. please specify your abstract type: research abstract background and objective: in addition to its anticonvulsive properties, valproate is also used as a mood stabiliser in bipolar disorder and as augmentation treatment of other psychiatric disorders. the unpredictable relationship between dose-plasma valproate concentrations and correlation between concentrations-efficacy suggest therapeutic drug monitoring (tdm) of plasma valproate concentrations might be useful. the aim of our study was to evaluate the rationale of a new protocol for measuring valproate concentrations and the incorporation of a clinical pharmacist in the process of valproate tdm service, compared to pre-existing standard measuring. setting and method: in the retrospective study we analysed the process of measuring plasma valproate concentrations at the department of psychiatry and at the unit for forensic psychiatry of a large teaching hospital in slovenia before the enrolment of a clinical pharmacist. for the prospective study we created a protocol for tdm of valproate in adults based on literature research. the protocol included reference range, sampling time, indications for sampling and schedule of other laboratory tests that have to be monitored during valproate therapy. main outcome measures: percentage of plasma valproate concentrations in reference range (c trough = - mg/l) before/after the enrolment of a clinical pharmacist, percentage of measured valproate c trough . results: in the retrospective study randomly chosen patients with measured plasma valproate concentrations were included ( % male, age ± years, length of hospital stay ± days). plasma valproate concentrations were measured . ± . times per patient, % were in the reference range (other % subtherapeutic), % were drawn at c though , . % were drawn for assessing compliance (nontrough). in the prospective study patients were included ( % male, age ± years, length of hospital stay ± days). plasma valproate concentrations were measured . ± . times per patient, % were in the reference range (other subtherapeutic), % were drawn at c trough , . % were drawn for assessing compliance. conclusion: the inclusion of a clinical pharmacist in valproate tdm service increased the number of valproate plasma concentrations in the reference range by almost % and increased the number of concentrations drawn at c trough , when indicated. including a clinical pharmacist in valproate tdm is beneficial and the new protocol is useful for optimising valproate therapy. concurrent and predictive validity of a self-reported measure of medication adherence the effect of pharmacist-led interventions in optimising prescribing in older adults in primary care: a systematic review aflibercept: . neovascular membranes with visual acuity higher than . . one eye affection severe cardiovascular pathology (severe episodes in the last months) non-responders to other anti-vegf bevacizumab: . diabetic macular edema macular edema secondary to vascular pathology setting and method: a longitudinal study was carried out in primary care centres. participants: patients aged c , under treatment with or more drugs and belonging to primary care areas in different towns. patients should have at least one of the following potential safety problems: (a) concomitant use of a non-steroidal anti-inflammatory drug (nsaid) with an antihypertensive drug, anticoagulant or antithrombotic drug; (b) use of two or more benzodiazepines. two clinical management units (cmu) were randomized per area to be included in the study. thirty patients per cmu were randomized to be enrolled and monitored during months number of adverse effects ( . ; p \ . ) and number of clinical problems ( . ; p \ . ).with each year increase in age ) and a significant rise in physician ( . ; p \ . ) and nurse ( . ; p \ . ) home visits. women compared to men resulted in a significant decrease ) but a significant increase in visits to nurses ( . ; p \ . ), hospital admissions ( . ; p \ . ) and hospital visits ( . ; p \ . ). age, sex and npsp had no significant effect on falls, fractures or cardiovascular events. conclusion: the npsp in elderly patients contributes to an increase in the use of health services and comorbidity. effective interventions should be addressed to general practitioners to reduce inappropriate prescriptions bpa was found in the dialysate ( ng/l) and ls ( ng/l) wherein the concentration of bpa decreases over time to reach ng/l at the end of a session. finally, bpa was present in all tested dialysis at concentrations of up to . ng/dialyzer in the compartment mimicking the blood and to . ng/dialyzer in the dialysate despite prior rinsing with l of . % nacl. conclusion: our study is the first one to show the risk of exposure to bpa and bpa-clx hdf-ol. while assessment of the impact of this exposure in a patient under treatment remains to be done, it is now possible to better master contamination by bpa and its four chlorinated derivatives through better practices (choice of medical devices) and improvement of the overall water treatment process san cecilio university hospital, genomic unit san cecilio university hospital, genomic unit, genyo, centre for genomics and oncological research the aim of this study is to compare the apparition of stroke, acs, cardio-vascular death and the need of surgery in patients after percutaneous transluminal angioplasty (pta) or stroke depending on the presence of cyp c * * polymorphisms. setting and method: retrospective cohort study. we recruited patients treated with clopidogrel after a pta of the lower limb or stroke (without surgery) from to in our hospital. data collected: age, sex, cyp c * (rs ) and cyp c * (rs ) genotypes and the primary end-point: stroke, acs, cv death and surgery of the affected vessel during months after discharge. polymorphisms were genotyped using taqman Ò genotyping assays technology. main outcome measures: we recruited patients with stroke ( . % men; mean age . ) and patients after pta ( . % men; mean age . ) treated with clopidogrel after discharge %) suffered the primary end-point during months after discharge; of these patients had the cyp c * allele. among patients with pta of the lower limb: % of them had the cyp c * allele and no one a cyp c * allele; ( . %) of these patients suffered the primary end-point during months after the discharge and of these had the * allele of the cyp c isoenzyme * allele and treated with clopidogrel have a higher risk of the primary end-point than those patients not carrying it spain please specify your abstract type: research abstract background and objective: the engagement of fcgrs by tnf antagonists could affect to macrophage-mediated clearance of immune-complexes. the aim of our study was to evaluate the potential role of fcgr a (a[c) (rs ) single nucleotide polymorphism (snp) as a predictor of tocilizumab efficacy in rheumatoid arthritis (ra) patients. setting and method: the fcgr a (a[c) (rs ) snp was genotyped using predesigned taqman Ò genotyping assays technology and analysed on a viia Ò real-time pcr system. the statistical analysis was performed using spss v the mean age of the patients was . ± . years and % were women. the mean das at baseline was . ± . . we found no statistically significant association between our end-point and the genetic polymorphisms studied tdmp : therapeutic drug monitoring of infliximab biosimilar and anti-infliximab antibodies in inflammatory diseases patients with dermatological conditions and inflammatory bowel disease being treated with ifx-b ( mg/kg/ weeks after the induction dose) were included. the concentrations of ifx-b and ati-b were quantified by two sandwich-type elisa immunoassays (triturus Ò analyser). main outcome measures: plasma levels of ifx-b and ati-b, clinical response and infusion reactions. the clinical response was assessed according to pathology of each patient (based on specific clinical variables for the pathology into the electronic history) pharmacokinetic results (% assessments): (a) . % no ifx-b detection (c \ . mcg/ml) and positive ati-b (c [ ua/ml) ( assessments/ patient). atis = , y ua/ml. no clinical response (nr) in . % assessments. (b) . % ifx-b and ati-b (c b ua/ml) no detection ( assessments/ patient). nr %. (c) . % ifx-b detection (c [ . mcg/ml) and negative ati-b ( assessments/ patients) weight: ( - ) kg. twenty assessments, . ( - ) assessments per patient, ( - ) ifx-b doses, % concomitant treatment ( / -azathioprine, / -corticosteroid) the incidence of ati-b was low. a correlation was observed between the presence of ati-b and loss of clinical response, as infliximab original. tdmp : serum concentration of non-vitamin k antagonist oral anticoagulants (noacs) in older hip fracture patients ina linnerud , mette i martinsen estimation of t of noacs by t / = ln /kel [kel; elimination constant] using two s-concentration measurements. results: we included patients (median age years, . % women). noac use was detected be serum analysis in patients ( . %; % coherent with mr), while patients ( . %) used warfarin. of the noac users ( . %) had s-concentrations of noacs above the reference range at admission, and five patients ( . %) had s-concentrations within the reference range before surgery. patients using noac had significantly longer median waitingtime for surgery than warfarin-users ( vs h, p = . ). blood transfusions were given to . % of noac-users vs . % of warfarin-users (p = . ). mean estimated t of noacs were , . and . h for dabigatran (n = ), apixaban (n = ) and rivaroxaban (n = ), respectively. conclusion: mr is effective in detecting noac use in older hip fracture patients, but importantly s-concentrations are higher than expected in this population. this might reflect the significantly longer waiting-time for surgery this column is supplied with packing material made of totally porous spherical silica coated with a silicone polymer monolayer containing octadecyl (c ) groups. the mobile phase was composed of . % na po h o (ph . ), acetonitrile, and methanol ( : : , v/v/v), which was degassed in an ultrasonic bath prior to use. the flow rate was . ml/min at ambient temperature and sample detection was carried out at nm. plasma samples were obtained from patients with cml receiving nilotinib treatment. sampling was performed at the steady state. blood samples were collected by venipuncture h after oral administration of nilotinib. plasma was separated by centrifugation at g for min and stored at - °c until analysis. plasma samples ( ll) were then extracted as described above. the same samples were also sent to a commercial laboratory (bml, inc.) for assaying nilotinib concentration by liquid chromatography-tandem mass spectrometry (lc-ms/ms). in addition, we applied this method to tdm of cml patients receiving nilotinib at our hospital. main outcome measures: the calibration curve exhibited linearity over the nilotinib concentration range of - ng/ml at nm, with relative standard deviations (n = ) of . , . , and . % for , , and ng/ml, respectively. the detection limit for nilotinib was ng/ml due to three blank determinations (q = ). in addition, we compared the results with those measured by lc-ms/ ms at bml, inc. (a commercial laboratory). as a result, a strong correlation was observed between the nilotinib concentrations measured by our hplc method and those obtained by lc/ms-ms (r = . , p \ . ). in addition, tdm of nilotinib was performed to six cml patients. there was the case which participated in dosage adjustment of nilotinib in hepatic dysfunction and poor glycaemic control. results: we have developed a simple ultraviolet detection method for the determination of nilotinib, which has high sensitivity and large dynamic range please specify your abstract type: research abstract key: cord- -t x gknd authors: nan title: abstract presentations from the aabb annual meeting san diego, ca ctober ‐ , date: - - journal: transfusion doi: . /trf. sha: doc_id: cord_uid: t x gknd nan background/case studies: zika virus (zikv) is associated with severe neurological consequences in fetuses and adults and potential for transfusion transmission (tt). rna persistence has been reported in whole blood (wb) long after clearance of viremia in plasma, raising concerns over the risk of tt with plasma based nucleic-acid amplification testing (nat). the dynamics of zikv persistence in asymptomatic infection are not well understood and are needed for understanding of the natural history of zikv infection. we sought to characterize the dynamics of infection through prospective enrollment of zikv rna blood donors. study design/method: donors identified through investigational zikv nat screening were enrolled into longitudinal follow up and assessed for viral and serological persistence and clinical outcomes. plasma and rbc were obtained from index donations and blood, urine, saliva and semen samples were collected prospectively at weeks , , , and following index donations from donors and detailed symptom questionnaires were administered at each study visit. blood compartments and body fluids were tested for zika rna by real time rt-pcr. plasma samples were tested for zika specific igm and igg antibodies results/finding: the percent of zikv rna samples, followed by the number of samples tested in parenthesis, for each sample type during each sampling interval is summarized in the table. plasma viremia declined rapidly after index donations whereas rbc-and wb-associated viral rna persisted for up to months and peripheral blood mononuclear cell (pbmc) associated virus was detected intermittently at low levels and waning by months. urine and saliva detection decreased significantly after weeks and was undetectable by months. of donors who were enrolled in the acute pre-seroconversion stage of infection % ( / ) developed multiple zikv related symptoms week post index donation, compared to only % ( / ) for donors detected post-seroconversion. conclusion: zikv rna persists in cellular blood compartments for several months following clearance from plasma and body fluids, with higher rates of symptoms than previously reported. the persistence of zika rna in rbcs has unknown implications for blood screening, which currently relies on plasma testing; infectivity studies are in progress. wb testing may be of value to extend detection of acute infection and for diagnostics and monitoring of pregnant women. iron status and novel risk factors for iron depletion in a diverse donor population bryan r. spencer* , yuelong guo , ritchard g. cable , joseph e. kiss , michael paul busch , grier page , stacy endres-dighe , steve kleinman , simone glynn , alan mast and for the nhlbi recipient epidemiology and donor evaluation study-iii (reds-iii) . american red cross, rti international, american red cross blood services, blood systems inc., blood systems research institute, university of british columbia, nih/ nhlbi, blood research institute, nhlbi background/case studies: blood centers and regulators in the united states (us) are evaluating strategies for minimizing iron depletion in blood donors. the logistics of donor management might differ across blood centers, but the optimal approach may also vary according to biological or behavioral differences across sub-populations of donors. studies donors have been conducted in predominantly caucasian populations, which may differ from racial/ethnic minority donors in iron metabolism and capacity to undergo repeat phlebotomy. study design/method: over , donors were enrolled from us blood centers for ferritin testing. the study population was enriched for racial minorities [ african-american (aa), asian (as), hispanic (hisp)] and for "super donors" ( , who had completed donations in two years without low hemoglobin deferral). the minority donors and the remaining non-hispanic white (nhw) donors were an unselected population with no specific eligibility criteria. subjects completed questionnaires on risk factors for iron depletion. logistic regression was used to identify demographic and behavioral predictors of absent iron stores (ais, ferritin < ng/ml) and low ferritin (lf, ferritin < ng/ml). results/findings: across all subjects, % had ais and % had lf, with a high degree of variability based on demographic factors and donation behavior. in models stratified by race, expected patterns common to all groups included a sharp increase in risk with increasing donation intensity, and a large decrement in risk for females > years old. in models including all subjects, race was an independent predictor of both ais and lf controlling for age, sex, body weight, donation frequency, and other factors (table) . aa and as donors showed % % decreased risk for ais compared to nhw, while hisp donors had % higher risk. daily use of exogenous iron reduced risk for lf and ais by to %, respectively, while the estimated benefit from less-than-daily use was lower ( to % protection). regular use of antacids was associated with a % or greater increment to risk. reported use of hormone supplements showed opposing effects in males and females. use of oral contraceptives or estrogen in females reduced risk by % - %, while males who reported current use of supplemental testosterone had twice the estimated risk for ais. conclusion: this large study confirms the high prevalence of lf and ais in us donors and the principal risk factors of age, sex, and donation frequency. the diverse population studied and the questionnaire data from donors identify additional demographic and behavioral risk factors of secondary importance. in developing iron mitigation strategies, practices based on age and gender could be further refined depending on a given blood center's operational context and donor population. data are reported as mean ( sd) *p < . compared to batf / , ul hod rbcs mfi, median fluorescence intensity background/case studies: during storage, red blood cells (rbcs) undergo multiple morphological, biochemical and molecular modifications, collectively called the storage lesion. the proportion of cleared rbcs is correlated with storage duration, which may be responsible for the rapid clearance of up to % of transfused rbcs, reducing transfusion yield. it has been shown, using imaging flow cytometry that a subpopulation of morphologically altered rbcs accumulates during storage. the reduced surface area of these small rbcs (srbcs) suggests their rapid elimination by the spleen in the hours following transfusion. this hypothesis remains to be clarified, since the physiological mechanisms of rbc clearance remain to be precisely identified. study design/method: murine "young" and "old" rbcs (respectively on d and d of storage) were transfused into different models including splenectomized or macrophage-depleted mice. flow cytometry was used to determine the kinetics of clearance, the transfusion yield and to quantify rbcs retention in organs. the accumulation, during storage, and the posttransfusion disappearance of srbcs were analyzed by imaging flow cytometry. results/finding: using a murine model of transfusion, we confirmed that the post-transfusion yield decreases with storage duration ( % on d vs % on d of storage). a clearance of the storage-damaged rbcs mediated by spleen and macrophages is shown by significant improvements in post-transfusion yield observed in the splenectomized ( %) and macrophage-depleted ( %) groups. as in humans, we observed the accumulation of a subpopulation of small rbcs (mouse small rbc: msrbc) of reduced projected surface area with altered morphology. these msrbcs disappear rapidly from the circulation in control or splenectomized mice with a decrease of more than % at h post-transfusion. in contrast, in macrophage-depleted mice, msrbcs are kept in circulation at h posttransfusion. at h, these msrbcs completely disappear in all models, suggesting the importance of their elimination and the presence of compensation clearance mechanisms. in control mice, storage-damaged rbcs are mostly retained in the spleen but also in the bone marrow (bm) . no retention is observed in the liver, kidney or lung. in macrophage-depleted mice, retention is decreased in the spleen and bm. conversely, elevated retention is observed in the bm of splenectomized mice, associated with a transient retention in the kidney and liver. conclusion: during storage of murine rbcs, damaged rbcs accumulate, and are eliminated following transfusion via spleen/macrophage-mediated mechanisms. they include, as observed in humans, a subpopulation of small rbcs which undergoes a rapid macrophage-mediated clearance. the increase in transfusion yield in the absence of spleen or macrophages suggests that the recipient's functional state is one of its determining factors. age dependent relapsing and remitting autoimmune hemolytic anemia in a murine model andrea sut ling wong* , amanda l richards and krystalyn e hudson . background/case studies: breakdown of tolerance to rbc antigens may result in development of pathogenic autoantibodies (autoab) and lead to autoimmune hemolytic anemia (aiha), a severe and sometimes fatal disease. aiha in humans has a number of known features, including increased frequency with age, and tendency to relapse and remit. however, the mechanisms behind such observations are not understood. to gain insight into tolerance (or loss thereof) to an rbc autoantigen, we utilized the hod mouse, which expresses an rbc-specific triple fusion protein consisting of hen egg lysosyme (hel), ovalbumin (ova), and, duffy (hod). hod mice were bred to a transgenic mouse that expresses a t cell receptor specific for an ova peptide in hod presented by mhcii (otii mice). thus, hod otii mice are predisposed to have autoreactive cd t cells. study design/method: four cohorts of hodxotii f mice ( - mice/ cohort) were bled monthly for months to assess for autoab production. peripheral rbcs were stained with anti-complement (c ) and mouse immunoglobulin ab. spleens were weighed and splenocytes were stained with anti-cd and ter to assess for the presence of rbc progenitors. statistical analysis between hod otii autoab vs. hod otii autoabvs. hod -otii was performed using kruskal-wallis test and corrected for multiple testing with dunn's test. results/finding: otii cd t cells were not deleted in the thymus of hod otii mice; rather, they matured to the periphery. despite these peripheral autoreactive t cells, no detectable autoab were observed in hod otii . however, as they aged, - % of hod otii were positive for rbc autoab by months. thereafter, $ % of the autoab mice stopped producing autoab within two months after onset and remained autoab free throughout the study. in of the cohorts, - % of autoab mice were female. hod otii autoab mice also had enlarged spleens compared to hod otii autoaband hod -otii mice ( . g vs. . g and . g, resp., p< . ). this may due to rbc consumption, extramedullary erythropoiesis, or both. consistent with increased erythropoiesis, elevated numbers of rbc progenitors (cd hi ter inter ) were observed in the spleens of hod otii autoab mice but not in hod otii autoaband hod -otii ( . % vs. . % and . % resp., p< . ). moreover, autoab and c deposition were found ( . - % and - %, resp.) on ter rbcs in all of the hod otii autoab mice analyzed. conclusion: several features known to exist in human aiha were observed, including age-dependant autoab production, relapsing of autoimmunity after onset, and an increased frequency in females. this model may serve as an experimental system to investigate the mechanisms of aiha. b -a a reduction in neutrophil numbers is a risk factor for rbc alloimmunization amanda l richards , christopher a tormey and krystalyn e hudson* . background/case studies: red blood cell (rbc) alloimmunization occurs in up to % of transfusion recipients (excluding abo and rhd). the underlying factors that influence alloimmunization are poorly understood; thus, there is currently no reliable way to predict who will make an alloantibody and who will not. patients who receive multiple rbc units or several separate transfusions are at higher risk of alloimmunization; likewise, certain disease states have higher rates of alloimmunization, such as myelodysplasctic syndrome (mds) and sickle cell disease patients. however, despite chronic transfusions, some patients never develop rbc alloantibodies. it has been recently reported that poly (i:c)-elicited inflammation leads to enhanced alloimmunization rates and is correlated with increased splenic neutrophil (pmn) numbers. additionally, rbc transfusion into an inflamed recipient leads to enhanced erythrophagocytosis by pmns. here, we test the hypothesis that pmns regulate rbc alloantibody generation. study design/method: mice: c bl/ (b ) mice were treated with pbs, or anti-ly g to deplete pmns, followed by poly (i:c) to elicit inflammation, and finally a transfusion of allogeneic dio-labeled rbcs expressing a synthetic antigen, hod (hel-ova-duffy). multiple splenic cellular subsets were evaluated for dio fluorescence, an indirect measure of rbc consumption, at - hours post-transfusion. anti-hod alloantibody generation was assessed days post-transfusion by flow cytometry. humans:retrospectively, mean white blood cell (wbc) and pmn counts were collected on chronically transfused mds patients at va connecticut healthcare. for alloimmunized patients (n ), wbc and pmn counts were assessed on the day of exposure to the alloimmunizing rbc unit, whereas counts were averaged for the entirety of rbc therapy for non-alloimmunized patients (n ). patients were matched for numbers of rbc transfusions. results/finding: mice: the mfi of anti-hod antibodies was significantly increased in pmn-depleted mice, compared to controls ( / experiments, p< . ). while many control mice made no alloantibody (non-responders), all pmn-depleted mice made detectable anti-hod. pmn depletion also led to a significant reduction in dio leukocytes, suggesting a lack of compensatory mechanism(s) for rbc consumption. absence of pmns also shifted rbc consumption from macrophages to immune-stimulating dendritic cell subsets. flow cytometric analysis revealed that pmns with internalized rbcs upregulated expression of co-inhibitory molecules (e.g. pd-l ), compared to pmns (without internalized rbcs) from the same mouse; thus, pmns may regulate alloimmunization through antigen presentation and/or inhibitory signals. humans:. alloimmunized mds patients had a significant decrease in pmns, compared to non-alloimmunized (p< . ); no significant differences were detected in mean wbc counts between the two arms. conclusion: these data demonstrate that in both murine and human settings, pmns may play a significant role in regulating rbc alloimmunization and may provide key insights into predicting which patients will become alloimmunized. b -a b cxcr pd and ccr expressions characterize responders to rbc immunization benoît vingert* , , , marie tamagne , , , sadaf pakdaman , , , anoosha habibi , , , philippe bierling , , , , , rachid djoudi and france pirenne , , , . efs ile de france, laboratory of excellence gr-ex, imrb u -eq , ap-hp, universit e paris est background/case studies: post-transfusion alloimmunization can induce life-threatening hemolytic transfusion reaction. in human, mechanisms responsible of rbc alloimmunization are not fully defined. cd t cells are major for antibodies production. we have already shown in responder patients that the majority of anti-rbc cd t cells have a th profile. in contrast, in whole blood of non-responder patients, there is an unexpected expression of circulating cd t cells with a cxcr pd phenotype. this phenotype is usually associated with the presence of tfh cells, specialized in the production of antibodies. it has been suggested that some of the activated circulating tfh could have a cxcr pd hi profile, with a differentiated expression of ccr . ccr is essential for t cells domiciliation in lymph nodes where the encounter t and b cells is major for b cell differentiation and antibody production. others chemokines receptor like ccr and cxcr can also differentiate circulating tfh subpopulations. in this study, we were interested in the phenotype and function of these cxcr pd lymphocytes which were paradoxically highly represented in non-responder patients. study design/method: the membrane and functional phenotype of the circulating cxcr pd cells were compared in groups of transfused sickle cell patients : alloimmunized (n ) and non-alloimmunized patients (n ). the analysis was also performed in non-transfused healthy controls (n ). all assays were performed on whole blood without separation procedures that are known to alter the expression of chemokine receptors results/finding: the cxcr pd hi subpopulation expression was identical between transfused groups and controls. ccr and cxcr expressions show no difference between the transfused groups or the controls. however, in non-responder patients, ccr expression was very strong independently of the expression of pd . in the aim to determine the help of the circulating cxcr pd cells in the production of antibodies, these cells were purified by flow cytometry and co-cultured for days with b cells, and in the presence of seb protein. the levels of antibodies after seb stimulation were identical with the cxcr pd subpopulations from transfused groups or controls. conclusion: the paradoxical presence of circulating cxcr pd cells in non-responder transfused patients do not appear to have any particular functions that can promote the absence of a humoral response. however, in responder patients, the high expression of ccr on circulating cxcr pd cells suggests remarkable migratory properties towards secondary lymphoid organs, and could facilitate allo-immune responses. in conclusion, the study of the cxcr pd profile and the ccr expression in these cells could help to differentiate responder and non-responder patients to rbc immunization. primed cd t cells to one rbc alloantigen can enhance subsequent alloimmunization seema r patel* , ashley bennett , kathryn girard-pierce , connie arthur , amanda mener , patty zerra , christopher a tormey , jeanne hendrickson and sean stowell . emory university, yale-new haven hospital, yale university, emory university school of medicine background/case studies: while red blood cell (rbc) alloantibodies can increase the probability of transfusion-related complications, not all patients become alloimmunized following transfusion. however, individuals that do generate alloantibodies appear to experience an increased rate of additional alloantibody formation following subsequent transfusion. however, how immunity to one rbc alloantigen primes immunization to a completely distinct alloantigen remains unknown. though cd t cell help classically occurs through direct recognition of a peptide that resides within a target b cell antigen, individuals who develop antibodies toward one rbc alloantigen experience increased rates of antibody formation against completely distinct rbc alloantigens. these observations suggest that cd t cells that respond to one alloantigen may directly facilitate immunity to a completely distinct rbc alloantigen. study design/method: b recipients were transfused with kel rbcs in the presence or absence of poly i:c (pic), followed by transfusion of hod rbcs, kel rbcs, rbcs expressing hod and kel (hod x kel), or a mixture of hod and kel rbcs (hod kel). to examine the role of cd t cells, pic/kel primed b recipients were cd t cell depleted prior to transfusion. in addition, b recipients were adoptively transferred with cd t cells from na€ ıve or pic/kel primed donors, followed by transfusion of hod rbcs or (hod x kel) rbcs. anti-hod and anti-kel alloantibody formation was evaluated using indirect immunofluorescence staining. results/findings: kel rbc transfusion in the presence of pic (pic/kel) not only enhanced anti-kel antibody production through a cd t celldependent process, but this same priming event directly facilitated anti-hod antibody formation following subsequent (hod x kel) rbc transfusion (p < . ); pic/kel primed recipients transfused with (hod kel) rbcs or hod rbcs alone failed to impact anti-hod antibody formation. the ability of immunity to kel to boost a humoral response to the hod antigen following (hod x kel) rbc transfusion required kel priming in the presence of pic. cd t cell depletion prevented pic/kel primed recipients from boosting an anti-hod antibody response (p < . ) and transfer of cd t cells from pic/kel primed recipients likewise directly facilitated anti-hod antibody formation following a (hod x kel) rbc transfusion (p < . ). conclusion: these results demonstrate that cd t cells primed to one rbc alloantigen can directly enhance the immune response to a completely distinct rbc alloantigen, suggesting a mechanism whereby alloantibody responders may exhibit an increased rate of additional alloantibody background/case studies: platelet refractoriness remains a significant clinical problem, yet the mechanisms by which it occurs are incompletely understood. immune-mediated platelet clearance by anti-platelet alloantibodies plays a significant role, and patients with detectable alloantibodies can be managed with transfusion of hla-matched platelets. still, many patients are refractory even after receiving hla-matched platelets. it was shown previously that cd t cells can play a direct role in platelet clearance, as allogeneic platelets are cleared within hours post transfusion in b celldeficient mmt recipient mice (ie in the absence of anti-platelet alloantibodies) and depletion of cd t cells prevents such clearance. since minor antigenic differences still exist between donor hla-matched platelets and a recipient, we hypothesized that minor antigens alone may mediate clearance of otherwise hla-matched platelets. study design/method: to test whether minor antigens can stimulate cd t cell-dependent platelet clearance we examined platelet refractoriness using mova and oti transgenic mice. leukoreduced donor platelets from mova mice, which express a membrane-bound form of chicken ovalbumin and thus present ovalbumin peptides complexed with murine mhc class i h kb, were labelled in vivowith the fluorescent dye cfse and transfused into wildtype (wt, c bl/ ) mice or oti mice, whose cd t cell receptors recognize a specific ovalbumin peptide in the context of mhc class i h kb. in some experiments oti mice were primed with mova or wt splenocytes one week prior to mova platelet transfusion, and in others wt mice were adoptively transferred with oti splenocytes hours before mova platelet transfusion. platelet recovery was measured immediately after transfusion as well as after , , , , and hours and on days - . results/finding: transfusion of mova platelets into oti mice results in significant platelet clearance as compared to transfusion with wt platelets. clearance kinetics demonstrate platelet loss starting after hours and peaking at hours, and are similar whether oti mice are na€ ıve or previously primed with mova splenocytes. specifically, mova platelet recovery in oti recipients is - % versus > % in wt recipients at hours (p< . ), whereas transfusion of wt platelets into either oti or wt recipients is approximately % at hours after transfusion. adoptive transfer of oti cd t cells into wt mice recapitulates the effect, with significant mova platelet clearance at hours compared to wt platelet clearance (p< . ). conclusion: this work extends the ability of cd t cells to mediate platelet clearance to a minor antigen, providing insight into the potential etiology of platelet refractoriness in patients receiving hla-matched products. this study also holds implications for the clinical management of any nonantibody-mediated platelet refractory patient, as therapies directed toward immunomodulation of t cell responses may prove beneficial. background/case studies: alloimmunization against major histocompatibility (mhc) antigens is a common complication of transfusion, and can negatively impact subsequent transfusions and transplants. we have previously demonstrated that pathogen reduction with riboflavin and uv light (uv r) is effective both at rapidly killing donor white blood cells (wbcs) and at blocking their ability to stimulate an allogeneic response in vitro. furthermore, uv r treatment of allogeneic platelet rich plasma (prp) prevents alloimmunization in mice, and provides partial antigen-specific tolerance to subsequent transfusions. as cells that die through different pathways can be either tolerizing or inflammatory, we sought to determine which cell death pathways are triggered by uv r, as well as evaluate the immunogenicity of prp containing wbcs killed by other methods. study design/method: wbc-rich prp was prepared from c bl/ mouse blood and treated with uv r, and wbcs prepared in parallel from the same blood were treated with known inducers of either apoptosis or necrosis. membrane integrity, phosphatidylserine exposure, caspase activity, and chromatin condensation were evaluated by flow cytometry. balb/c recipients were transfused with either uv r treated wbc-rich prp, or uv r treated wbc-poor prp either alone or with added untreated, apoptotic, or necrotic wbcs, all generated from allogeneic c bl/ donor blood. a second transfusion of untreated wbc-rich c bl/ prp was given weeks later, and alloresponses were compared against mice given no transfusion or only the second untreated transfusion. results/finding: uv r treated wbcs have a pattern of phosphatidylserine exposure and loss of membrane integrity consistent with early apoptosis, but fail to demonstrate significant caspase activity or clear chromatin condensation. alloantibody responses to transfusion were significantly higher in mice previously exposed to untreated (p< . ) or necrotic (p< . ) wbcs, but not those given uv r treated or apoptotic wbcs. ex vivo cytokine responses to stimulation with c bl/ wbcs were reduced in recipients of either uv r or apoptotic wbcs, and enhanced in recipients of untreated or necrotic wbcs. conclusion: the mechanism of wbc death following uv r treatment shares some membrane characteristics of early apoptosis, but is distinct from classic apoptosis. however, both uv r treated and apoptotic wbcs fail to trigger an alloresponse, and offer some protection against subsequent alloexposures. background/case studies: in mitochondria-less red blood cells (rbcs), oxygen is the main substrate for oxidative reactions and resulting oxidative damage is considered as one of the major causative factors in the development of rbc storage lesion. oxygen saturation (so ) of venous blood is generally assumed to be around - % as measured from a central venous line. however, a recent investigation of so levels in freshly prepared leukocyte-reduced red cell concentrates (lr-rccs) revealed unexpectedly wide so distribution (mean . % . % [yoshida et al. ; blood transfusion , ] . the present study was undertaken to determine the distribution of so in lr-rcc produced at a medium-size blood center using a novel non-invasive so probe. additionally, quantitative metabolomics were carried out to examine the redox status of the stored rbc under various so levels. study design/method: the so from units of lr-rcc were examined on five consecutive days representing % of the collected units during the period at a regional blood center where all the units were processed at room temperature within hours of blood collection. so was measured noninvasively through the pvc bag immediately prior to refrigeration by employing a resonance raman spectrometry (pendar microvascular oximeter a u ; pendar technologies, cambridge ma). in addition to so , process methods, rcc volumes, blood types, gender and process times were recorded for analyses. in a separate study, lr-rcc (n ) from human volunteers were stored in as- under normoxic, hyperoxic, or hypoxic conditions for up to days (so ranging from < to > %) prior to uhplc-ms metabolomics analyses in presence of c, n or deuterated internal stable-isotope labeled standards for absolute quantitation. results/finding: measurements of so carried out non-invasively at a blood center yielded a similar wide distribution as previous study from units of lr-rcc procured and sampled invasively within hours after blood collection [yoshida ibid]. the shape of the so distribution appeared near normal with the mean of . % . %, median . %, range < % to > % and inter-quartile range (iqr) of . %- . %. male donors showed higher so compared to female donors (p< . ). no correlations were observed between so levels and processing time, donor age or blood types. metabolomics workflow indicated that lower so levels ameliorate the energy and oxidative metabolic lesion. lower so levels yielded higher rate of gsh synthesis, higher nadph concentration, higher gsh / gssg and nadph/nadp ratios, lower supernatant urate consumption and lower purine oxidation. the surprisingly wide distribution of starting %so levels was observed from lr-rcc manufactured at a blood center using -hour room background/case studies: cellular prion protein (prp c ) is a gpi-anchored cell surface glycoprotein that is expressed mainly in the brain but also in peripheral organs including blood, bone marrow (bm), and lymphoid tissue. prp c can be converted post-translationally into scrapie-prp (prp sc ), which is involved in the pathogenesis of neurodegenerative diseases including creutzfeldt-jakob disease, kuru in humans, and scrapie and bovine spongiform encephalopathy in animals. however, biological functions of prp c have yet to be conclusively elucidated. study design/method: in this study, prp c knockout mice (ko) are utilized to investigate the role of prp c in the hematopoietic system with controls of age and sex-matched prp c transgenic mice harboring a slightly augmented prp c expression. peripheral blood was examined by hematology analyzer to establish counts. bone marrow, thymus, spleen, lymph nodes, and peripheral blood were harvested and analyzed by flow cytometry using a comprehensive panel of fluorochrome-conjugated antibodies specific for all hematologic cell precursors/ lineages. histology of bone marrow, spleen, thymus and lymph nodes were evaluated by light microscopy. results/finding: complete blood count (cbc) showed a significant increase of wbc in ko mice. closer analysis of wbc differential revealed that the elevated number of wbc in ko mice was due to lymphocytosis. specifically, ko mice had a -fold increase in the absolute lymphocyte count (ko . . x /l vs. wt . . x /l, p . ), as well as a higher lymphocyte percentage compared to controls. ko mice also had a trend toward higher hemoglobin, rbc, and hematocrit compared to wt mice. additionally, platelet count in ko mice was higher than control mice. of interest, the mean platelet volume indicating platelet size was significantly increased in ko mice compared to controls (ko . . fl vs. wt . . fl, p . ). a comprehensive flow cytometric analysis of all cell lineages revealed no significant differences in the numbers of rbc and megakaryocyte in bm, and of lymphocytes in the thymus, spleen and lymph nodes. histological analysis of bm, thymus, spleen and lymph node tissue from ko and wt animals failed to show morphological differences between the two groups. conclusion: absence of prp c resulted in significant leukocytosis and specifically higher absolute count and percentage of lymphocytes, as well as larger platelets in peripheral blood, but does not appear to affect hematopoiesis and lymphopoiesis. our findings indicate that prp c might be critical in the survival and trafficking of lymphocytes in peripheral blood. the molecular mechanisms underlying the observed changes in lymphocytes and platelets, and whether these involve functional changes in these cells will be subject of future studies. potential role of cd foxp regulatory t cells derived exosomes in their immune modulation yiming yang*, rufeng xie and jie yang. blood engineering laboratory, shanghai blood center background/case studies: exosomes are defined as one type of membrane vesicles secreted into extracellular space by most types of cells and are reported to involve in intercellular communications, mediate biological process. human periphery blood cd foxp tregs cells are reported as more stable regulatory cells with greater inhibition effects. however, cd foxp tregs derived exosomes and their functions involved in cd tregs mediated immune-modulation were seldom reported. study design/method: cd t cells were freshly purified from pbmcs, cultured with anti-cd /cd antibody packaged beads and il- , and then polarized with tgf-b and rapamycin into cd foxp treg cells. the harvest cells were co-cultured with cd /cd beads stimulating cd cd effector cells in the transwell plate. the supernatant derived from cd tregs was collected and ultrafiltrated by centrifugation and the remaining solution was precipitated with peg. the harvest precipitation was resuspended in pbs and exosomes were analyzed by sem and nta. exosome surface marker cd , cd , tsg and other proteins expression were evaluated by flow cytometry and western blot. microrna was isolated with mircute mirna kit and mir- , let- b, let- d were measured by qpcr. the precipitated exosomes were further purified by cd immunoaffinity capture and co-cultured with effector cells to investigate their function in immune modulation. results/finding: as compared with direct contact co-culture, separated cd treg cells could suppress the proliferation of effector cells with a small decline (p> . ), which means some non-contact factors involved in the cd treg mediated immune modulation. a total number of . . / cells exosomes were harvest. electron microscope analysis demonstrated a kind of round-shaped membrane vesicle - nm in diameter ( . . nm by nta). cd and cd were expressed on these background/case studies: regulatory t cells (tregs), containing cd and cd subtypes, play an essential role in immune regulation and autoimmune disease prevention which makes it a potential candidate for cell therapy on autoimmune disease (aids). unfortunately, due to the instability of natural cd foxp regulatory t cells (ntregs) in inflammation conditions (including instability of foxp , conversion to pro-inflammatory effector cells and was unable to modify established disease), thus, it is needed to investigate cd regulatory t cells stability both in vitro and in vivo. in our previous works, we found that cd treg has an effective therapeutic function on cia mice. in this study we aim to investigate the stability of induced polyclonal human cd regulatory t cells in inflammation and transfusion. study design/method: human cd tregs were induced with tgf-b and rapamycin from cd t lymphocytes in vitro. collagen-induced arthritis (cia) mice were induced with type-two collagen as an autoimmune disease model. in vitro the stability of cd tregs when encountering with inflammation were test by foxp expression, th and th cells conversion in inflammations conditions (il tgf-b il il and il tgf-b il b il ) on day , day and day . in vivo, cd tregs were transfused into cia mice and then their survival in mice and foxp express were evaluated to reveal the stability of cd tregs in an inflammation condition model. additionally, we also investigate the stability maintenance of cd tregs when induced factor tgf-b and rapamycin were removed by testing the foxp expression on day , day and day . results/finding: ex vivo induced human cd treg were foxp ( . . %) and did not secret il a (both in supernatant and % of cells). foxp express in cd tregs were maintained after induced factor tgf-b and rapamycin were removed on day , day and day . in vitro, foxp , il and ifn-c expression has no significant difference when compared with controlled tregs on day , day and day and did not secret il a when encounter with inflammation conditions (il tgf-b il il and il tgf-b il b il ). in vivo, cd treg cells were transfused into cia mice on the peak of disease onset ( days after the first collagen immunization, has inflammation condition in vivo) to test cd tregs survival. cd tregs were found in cia mice foot ( . . %), blood ( . . %) and spleen cells ( . . %) hours after transfusion and their % of foxp were remained. conclusion: the results revealed that ex-vivo induced and expanded human cd tregs are stable in inflammation and transfusion and can maintain foxp expression when induced factor were removed, these make cd treg a novel and stable cell for potential cell therapy on aids. this research can provide some instructive reference and improve the utilization of blood components. tolerogenic dendritic cells induced by mtor suppression and control inflammation in chs model through s k related proteins translation inhibition. li gao*. shanghai blood center background/case studies: tolerogenic dendritic cells (tdcs) adoptive cellular immunotherapy is a cutting edge strategy for treating hypersensitivity response disease, in which immune responses are directed against selfantigens, such as atopic dermatitis, systemic lupus erythematosus (sle), rheumatoid arthritis (ra),et al. however, the traditional strategy base on the tdcs was usually unstable and inconspicuous through cytokines inducing processing so that might be the limitations on tdc adaptive cell therapy in future clinical use. study design/method: human tdcs were derived from fresh purified monocytes from pbmncs isolated from buffy coat and induced by mtor inhibitors (rapamycin and temsirolimus) in safe concentrations confirmed by apoptosis assay when the cells were completely differentiated. the mature markers and endocytisis were detected by flow cytometry. the production of cytokines and chemokines was measured using elisa. mechanism investigation was analysis by real-time pcr and western blotting. contact hypersensitivity (chs) model, an atopic dermatitis animal model, was treated with tdcs induced via mtor suppression and analyzed by ear thickness and tissue leukocytes number calculating. results/finding: human tdcs treated with mtor inhibitors had a lower mature marker cd /cd /cd expression after tlr signaling activation, accompanied with a set of cytokines and chemokines remarkably downregulated in a concentration dependent manner but not the lps absent group. moreover, mtor suppression extremely reduced the capacity of lps treated human dcs to stimulate autogenic na€ ıve t cell proliferation, which is one of the most important characteristics of tdc. beyond expectation, the common signal transduction pathway, mapk and nf-jb pathway, were not the signal target so that it could hardly be the explanation for the tolerogenic performance of tdc when exposure to lps stimulation. however, the p s k and its downstreanm proteins, especially the protein s , which controls the protein translation, were shown in charge of the tolerogenic induction mechanism. the data were also supporting the suggestion that rare difference on mrna transcription of the related functional proteins in tdcs induced by mtor inhibitors when exposure to lps stimulation from the non-induced cells, although there was more transcription of ido induced by mtor inhibitors. more important, edema responses of ears were clearly weakened in the chs model and recruited less leukocytes to the tissue when co-sensitized with mtor inhibitors or with tdcs induced by mtor inhibitors suggested that the tdc induced by mtor suppression were able to control hypersensitivity inflammation response in vivo. conclusion: accordingly, tdc induced by mtor suppression is a potent adoptive cellular immunotherapy strategy for treating hypersensitivity response disease and the induction mechanism of it might be through suppressing systematically effective function proteins by mtor-s related protein translation inhibition. xiaoyun fu* , , mikayla anderson and james c zimring , . bloodworksnw research institute, university of washington school of medicine background/case studies: red blood cells (rbcs) undergo many changes when stored under blood banking conditions, collectively known as the storage lesion. bioactive lipids generated during rbc storage have been implicated in certain adverse outcomes. recently, we reported that bioactive lipids, especially polyunsaturated fatty acids (pufas) and their oxidized products (oxylipins) accumulate during rbc storage despite leukoreduction. to evaluate the extent of membrane lipid degradation and oxidation in stored rbc units among the donor population with different blood groups, we quantified pufas and lysophospholipids (lpls) in leukoreduced rbc units. study design/method: rbc units from different donors were acquired and processed on day (one day past their expiration). bioactive lipids including common fatty acids, oxylipins, and lpls were analyzed by liquid chromatography-tandem mass spectrometry with multiple reaction monitoring (lc-ms/ms-mrm). total fatty acid concentrations of selected units were also analyzed. a one-way anova test was used to determine significant difference of analytes amongst the different blood groups. results/finding: we observed a wide distribution in concentration of major pufas in stored rbc units. for example, arachidonic acid (aa) ranges from . - . mm, linoleic acid (la) ( . - mm), dihomo-c-linolenic acid (dgla) ( . - . mm), eicosapentaenoic acid (epa) ( . - . mm), docosahexaenoic acid (dha) ( . - . mm), and alpha-linolenic acid (ala) ( . - . mm). ten oxylipins including hetes, hodes, and dihomes, and lpls including lpcs, lpss, and lpes all showed a large variation in concentration among donors. of analytes quantified, showed a significant difference in concentration among different blood types by one-way anova testing (fdr< . ). the ab rh blood group consistently exhibited the lowest concentration of major pufas, while the o rh-blood group showed the highest, averaging a two-fold difference in concentration (o rh-/ab rh ). the fold increase of o rh-/o rh among pufas ranges from . to . , suggesting the rh blood group, independent of the abo blood group, correlates with donor to donor variation in lipid metabolism. conclusion: the wide distribution in the concentration of bioactive lipids among stored rbc units suggests that lipid degradation is highly donor-background/case studies: to ensure availability of biological products to hospitals, blood banks have developed and validated multiple storage conditions for each of their products to maximize shelf life and quality. in the case of labile products, their metabolism is known to remain active during storage, leading to storage lesions. micrornas (mirnas) levels are modulated by these storage-related damages, which makes mirnas ideal candidates as potential biomarkers of quality monitoring. lately, nanoparticles have been widely studied and used for biosensing applications. the objective of this work is to develop biocompatible gold nanosensors for sensitive, selective and direct detection of biomarkers to characterize and assess the quality of blood products delivered to hospitals. study design/method: gold nanoparticles (gnps) surrounded by a fluorescent silica shell were prepared using a wet chemistry method. mirna- was chosen as a potential target, since it is strongly expressed in platelet concentrates and its concentration fluctuates according to storage lesions. custom rna and dna molecular beacons were designed and used as a probe for the specific detection of mirna- targets in pbs and human plasma. these fluorescent transducer probes were conjugated at the surface of fluorescent silica shell-gnps using an edc/nhs cross-linking reaction. the hybridization reaction between the target and the probe initiates an energy transfer mechanism which can be recorded by fluorescence. results/finding: gnps ( nm) surrounded by a thick fluorescent silica shell ( nm) were prepared and used as nanosensors because of their optimal luminescence properties and long-term stability. conjugation of the probe onto the nanoparticles was confirmed by fluorescence spectroscopy and microscopy, as well as nanoparticle tracking analysis. the fluorescent response of the molecular beacons was studied and showed a reproducible and linear relationship (r rnaprobe . and r dnaprobe . ) with mirna- concentration, down to a -nm limit of detection. hybridization assays in % human plasma appear to demonstrate denaturation of rna probes and targets. conclusion: biocompatible fluorescent gnps were prepared and used as tools for blood product characterization. the conjugation of a molecular beacon at the surface of nanoparticles was achieved and characterized using spectroscopic and microscopic techniques. the functionalization of the probe is still being optimized. the fluorescence response of the molecular beacon was characterized for the detection of a model mirna target in pbs and in % human plasma. energy metabolism profile of erythrocytes during storage suping ren*, qun yu, yanbing wang, changlan li and yu wang. background/case studies: the moment the mature red blood cells (rbcs) leave the bone marrow, it is optimally adapted to perform the binding and transport of oxygen and its delivery to all tissues. red blood cells modulate oxygen transport, protect hemoglobin from oxidant-induced damage, and maintain the osmotic environment of the cell. glycolysis is the only energetic metabolic pathway for mature rbcs to obtain atp which is the energy for rbcs to maintain a number of vital cell functions. generally, the current methods used to measure rbcs glycolysis are not in living state in realtime, or are destructive to cells or require radioactivity.xf technology can be applied to different types of cells, in which the red blood cells are suspended and the cell shape and size are different from other cells, and more importantly, rbcs have no nucleus, mitochondria and other organelles, so application of the xf technology in erythrocytes and exploration of the assay conditions are necessary. . . a . . . . a . . . . a . . total atp,lm/ghb . . a . . . . a . . . . a . . extracellular lactate,mm a extracellular glucose,mm a a a extracellular na ,mm a extracellular k ,mm a a a a p< . , paired t-test b intercept blood system for red blood cells is not approved for commercial use. c this project has been funded in whole or in part with federal funds from the dhhs; aspr; barda; contract no. hhso c. background/case studies: pathogen inactivation methods for platelet concentrates are increasingly being used in blood banks worldwide to make transfusion safer. in vitro studies have demonstrated the effects of pathogen inactivation on storage lesion, but little routine quality control data on blood banking outcomes have been reported. study design/method: swirling of distributed products was monitored one year before and one year after implementation of intercept pathogen inactivation. metabolic parameters like ph, glucose and lactic acid were determined in a random sample of expired pathogen inactivated products. furthermore, indicators of platelet storage lesion were measured in apheresis concentrates with premature low swirling and compared to controls with normal swirling. results/finding: in an experimental phase on a limited number of products (n ) to validate the intercept pathogen inactivation method, ph and glucose levels decreased faster in apheresis platelet concentrates with high platelet content than with low platelet content or than in pooled buffy coat derived products. once pathogen inactivation was implemented, routine products showed glucose exhaustion more often when prepared by apheresis compared to buffy coat derived platelet concentrates despite more plasma carryover in the former. furthermore, the number of apheresis products with premature low swirling increased by % ( / , ) compared to the previous year without pathogen inactivation ( / , , p . , chisquare) . in contrast, the incidence of premature low swirling in platelet concentrates prepared by the buffy coat method decreased ( / , vs / , ). of note, apheresis concentrates with premature low swirling had a significantly higher median platelet count ( . x ) than unaffected controls ( . x ) and showed signs of increased storage lesion compared to controls expiring on day five without swirling defects. these signs included lower ph, higher lactic acid concentration, increased mean platelet volume, phosphatidylserine exposure and alpha-degranulation. conclusion: the risk of increased storage lesion rates following intercept pathogen inactivation is higher for apheresis than for buffy coat derived platelet concentrates, especially when platelet content is above . x . in vitro quality of single dose amotosalen/uva treated platelets in % plasma/ % pas- after days of storage crystal stanley , marguerite kelher , nero evero , melissa vongoetz , betsy donnelly and anna erickson* . belle bonfils memorial blood center, university of colorado, cerus corporation background/case studies: the interceptv r blood system for platelets is fda approved for the ex vivo preparation of pathogen-reduced amicus o apheresis platelet components (pc) in pas- to reduce the risk of tti, including sepsis, and to potentially reduce the risk of transfusion-associated gvhd. registration studies (clinicaltrials.gov nct ) are in progress to support approval of the trima o apheresis platform for collection of platelets components (pc) suspended in pas- and plasma. the objective of this study was to evaluate in vitro function of platelets suspended in % plasma/ % pas- , collected using the trima platform, after treatment with the intercept blood system for platelets. study design/method: double dose apheresis pc, . . platelets in ml, were collected on the trima apheresis platform in % plasma/ % pas- . a sample was taken from each donation prior to dividing the donation to produce intercept treated apheresis pc (t), using the small volume (sv) set, and an untreated control pc (c). input volumes for replicates, n , were ml (t) and ml (c) with doses of . . (t) and . . (c). all pc were stored under the same conditions and evaluated on day and day for physical/metabolic characteristics. results/finding: on days and all t and c pc had ph c ! . . the dose recovery for t was % %. on day , t had lower count, volume, dose, bicarbonate and glucose compared to c pc; however, parameters predictive of in vivo function (atp, morphology score, hsr, and esc) were equivalent between t and c (table ) . conclusion: trima pc in % plasma/ % pas- treated with the inter-cept blood system for platelets using the sv set and stored for days retained in vitro metabolic and functional properties consistent with in vivo functionality. induction of pluripotent stem cell-derived cardiomyocyte toxicity by supernatant of long term-stored red blood cells in vitro feng-yan fan , , yang yu , li-ping sun , shu-fang wang , rui wang , lei-ying zhang and deqing wang* . the department of blood transfusion, the pla general hospital, the department of blood transfusion, air force general hospital, pla background/case studies: recently, multi researches have reported that longer term-stored red blood cells(rbcs) units were associated with increased risks of clinically adverse events, especially in critically ill patients. however, other studies have concluded the negative results. whether rbcs storage duration was associated with increased risks of clinically adverse events is uncertain and had become a popular topic. to study the adverse effects of longer term-stored rbcs directly, we aim to look at the pluripotent stem cell-derived cardiomyocyte toxicity induced by supernatant of suspended red blood cells(ssrbcs), and study the possible mechanism. study design/methods: five doses of leuko-reduced rbcs were prepared, and supernatant was isolated by centrifugation on d , d and d . we looked at the cardiotoxicity of ssrbcs on human-induced pluripotent stem cell-derived cardiomyocytes (hips-cms). hips-cms were treated with ssrbcs in % final volume simulating the large volume blood transfusion. using real-time cellular analysis (rtca) technology the beating of hips-cms was recorded in real time in detail. levels of k and lactic acid (la) were tested using automatic biochemical analyzer. k and la solution with concentrations being consistent with ssrbcs were prepared and cocultured with hips-cms. we analyzed the cardiotoxicity of k and la solution on hips-cms. treated hips-cms with d ssrbcs, d k and cell culture media for h. the nuclear shape and integrity of filament and sarcomere was examined by immunofluorescence. total rna of hips-cms was isolated and mrna analysis microarray was implemented. screened for toxic effects related signaling pathways through bioinformatics analysis. results/findings: d ssrbcs had no obvious influence on beating state of hips-cms-hips-cms treated with d ssrbcs stop beating, but beating patterns restored at h. hips-cms treated with d ssrbcs stop beating, and beating patterns did not restored at h. levels of k and la in ssrbcs changed most obviously. only d k solution made hips-cms stop beating and can restore in h; d k, d k and la solution did not influence the beating pattern in at the end of the treatment for h, hips-cms treated with d ssrbcs show obvious shrinkage. at the end of the treatments for h, cells treated with d k and d ssrbcs both show obvious shrinkage, the shrinkage in d ssrbcs group was more serious. the immunofluorescence results show the integrity of filament and sarcomere was complete and no nuclear pyknosis was detected. gene expression array results show a total of genes were differentially expressed in d ssrbcs group compared with naive group. there was no consistent separation within the d k and naive group. fifteen differently expressed genes were selected with bioinformatics method which were likely to play an important role in the cytotoxic effect. under the condition of simulating the large volume blood transfusion, ssrbcs of long term-stored rbcs have toxic effect on myocardial cells. in addition to high potassium that induced cardiotoxicity, there must be other elements are involved in the toxic effects. further study should be applied to signal pathways on ssrbcs induced cytotoxicity. large volume transfusion of long term-stored rbcs may be a risk factor for adverse clinical outcomes, and clinical should pay attention to it. background/case studies: processing thawed, deglycerolized red cell concentrates (rcc) in a functionally closed system allows for a prolonged storage after thawing. thawed cells are better maintained in as- as compared to sagm. the presence of citrate in as- seems to be necessary to prevent hemolysis of thawed cells. during storage in as- , atp and , -dpg levels rapidly decline. recently developed additive solutions like pag m and as- have shown to better maintain , -dpg and atp levels during storage of normal, unfrozen, rcc. however, most probably due to the absence of citrate, these solutions are not suitable for storage of thawed cells. we therefore designed pag c in which the mannitol of pag m was replaced by citrate. the aim of this study was to investigate the in vitroquality of thawed, deglycerolized rbc during storage at - c in pag c. study design/method: leukoreduced rcc (n ) in pag c (phosphate, adenine, glucose, guanosine, gluconate, citrate) were stored at - c. on day , rccs were glycerolized using acp (haemonetics v r , braintree, ma) to a final concentration of % (w/v), frozen and stored for at least two weeks at - c. after thawing and deglycerolization using acp , rcc were resuspended in pag c. during storage at - c, stability (hemolysis), atp and , -dpg levels were determined. results were compared with thawed rcc (prefreeze storage in sagm, n ) resuspended in or sagm (n ). results/finding: pre-freeze storage in pag c resulted in increased , -dpg levels at day as compared to storage in sagm, resp. . . mmol/g hb and . . mmol/g hb. hemolysis during post-thaw storage in pag c remained below . % for days and was comparable with storage in as- . in sagm, hemolysis remained below . % for days. during the first weeks of post-thaw storage in pag c, both atp and , -dpg levels increased, followed by a gradual decline during prolonged storage. during the whole postthaw storage period, rccs in pag c showed significantly higher atp and , -dpg levels compared to as- or sagm. while in sagm and as- , , -dpg levels were undetectable after days post-thaw storage, in pag c, , -dpg levels only decreased to . lmol/g hb after days of storage. conclusion: pre-freeze storage in pag c resulted in increased , -dpg levels. as compared to as- , post-thaw storage in pag c showed comparable hemolysis while atp and , -dpg levels were much better maintained. based on a maximum allowed hemolysis of . % and an atp content of > . mmol/g hb, thawed rcc can be stored at - c for days in pag c. background/case studies: platelets (plts) are vital for effective treatment of hemorrhage. cold ( c, c) storage of plts in platelet additive solution (pas) is a promising alternative to conventional storage at room temperature (rt) due to a lower risk of bacterial concerns, preservation of plt function, and mitigation of plt activation. currently only apheresis (ap) and pas systems are fda-approved for use in the us: trima and isoplate-pas (iso; terumo) and amicus and intersol (int; fenwal) . the goal of this study was to assess the adhesive function of long-term cold-stored plts collected by fda-approved ap/pas methods. study design/method: plts were collected (n - ) in % iso using a trima or in % int using an amicus and stored for days at rt and c. samples were tested on day (baseline, bl), , , and of storage to assess plt adhesion under shear flow (bioflux). acd vacutainer tubes were collected from donors and centrifuged to obtain red blood cells (rbcs) for all bioflux runs. simulated whole blood was created by combining plts labeled with calcein-am with rbcs at % hct. labeled blood was perfused through microfluidic channels (fluxion) coated with ug/ml type- collagen at s - shear rate. images were acquired every sec for min using a fluorescent microscope and % surface coverage was reported. data were analyzed using two-way anova and posthoc tukey test with significance at p< . . results/finding: both rt-int and rt-iso plts showed significantly decreased adhesion by day of storage compared to bl (bl: . . %, rt: . . %; p< . ). c-int samples showed no difference in adhesion at any timepoint compared to bl-int but significantly enhanced adhesion compared to both rt-int and rt-iso. in contrast, c-iso plts showed significant enhancement of surface coverage compared to bl-iso by day (p . ) and compared to c-int by day (p< . ). conclusion: our work suggests that c storage of plts collected with a trima ap system in iso for up to days offers a significant enhancement in adhesive function compared to plts collected with an amicus system in int and stored at c. these results are surprising since both c-int and c-iso have been shown to express similar levels of cd p, pac- , and phosphatidylserine and may suggest differences in pas plt intracellular signaling. as expected, storage at c of plts collected on either platform demonstrated superior function to rt storage. a plt product with superior hemostatic function and a shelf-life x longer than the current standard-ofcare provides the potential for shipment of products to underserved areas and may bolster plt availability for trauma care in the us. table . comparisons of white blood cell counts and percentages of apoptotic cells in whole blood components after -week storage between unirradiated and irradiated groups (n ) tang, is an anti-inflammatory agents and has a good safety records in clinic. it could reduce the severity of experimental autoimmune encephalomyelitis (eae), asthma, colitis, systemic lupus erythematosus(sle) and other immune diseases.however,its potential in inducing transfusion tolerance remains to be explored.the aims of our study are to find if baicalin could inhibit red blood cell (rbc) immunization and to elucidate the possible mechanism of yin-chen-tang in preventing hdn. study design/method: we used human red blood cells with adjuvant lipopolysaccharide (lps) and transfused mice to induce antibodies, as an experimental system to study the effect of baicalin on rbc immunization. mice were divided into a normal control group, a human rbc transfused positive control group receiving human rbc and lps intravenously weekly for five weeks, a control group receiveing dexamethasone ( mg/kg/day) intraperitonealy daily for five weeks,a treatment group receiving baicalin ( mg/kg/day) intraperitonealy daily for five weeks. assessment of human rbc immunization was performed by measuring serum immunoglobulin g (igg) and immunoglobulin m (igm) against human rbc weekly. and the lymphocyte changes in spleen are also monitored by flow cytometry. results/finding: we found that baicalin treatment decreased serum igg but not igm production significantly since the second week, with a concomitant reduction in th cells and increase in cd regulatory t cells in both spleen and mesenteric lymph nodes. and there are no significant differences in the percentage of th ,th ,tfh and tfr cd subpopulation among all groups.in addition, baicalin treatment didn't decrease the size of spleen and the percentage of cd positive cells in spleen in baicalin treatment mouse but in dexamethasone treated mouse. our results indicate that baicalin could inhibit rbc immunization especially igg production without the damage to the function of spleen,while dexamethasone as a wildly used immune-suppressive drug in blood transfusion could damage the function of spleen.considering its good safety records in clinic, it may be exploited for suppressing transfusion immunization events. in addition, our results elucidate the inhibitory effect in antibody production of baicalin may be a possible mechanism for yin-chen-tang as a widely used chinese herbal medicines in preventing hdn. comparison of immucor's pak plus and pak lx assays for the detection of human platelet alloantibodies randy m schuller* , sarah kloss , sara crew and sandra j nance . american red cross, american red cross and american rare donor program background/case studies: alloantibodies directed against human platelet membrane glycoproteins (gp) ia, iia, iib, iiia, ib, ix, iv, and cd have been implicated in several clinically significant disorders such as fetal and neonatal alloimmune thrombocytopenia (fnait), post-transfusion purpura (ptp), refractoriness to platelet transfusions, and passive transfer of antibodies in donor plasma. polymorphic epitopes on these gps give rise to unique human platelet antigens (hpa). identification of the specific platelet alloantibody is crucial in diagnosing and treating these bleeding disorders. currently the only k fda approved test permits the identification of these hpa antibodies to the glycoprotein level. immucor has recently released pak lx, a research use only (ruo) assay in the united states that has the ability to identify hpa antibodies to a single nucleotide polymophism (snp). we compared the performance of pak lx to the fda approved immucor pakplus. study design/method: we compared pakplus and pak lx results from plasma and serum clinical specimens. group contained a single hpa alloantibody specificity with or without hla antibodies (n ). group included specimens with hla antibodies alone and group consisted of patient samples that were negative for both hpa and hla antibodies. pak lx utilizes a luminex bead based assay which allows the user to report antibodies to the platelet specific antigen (hpa- , hpa- , hpa- , hpa- , hpa- , gpiv) and hla class i. pakplus uses an elisa method and results can only be reported to the glycoprotein location (gpiib/iiia, gpia/ iia, gpib/ix, gpiv) along with hla class i. however, based upon the pattern of reactivity observed in the pakplus and pak lx assays it is possible to determine the most probable hpa antibody specificity to the hpa snp. results/finding: conclusion: when analyzing hpa antibody specificity, there is % concordance observed for hpa- a, hpa- b and hpa- b antibodies. the pak-plus assay had difficulty discriminating hpa- b from hpa- a antibody when hpa- a antibody was present ( false positive samples) although the pak-plus signal od to cutoff od ratio was significantly higher for hpa- a when compared to hpa- b in these samples. the discordant hla class i antibody results between the assays was isolated to very weakly positive antibody (within % of the cut-off for pakplus and < . adjusted ratio for pak lx). we conclude that pak lx is an easy to use platelet alloantibody screening method that has the ability to differentiate hpa antibodies to the allele level. histo-blood group antigen lewis y promotes cell migration via regulation of microtubule acetylation huijun zhu* and ping lu. shanghai blood center background/case studies: blood group antigens are critical for transfusion practices as antibodies raised against them can cause severe transfusion reaction. beside this, blood group antigens themselves are composed of sugar chains, proteins, lipids, etc, which may be involved in various biological processes. lewis y is a histo-blood group antigen belonging to abh family. ley consists of carbohydrate chains which may play important roles in cell recognition, adhesion as well as migration, which are all critical steps in tumor progression and thus attracts wide researches focusing on its relevance in tumor biology. ley is demonstrated to affect cell mirgration via various mechanisms. however. although changes in cytoskeleton organization is the basis for cell motility, little is known about the association between cytoskeleton and ley. as microtubule and its construction unit tubulin participate in various steps of cell migration, we aim to explore the role of ley in microtubule and cell migration using breast cancer cells, which may provide reference to clinical study of other histo-blood group antigens and change the way of thinking in transfusion practice. study design/methods: we first manipulate ley expression in breast cancer cells by overexpression or sirna knockdown of fucosyltransferases, and block ley activity in mda-mb cells using anti-ley antibody, to verify the effect of ley on cell migration. then, we detect acetyl-a-tubulin level change as microtubule acetylation is a sign for stability. to establish the role of ley in cell migration via microtubule modification, we use hdac specific (tubacin) and nonspecific (tsa) inhibitors to minimize deacetylation of acetyl-atubulin and test again the effect of fut overexpression on cell motility. results/findings: fut overexpression increases both ley expression and cell migration, while fut knockdown leads to the opposite. ley activity blockade by anti-ley antibody also significantly inhibits cell migration. western blot and immunostaining results show a-tubulin acetylation level is negatively related with ley expression. tubacin or tsa treatment increases the acetyl-a-tubulin level while inhibits cell migration; in the meantime, the significance of fut overexpression in promoting cell migration is eliminated. conclusion: it can be concluded from the results above that ley can promote cell migration via regulation of a-tubulin acetylation, wherein ley may have interaction with deacetylase hdac . as tumor promoter, hdac becomes the target of many anti-cancer drugs. we demonstrated the potential association of ley and hdac function in this study. many blood group antigens are also carbohydrate chains, which are not only critical in blood group determination, compatible transfusion and immunological reaction, but may also have an effect in the initiation and development of diseases as tumor, similar to ley; they can even be components in a network with other important molecules and contribute to the destiny of diseases. transfusion of blood products is frequently needed by tumor patients. most attention is focused on the search of compatible blood for reducing transfusion reaction. however, it may lower the chance for the disease to advance to take account background/case studies: reducing the risk of bacterial contamination in platelet (plt) products is of great concern since plt storage occurs at room temperature (rt). pathogen reduction technologies (prt) were developed to inactivate pathogens prior to transfusion; however, studies have shown that prt may damage plts over the course of extended storage at rt resulting in a greater loss of function than what is normally concomitant with platelet storage lesion. storage of plts in platelet additive solution (pas) at c helps to preserve plt function and reduces the risk of contamination. in this study, we established the impact of prt performed after long-term coldstorage of plts in pas, instead of before storage, on plt function, mitochondrial respiration, and cell death parameters. study design/method: plt units were collected in pas (n ) and stored at c for up to days. after this time period, the bag was treated using mirasol prt (riboflavin and uv). samples were obtained and tested on the day of collection (baseline, bl), pre-mirasol (pre), post-mirasol (post), and minutes post-mirasol . aggregometry (adp, collagen, trap), rotem, flow cytometry (cd p [p-selectin] , lactadherin [ps] , , and gpib), high-resolution respirometry (oroboros), and imaging flow cytometry (amnis) were used for analysis. data are reported as means sem, and paired student's t-tests were used to determine statistical significance (p< . ). results/finding: on day , p-selectin levels were significantly higher in pre than bl (p . ). mirasol treatment caused a significant increase in pac- expression compared to pre (pre: . . %, post: . . %; p . ), which remained after incubation. a significant drop in both collagen and trap aggregation was observed in post samples compared to pre, but adp aggregation response was preserved. no differences in p-selectin, gpib expression, and mitochondrial respiration were observed between pre and post samples. post- samples displayed significantly less function, higher activation levels, and lower mitochondrial respiration compared to pre and post. conclusion: prt treatment of plt units in pas after day storage at c presents a unique alternative to prt treatment of plts prior to rt storage. in addition to providing a lower risk of bacterial contamination, c-stored pas plts may provide better preservation of hemostatic function than standard-of-care rt plts, even after mirasol prt treatment. however, we show here that mirasol prt of day c-stored pas plts followed by incubation ( minutes or more) results in widespread cell damage and should be avoided. safety evaluation of lyophilized canine platelets in a model of coronary artery bypass graft (cabg) todd m. getz* , arthur p. bode , anne s hale , michael stanton , mark johnson and g. michael fitzpatrick . cellphire, bodevet, inc, cellphire, background/case studies: cellphire has completed a micro dose clinical safety trial using lyophilized human platelets. cellphire also evaluated the safety of lyophilized canine platelets (lcp) in comparison to liquid stored canine platelets, following intravenous administration in a model of on-pump coronary artery bypass graft (cabg) in the canine. this safety study was in support of a future phase ii human clinical trial in cardiac patients. study design/method: three groups of eight mixed breed hounds underwent cabg to create an anastomosis and were administered lcps equal to , , and . % of the total circulating platelet count (tcpc). one group of four animals served as the vehicle group which received lyophilization platelet-formulation buffer, and another group of four animals received control ( -day old liquid-stored platelets). safety was assessed through the collection of blood loss data, evaluation of blood flow through the bypass graft, evaluation of the development of acute thrombosis, and maintenance of patency through the graft over the hr evaluation period. full necropsies with complete tissue analysis were also performed. efficacy signals were evaluated through the collection of blood loss data and coagulation endpoints (pt, aptt, fibrinogen, and teg). the results demonstrated that administration of the test article at doses up to % of the tcpc was not associated with any unexpected mortality, adverse changes in hematology or coagulation parameters, development of thrombosis at the anastomosis sites, or evidence of adverse thrombosis formation either clinically or microscopically regardless of group. the mortality noted on study was considered to be related to the surgical model and not a result of test article administration. the results also demonstrated that administration of doses of % and % of the tcpc produced a significant decrease in blood loss. the lcps at % and % tcpc were as effective in mitigating blood loss as -day old liquid-stored platelets and trended towards being more effective. no appreciable differences in coagulation parameters were observed between groups. conclusion: the results of the study demonstrate that administration of lcp up to % of the tcpc was safe in a canine cabg model. the data also demonstrate that administration of lcp at doses of % and % of the tcpc reduced blood loss. these results suggest a starting dose above . % tcpc may be required to achieve an effective dose in future human phase ii trials in cardiac patients. although the study was not powered for efficacy, these data indicate a level of safety, as % tcpc had similar efficacy signals as % tcpc with no observable severe adverse events. the starting effective dose may vary depending on the clinical indication. future studies will be required. this study was funded under barda contract hhso c. the study on pcr-ssp technique for the genotyping of cd - del.ac mutation and the genetic polymorphism of cd - del.ac in chinese population lilan li* and guoguang wu. nan-ning institute of transfusion medicine background/case studies: cd (platelet glycoprotein iv, scarb ) is an important and characteristic platelet antigen implicated in immune-mediated thrombocytopenia in chinese population. except anti-hla, anti-cd is the most common antibody of clinically relevant platelet antibodies in chinese population, which is associated with the high frequency of cd deficiency in china. cd gene mutation is the main reason that leads to cd deficiency. cd - del.ac (frameshift at aa ) mutation is one of the cd mutations that causes cd deficiency. have had natural mumps, measles and rubella infections, resulting in lower antibody levels in their blood. the recommendations may thus be unfounded and outdated, and prevent valuable vaccination opportunities for children with frequent blood transfusions. this places an already highly vulnerable pediatric population at risk for acquiring preventable infections. the primary aim of this project was to determine mmr vaccination immunogenicity in patients chronically transfused with rbc. study design/method: medical charts were reviewed for vaccination and transfusion histories. mmr-specific antibodies were quantified in pediatric patients who received both doses of the mmr vaccine at and months of age while they were on a chronic rbc transfusion program for sickle cell disease, b-thalassemia major, diamond-blackfan anemia or pyruvate kinase deficiency. there was no formal control group; long-term immunity rates in the literature are ! % for all mmr components. results/finding: table shows immunogenicity to vaccine components. delays between vaccination and serology testing averaged . years ( . to . years). thirteen patients ( %) were chronically transfused at the time of serology. twenty-three patients ( %) seroconverted to at least one of the vaccine components. conclusion: to the best of our knowledge, this is the first study designed to measure the effect of rbc transfusions on mmr vaccine immunogenicity. although lower than the rates reported in the literature, the results suggest a high rate of immunogenicity to each component of the mmr vaccine in chronically transfused patients immunized prior to months posttransfusion. weighing the risks and benefits of disease prevention in a highly vulnerable population, and taking into account the aforementioned results, a reevaluation of immunization delays post rbc transfusions is called for in chronically transfused infants. post-vaccination serology should be considered. cold stored uncrossmatched whole blood can be safely administered to pediatric trauma patients christine m leeper , , franklyn cladis , richard saladino , darrell triulzi , barbara a gaines and mark yazer* . university of pittsburgh, children's hospital of pittsburgh of upmc, institute for transfusion medicine background/case studies: the use of uncrossmatched cold stored whole blood (wb) is becoming increasingly popular in the initial resuscitation of trauma patients without a current abo group. wb has advantages over conventional component therapy including greater platelet and factor concentrations, as well as less saline and additive solution compared to an equivalent volume of reconstituted whole blood. this report details the initial use of wb in pediatric trauma patients. study design/method: pediatric trauma patients ! years old and ! kg with evidence of hemorrhagic shock were eligible to receive up to cc/kg of cold stored, leukoreduced group o negative wb during their initial resuscitation. all wb units had a low titer of anti-a and -b (< ) to reduce the likelihood of hemolysis in non-group o recipients. biochemical markers of hemolysis were measured on the day of wb transfusion and the following two days. admission thromboelastograms were obtained and repeated as necessary during the resuscitation. after receipt of the maximum quantity of wb, conventional components were utilized. results/finding: in approximately months, trauma patients received wb: group o and group a recipients, % male, median (iqr) age was ( . - ) and % blunt trauma mechanism. patients were severelyinjured with a median (iqr) injury severity score of ( - ) and % mortality rate. the median (iqr) quantity of wb transfused to group o recipients was . ( . - . ) ml/kg versus . ( - ) ml/kg to non-group o recipients. no transfusion reactions were reported. the mean standard deviation haptoglobin concentrations for non-group o recipients was . . mg/dl on day , . . mg/dl on day , and . . mg/dl on day ; the corresponding haptoglobin concentrations for group o recipients were . . mg/dl, . . mg/dl, and . . mg/dl, respectively (p> . for all comparisons). similarly there were no significant differences in total bilirubin, ldh, creatinine, and potassium at any time point. regarding evaluation of cold platelet function, we compared the subset of patients who received wb but no warm platelets (n ) to a historical group of pediatric trauma patients who received conventional components including warm platelets (n ). the mean standard deviation platelet volume administered was cc for whole blood recipients versus cc for warm platelet recipients. when pre-and posttransfusion teg and platelet counts were analyzed, there was no difference in median platelet count or teg maximum amplitude (ma) between cold and warm platelet groups. conclusion: use of cold-stored uncrossmatched whole blood for the resuscitation of pediatric trauma patients is feasible, acceptable, and appears to be safe. receipt of low titer group o wb did not lead to detectable hemolysis amongst the non-group o recipients. given this finding, the maximum quantity of wb per patients will be increased to ml/kg. identification of red blood cell antibodies in human breast milk by novel adaptation of serological method philippe p pary*, alexis leonard, lauren hittson, naomi lc luban, deepika s darbari, yunchuan delores mo, cyril jacquot, valli criss and jennifer webb. children's national medical center background/case studies: human breast milk contains immunoglobulins that are present in maternal serum and secretions. data in mice has demonstrated the potential for kell antibodies to be absorbed enterally from breast milk and impact the survival of transfused kell positive cells; however, methods to test and titer human breast milk for red cell antibodies are lacking. a two week old infant with a history of rh-d hemolytic disease of the fetus and newborn (hdfn), previously treated with intravenous immunoglobulin and phototherapy, was referred for anemia and reticulocytosis. patient was o positive, positive direct antiglobulin test (dat) with anti-human igg only, and a positive antibody screen by gel method. antibody identification showed anti-d in both the plasma and eluate. patient was transfused o negative red cells and discharged. over several weeks, the patient returned twice for persistent anemia requiring additional transfusions. at eight weeks of age, evaluation showed a persistent dat igg reactivity concerning for continued antibody exposure. maternal breast milk was evaluated as a potential source. study design/method: based on similar properties of human breast milk and plasma, testing to identify igg antibodies using a stantard tube saline method was performed with a minute c incubation, followed by automated washes prior to the addition of anti-human igg reagent. as a control, breast milk from an o positive, antibody screen negative mother was used to assess for interference by milk proteins. antibody screens were performed on the plasma of the patient, the patient's mother and the control concurrently using the same method. antibody identification and titers were also performed when indicated. only freshly collected breast milk stored at room temperature for less than days was found suitable for this technique. results/finding: the patient's mother showed plasma anti-d with a titer and the breast milk showed anti-d with a titer between and . the patient had a consistent plasma anti-d titer of . the patient's mother chose to stop breast feeding after weeks, and the patient's hemoglobin was improved at and weeks of age. using this method, we identified two additional cases of breast milk induced hemolysis: another anti-d and an anti-jka. conclusion: testing showed that it is possible to identify red cell igg antibodies in human breast milk using a standard tube saline method. we identified implicated antibodies in the breast milk received by infants with persistent anemia due to hdfn. breast milk titers were generally lower than maternal serum titers, but titers varied depending upon the timing and frequency of breast feeding. cessation of breast feeding correlated with improved hemoglobin in affected infants. background/case studies: red blood cell (rbc) transfusion is lifesaving for patients with sickle cell disease (scd), but is commonly complicated by rbc alloimmunization. despite transfusion protocols serologically matching for c,e, and kell antigens, alloimmunization to rh antigens continues. scd patients often exhibit a hybrid rhd-ce-d gene which is often characterized by the production of a partial c antigen. it has been previously documented that % of c scd patients from the west indies and west and central africa are partial c and at ( %) risk for alloimmunization to the c antigen through transfusion of c rbcs. this study sought to determine the prevalence within a cohort of children with scd at a u.s comprehensive scd center. study design/method: rbc genotyping results performed on all scd patients using precisetype hea array (immucor, norcross, ga) at children's healthcare of atlanta were reviewed and compared to the serologic type for rh (c/c, e/e) antigens. the prevalence of c-antigen positive patients (serologically) was determined overall, and compared to the prevalence partial c antigen based on the detection of the rhce*ce( g, t) allele in the absence of an rhce gene encoding a conventional c antigen in trans, since this allele is commonly linked to the hybrid rhd*diiia-ce( - )-d gene which encodes the partial c antigen. review of the blood bank information system was performed to identify the number of c-antigen positive transfusion exposures and frequency of alloimmunization to the c antigen. results/finding: out of a total of patients with genotype/rh phenotype data available, ( . %) were c antigen positive serologically. the allele frequency of rhce*ce( g, t) was . . in total, ( . %) patients possessed rhce*ce( g, t) in the absence of conventional c gene in trans. of the c antigen positive patients, individuals ( . %) were predicted to be partial c based on four molecular profiles [rhce*ce( g, t)/rhce*ce: ; rhce*ce( g, t)/rh*ce: ; rhce*ce( g, t)/rh*ce( g): ; rhce*ce( g, t)/rh*ce( g, t): ]. in these partial c patients, no anti-c alloantibodies (or other rh antibodies) were detected after transfusion exposures ( c-antigen negative units; mean: , range: - ), likely from placement of a c-negative rbc restriction upon detection of the rhce*ce( g, t) allele. conclusion: this report confirms previous data of a high prevalence of the partial c antigen in scd patients historically typed as c-positive serologically, and demonstrates the benefits of rbc genotyping to prevent alloimmunization to a highly immunogenic rh antigen by identifying individuals who should receive c-negative blood. all patients with scd should have rbc genotyping performed for determination of their rbc phenotype, preferably prior to receiving transfusions. investigational detection of zika virus rna in us blood donors paula p sa a* , megan l nguyen , melanie c proctor , david e krysztof , gregory a foster , erin k sash , sandy s dickson , joua yang , jeffrey m linnen , kui gao , jaye p brodsky and susan l stramer . american red cross, grifols diagnostic solutions inc., grifols diagnostic solutions, inc, quality analytics, inc background/case studies: zika virus (zikv), an emerging flavivirus, is primarily transmitted by infected aedes aegypti mosquitoes, but recent outbreaks have revealed non-vector transmission routes including the unprecedented sexual transmission of an arbovirus. acute zikv infection is mainly asymptomatic or presents as a self-limited disease but also includes severe congenital defects and neurologic disorders. the large proportion of asymptomatic cases, high numbers of returning travelers from zikv-active areas, severe clinical consequences to developing fetuses, the detection of rna in asymptomatic donors during the french polynesia epidemic, and suspected cases of transfusion transmission in brazil led fda to release guidance documents to minimize the risk of zikv transmission via blood/ blood components. study design/method: investigational testing by mini-pool (mp)-nat using the procleix zika virus assay (tma) was implemented on collections from five presumed high-risk us states on / / (fl, ga, sc, ms, al). following revised guidance on / / , testing was extended to all blood donations; conversion from mp-nat to individual donation (id)-nat was implemented in phases and completed on / / . travel history questions were discontinued on / / . confirmatory testing included repeat tma; in addition, rt-pcr, serology and red cell (rbc) tma were performed. estimates of viral loads were performed by end-point tma on plasma and rbcs. results/finding: as of / / , , , donations were tested including , ( %) in , mps. no reactive donations were identified by mp-nat. of the , , id-nat donations, were initial reactive (ir) of which ( %) confirmed positive (cp) by subsequent testing (cp rate of : , ; positive predictive value of %; specificity of . %). five ( %) cp donations were id-nat repeat reactive (rr); ( %) donations were id-nat ir only, igm positive and rna positive in rbcs. cp donors resided in ma, tx, ca, ny, wv and in fl, of which were local transmissions. six donors had traveled to a zikv-active area returning to the us from to days prior to donation. two donors with a travel risk reported clinical symptoms; cp donors ( %) remained asymptomatic. zikv rna was detected in rbcs from all cp index donations with estimated levels varying from less than copies (c)/ml to about Ê c/ml. at the time of writing, the longest period of detection in rbcs was days vs. days in plasma from the same tma-rr donor. zikv rna levels in plasma were obtained from ir and all rr donors, ranging from to c/ml. study design/method: plasma from blood donors were screened by individual donation (id-nat) for the presence of zikv rna with the cobasv r zika test. id-nat samples were repeated in duplicate and further tested by a second nat to confirm infection and estimate vl, and for anti-zikv igm. simulated mps of were prepared by diluting nat plasma : and tested to discriminate id-nat only detectable donations. nat yield samples for which simulated mp and conclusive igm results were available (n ) were sorted into categories corresponding to sequential stages of acute zikv infection: igm-/low vl; igm-/high vl; igm /high vl; igm /low vl. results/finding: of , donations collected april -december , were reactive for zikv rna. igm-index donations had higher vls (mean . x vs . x iu/ml) and higher proportions of simulated mp-detectable results ( % vs %) than igm donations. the distribution by stage of infection was evaluated as the epidemic evolved. over the course of the epidemic, the rates of id-nat only detectible and igm donations increased (table ) . conclusion: this study demonstrates how the viral and immunological profiles of zikv infection in the index donations shifted through the course of the pr epidemic. categorization of index samples into stages of infection is important for blood safety considerations, since infectivity and utility of mp vs id-nat screening likely correlate with vl and serological stages of infection. staging of infections also has implications for diagnostic testing and understanding the durations of zikv viral and immunological markers in blood and persistence of zikv in body fluids and tissues. cobasv r zika is not commercially available for blood screening. data generated under the cobasv r zika ind is preliminary and has not been reviewed by fda. this project has been funded in whole or in part with federal funds detection of zika virus rna in united states blood donations using cobas v r zika on the cobas v r / systems lisa lee pate* , phillip c williamson , michael paul busch , susan rossmann , scott jones , ann butcher , john duncan , jean stanley and susan a galel . roche molecular systems, inc., creative testing solutions, blood systems research institute, gulf coast regional blood center -sugar land, qualtex laboratories background/case studies: in february , the us fda recommended that all blood donations in areas with active zika virus (zikv) transmission be tested with an fda approved nucleic acid test (nat) for zikv rna or treated with an fda approved pathogen reduction technology. the cobasv r zika test was approved under an investigational new drug application on march , and testing of puerto rico donations began on april , . as a precautionary measure some blood centers in the us states also began nat testing for zikv. in august , the fda recommended universal screening of all blood donations. the aim of this study is to describe the detection of zikv rna in blood donations collected in us states between april , -february , using the investigational cobasv r zika for use on the cobas v r / systems. study design/methods: donations were screened with cobasv r zika by individual donation testing. all initial reactive (ir) results were repeated in duplicate. supplemental testing included an alternative nat (altnat) assay which is less sensitive than cobasv r zika and serology testing for anti-zika igm and igg. reactive donors were invited to enroll in follow-up, which included cobasv r zika and serology testing. a donor was considered to be zika confirmed positive if at least one replicate of the repeat testing by cobasv r zika was reactive on index donation or follow-up, reactive by altnat on the index donation, or positive for anti-zika igm on index or follow-up. all ir donations were also retested at a : dilution to simulate mini-pool testing. results/findings: a total of , , blood donations were screened using cobasv r zika. of ir donations, were repeat reactive (rr), non-rr and had no repeat testing. of the rr donations, were positive by altnat; of these were igm positive. all altnat negative donors were igm positive. one donor was alt-nat equivocal and igm negative. of the rr donors that were not igm positive on index, enrolled in follow-up and all seroconverted. of non-rr donations, were altnat negative and is pending supplemental testing. / donors were igm positive on index. donors were igm negative on index; / enrolled in follow-up; remained igm negative and was gm inconclusive. of donations without repeat testing results, met criteria for positive ( was altnat positive, igm negative and altnat negative, igm positive). donation is pending additional testing. altogether, / ir donations met the criteria for true positive on the index donation. / ( %) true positive donations were reactive when retested in a simulated minipool. / were igm positive. conclusion: . % of the , , donations in us states screened for zikv rna were confirmed as true positives. cobas v r zika is not commercially available for blood screening use. using monte carlo simulation luiz amorim* , marc germain , gilles delage , maria esther lopes and yves gr egoire . hemorio, hemaquebec, h ema-qu ebec background/case studies: zika virus was implicated in very large and recent outbreaks, in french polynesia ( ) , and in brazil ( / ), which was followed by outbreaks in south america, central america and caribbean. four probable transfusion transmitted cases were reported in brazil; since % of zika cases are asymptomatic, the actual transfusion rates can be much higher than reported. in this study, we used a monte carlo simulation for risk estimation during the brazilian outbreak. study design/method: the data feeding the monte carlo simulation were collected from january st , through november, th , , from brazil (the whole country) and for rio de janeiro state, one of the outbreak epicenters. the data came from brazilian epidemiologic bulletins and from brazilian blood donation figures. the risk assessment was performed separately for whole blood (wb) donation and for apheresis platelets (ap). the model took into account the following parameters: zika incidence in brazil and in rio; lognormal distribution symptomatic viremia (period: days, with % of the values lower than days); % of infected donors with symptoms lasting days; . donation/donor/year for wb and . for ap. the formula for transfusion risk calculation was: incidence x infectious period x average donation number per donor per year (wb, x/y; aph, z/y) x ( -proportion of refused donors) x ( proportion of discarded donations due to post donation -pd -information). results/finding: the table below shows the results. the estimated risk of transfusion transmitted zika is very important in brazil and in rio de janeiro, where it can attain : , , for apheresis platelets. the severe consequences of zika in vulnerable populations -pregnant women and newborn -indicate that interventions to reduce this unfavorable outcome, such as donor testing and pathogen inactivation, should be considered in brazil dengue (denv) arboviruses in the population are not available in brazil. the objective of this study was to assess the contemporaneous incidence of these agents in donors at large geographically dispersed blood centers located in the southeast and northeast of brazil. study design/method: in the brazil public blood bank system, nat screening for hiv, hcv and hbv is performed on minipools (mp from donations). the residual volume of mp plasma, . - . ml, is routinely discarded. beginning in april each blood center saved $ mps/week for retrospective testing using the triplex zikv, chikv, denv transcription mediated amplification (tma) assay developed by grifols/hologic. mps were shipped to the usa and batch tested at grifols. in the first two weeks (april - ) mp were combined into pools of donations; thereafter mp were tested without additional pooling. to estimate the percent positive donors, the denominator was adjusted to account for the number of donations included in each pool each month and % confidence intervals (ci) calculated using the method developed by biggerstaff. results/finding: the triplex assay performance was shown to have very high sensitivity ( % limit of detection < copies/ml for zikv/chikv/ denvs) and to accurately discriminate each of the arboviruses. testing of the first months of samples is complete for , mp, comprised of , donations collected from april to october , . a total of pools were positive, with detected between april-june . the table summarizes the highest monthly estimated percent positive donors for each virus in each city. months with highest percent postive donors were april or may. at the peak over . % of donors in belo horizonte and rio were viremic for zikv, whereas zika was not evident in donors in recife, but over . % of donors in that city were viremic for chivk during the peak. conclusion: during the latter part of the arbovirus outbreak season in brazil in , zikv, chikv, and denv were being transmitted by mosquitoes to donors with asymptomatic donors donating, indicating that blood recipients in brazil were extensively exposed to viremic blood components. the use of donor mps for surveillance may be one of the most efficient approaches for public health monitoring of the onset and magnitude of arbovirus infections. universal zika screening for blood donors in singapore sally lam* , sze sze chua , mars stone , michael paul busch and ai leen ang . health sciences authority, blood services group, blood systems research institute background/case studies: singapore reported its first locally transmitted zika case on august . the numbers rose rapidly to cases by the end september, with eight clusters (hotspots) of cases island-wide. zika virus (zikv) shares the same mosquito vector, aedes aegypti, as the dengue viruses and can caused microcephaly in unborn fetuses of infected pregnant women and guillan-barr e syndrome, which hastened singapore's blood services group (bsg) to look into securing the safety of blood supply from the zika threat. we aimed to assess the assay performance of usa-fda investigational (ind) procleix zikv nucleic acid technology (nat) assay for universal blood donation screening in singapore to prevent transfusion-transmitted zika infection. study design/method: all blood donations were screened for zika with the procleix zikv nat assay since october . zika nat reactive samples were tested at blood system research institute (bsri) for zika rna in plasma and red cells by pcr and for zika and dengue igm and igg antibodies. a zika confirmed case was defined by the presence of zika rna by pcr and/or zika antibodies. the analytical sensitivity was evaluated using blinded frozen samples consisting of replicates of half log dilutions of the who international standard for zikv and replicates of negative controls prepared by bsri. probit analysis was performed to determine the % and % limits of detection (lod) . clinical performance of the procleix zikv assay was also assessed with local patient samples obtained from institute of infectious disease and epidemiology, singapore and a member blinded zikv reference panel from the usa-fda. results/finding: a total of , donations were screened from october to march , with false positive case and zika confirmed donation detected. alternative zikv pcr tested positive in both the plasma and red cells with an estimated plasma viral load of . x copies/ml. zika igm was negative in the index donation sample but present in the -day post-donation follow up sample.. the donor reported no clinical symptoms. the analytical sensitivity for the procleix zikv assay was determined to be . copies/ml at % lod and . copies/ml at % lod. the procleix zikv assay detected rna in out of patient samples and provided . % agreement to the results of the usa-fda zikv reference material. conclusion: the investigational procleix zikv assay showed good analytical sensitivity and clinical performance, suitable for blood screening of zika infection especially in asymptomatic donor populations. bsg commenced universal zika nat screening by individual donation testing following the zika outbreak with confirmed zika donation (high-titer and seronegative) interdicted, which translates to a risk incidence of in , donations in singapore. background/case studies: a cap/aabb work group suggested that steps be taken to phase in rhdgenotyping for patients with a serologic weak d phenotype. weak d types , and express all the major rhd epitopes and these patients can be managed as rhd-positive, which may lead to a reduction in unnecessary rh immunoglobulin (rhig) administration and conservation of rhd-negative rbcs. study design/method: rhd genotyping was performed on all patient samples with weaker than expected or discrepant rhd typing results, utilizing a commercially available genotyping kit manufactured by immucor (rhd beadchip). initially, testing was performed at a reference lab while the rhd beadchip was validated and implemented at this institution. a serologic weak d phenotype is defined as weak to reactivity on initial gel testing. if genotyping demonstrated weak d types , or , the intent was to manage the patient as rhd-positive. if weak d types , or were not detected, the patient is considered at risk for alloimmunization and treated as rhdnegative. while rhd genotyping results were pending, rhd-negative rbcs were used and if pregnant, the patient was eligible for rhig. results were generally available in to weeks. results/finding: rhdgenotyping was performed on patient samples over months. of these patient samples, ( %) were weak d types or . the remaining samples demonstrated a variety of alleles including known partial d variants (see table) . one patient identified as weak d type required multiple transfusions over the study period, and refused rhd-positive rbcs. the remaining weak d types and patients have not received transfusions at this institution since they were genotyped. four of obstetric weak d types and patients received rhig while genotyping was pending. conclusion: testing and management of patients with serologic weak d phenotypes is not standardized. rhd genotyping may lead to more consistent, personalized patient care and appropriate management of resources. in this month study period serologic weak d patients were identified who could be managed as rhd-positive, however this did not result in withholding any doses of rhig nor conservation of rhd-negative rbcs. genotyping results pertaining to the management of an obstetric patient were discussed with each obstetrician and it is possible this information may impact management of future pregnancies. these outcomes highlight the limitations of current genotyping processes, including long turn-around-time background/case studies: the rh blood group is highly immunogenic and the most clinically significant blood group secondary only to abo. currently, in the united states, blood donors who type rhd-negative by serology undergo weak-d testing to identify some weak and partial states of rhd expression. however, not all rhd expression can be detected serologically. it has been suggested that investigation of serologic rhd-negative blood donors using genotyping methods can more accurately identify units that may lead to alloimmunization in rhd-negative recipients. study design/method: rhd genotyping of all serologic rhd-negative blood donors presenting to our blood donor center was implemented to identify units with altered rhd alleles that should be characterized as rhdpositive. repeat donations were not tested. initial serologic testing of blood donors was performed using fda approved anti-d reagents. when reactivity with all reagents was negative, rhd genotyping was performed using a commercially available genotyping kit manufactured by immucor (rhd beadchip). this assay detects over rhd variant alleles and additional dna sequencing was performed in selected cases. to maximize efficiency samples were batched for testing; testing was generally performed once a month. if an rhd variant known or suspected to be associated with an increased risk of alloimmunization was detected, recipients of previous donations were investigated for evidence of alloimmunization, and all future donations were restricted to rhd-positive recipients. results/finding: over a period of months we tested rhd-negative blood donors. there were ( . %) partial-d, weak d ( . %), and ( . %) del donors. in one donor sample a novel rhd allele was identified through dna sequencing (rhd*ivs - _ deltctc). the phenotype associated with this allele variant is unknown. investigation of previous donations from these donors showed that rhd-negative recipients received rbcs from of these donors. five of these recipients underwent antibody screening after an average follow-up period of months; anti-d was not detected in any sample (see table) . conclusion: serologic testing occasionally fails to identify some rhdpositive donor units, which could place rhd-negative recipients at risk for alloimmunization. dna-based testing can be used to identify donors who have the potential to sensitize rhd-negative individuals. in this limited study period a small number of serologic rhd-negative donors, whose genotype indicated potential to sensitize recipients, were found. however, review of recipient transfusion records indicated that prior exposure to these donors' rbcs did not lead to detectable immunization to date. future potential sensitizing events will be avoided by restricting these units to rhd-positive recipients. grifols diagnostic solutions labs, grifols immunohematology center background/case studies: pregnant women with rhd variants may be candidates for rhig prophylaxis if molecular analysis reveals a genotype associated with possible anti-d formation. proposed testing algorithms advocate molecular characterization of weak d types but if a patient types as rhd-positive, no further action is proposed. women with partial d variants who may also be at risk of anti-d formation have not been included in algorithms proposed to date yet molecular testing may unmask this hidden subpopulation of women who type as d-positive but who may be candidates for rhig prophylaxis. our hospital is in an urban setting in which % of deliveries are to african-american patients. we initiated routine, full-gene rhdsequencing for obstetric patients whose serology demonstrated not only weak d, but also those who were categorized as "d " with reactivity to determine the prevalence of partial d patients in an ethnically-mixed population who may be at risk of anti-d formation. study design/methods: from october to march , we performed routine d typing (neo, immucor) on obstetric specimens followed by rhd sequencing on samples with either a serologic weak d phenotype or anti-d testing strength of using at least antibody. solid phase and manual testing used the series and series reagents. four additional anti-d reagents manufactured by grifols (dg gel anti-d), quotient (anti-d blend), biorad (anti-d (rh ) blend), and ortho (bioclone anti-d) were also used for supplemental testing. rhd sequencing was performed by sanger methodology using routine clinical protocols. results/findings: rhd polymorphisms or variations were identified in all samples. two of ( . %) were d with an rhd gene with only common, known intronic variants that is predicted to produce the "reference" rhd protein (ivs - c, rs ; ivs c, rs ; and ivs a, rs ). two ( . %) were d and heterozygous for two apparently new rhd coding variations which we are confirming by further testing. four ( . %) patients had rhd alleles with known potential to make anti-d (rhd*dol , rhd*dar . , and with weak d type . ). one had weak d type , which has uncertain susceptibility to alloimmunization and one was weak d type , which has not yet been associated with anti-d. interestingly, two ( . %) had variable d expression associated with apparently new alleles, pending ongoing confirmatory testing and cloning. one patient background/case studies: a weak d type is a variant of the rhd protein that comprises an amino acid substitution located in the th transmembrane segment and expresses a reduced amount of the d antigen. this variant is known to be associated with the missense mutation c. g>c which is the first nucleotide of the exon of the rhd gene and thus could be implicated in exon skipping when it is mutated. when performing ngs (next generation sequencing) analysis to fully genotype known patients, we identified an additional variant. study design/method: dna samples were studied by beadchip technology (immucor/bioarray solutions) and ngs using the sureselect human all exon v (agilent) and the nextseq platform. in silico analysis with different bioinformatic tools was used to predict splicing events. furthermore, a functional splicing assay was performed to determine the impact of the nucleotide variations on exon skipping of rhd gene. this study was completed by the comparative modeling between the wild type and the weak type rhd proteins. results/finding: by a targeted analysis of full exome sequencing, we have confirmed the blood group genotype of patients previously characterized by beadchip technology. interestingly, out of carry the c. - c>t intronic variation on the rhd gene, already described and associated with a del allele. among these last patients, one has been previously characterized as rhd weak type carrying the c. g>c (p.gly ala). independently, sanger sequencing on unrelated rhd weak type samples pinpoint to a linkage disequilibrium between c. g>c (exac, maf . ) and the c. - c>t (exac, maf . ). in silico analysis of both mutation located close to the splice acceptor site of the exon does not predict a significant reduction of its strength score. with minigene vectors harboring rhd wildtype exon , mutant rhd c. g>c, mutant rhd c. - c>t and double rhd mutants c. g>c plus c. - c>t, we showed no influence on skipping of exon due to these mutations. comparative modeling of rhd proteins pointed out an additional hydrophobic interaction on the rhd weak type between ala (transmembrane helix ) and val (transmembrane helix ) hampering membrane insertion. conclusion: the c. - c>t variation is always associated in cis with the missense mutation c. g>c on the allele rhd weak type . the c. - c>t can be found alone on the rhd gene as a neutral polymorphism. we assess that these two mutations isolated or combined do not lead to abnormal rhd transcripts. our results clearly demonstrate that the weak d antigen reactivity observed with rhd type red blood cells is due to the substitution of alanine at amino acid position to glycine. topology of jk-weak or jk-negative single-nucleotide missense variants in the kidd protein glenn ramsey*. northwestern university background/case studies: the human urea transporter-b (hut-b) protein carrying the kidd blood group has transmembrane (tm) and tilted ureapore a-helices, a long extracellular connector segment, and cytoplasmic segments at each end. numerous single-nucleotide missense variants (snmvs) weaken or abolish expression of jk a/b antigens determined at p. . we mapped all reported jk-weak or jk-negative (jk-neg) snmvs onto the hut-b structure to explore topological correlates of jk antigen expression. study design/methods: jk*a and jk*b snmvs affecting jk expression were compiled from dbrbc and isbt registries, literature searches and - aabb, isbt and british blood transfusion society meeting abstracts. snmv locations were correlated with the human homolog of the x-ray-crystallographic structure of mammalian ut-b derived for analysis of ut function (levin ej, ) . results/finding: seven snmvs located within amino acid (aa) from the exofacial or internal end of a tm helix are mostly weak variants (table) . all at the exofacial ends (p.a t, p.w r, p.v d) are jk-weak; the two jkneg exceptions p.g e and p.g e are at the internal end of the tm helix bearing jk a/b . four snmvs in the cytoplasmic n-terminal segment are mostly weak variants. in contrast, snmvs within membrane helices are mostly jk-neg variants. three jk-weak snmvs (p.v m, p.e k, p.v i) have been associated with allo-anti-jk a/b to the antigen on their alleles ("weak partial"). six of the jk-neg variants are within aa (p. -p. ) of jk a/b at p. . none of these snmvs are in the long extracellular connector region or the cytoplasmic c-terminal segment. jk-neg variants p.n s and p.s p are adjacent to p. f and p. l which line part of the urea transporter pore. conclusion: in the transporter-structured rhd and rhce proteins, snmvs with weak d, c, c, e or e expression are mostly within the rbc membrane, and non-canonical antigen-negative snmvs are unusual. in the structurally similar kidd hut-b, most jk-weak snmvs are at the ends of the tm helices or in the n-terminal cytoplasmic segment. among jk-neg snmvs, most are in membrane helices. however, whether a variant appears jk-weak or jk-neg may depend on the extent of testing. next-generation sequencing may provide more complete structure-antigen correlations. background/case studies: the kidd-null blood group is most often inherited as a recessive genetic trait due to biallelic mutations in the slc a gene, which encodes the urea transporter ut-b . the kidd-null phenotype is associated with transfusion risk and also is associated with abnormalities in the ability to concentrate urine. the cause of the identical kidd-null phenotype with dominant inheritance [in(jk)] has not yet been defined, though it was first described in . in contrast to recessively inherited kidd-null phenotype, this is not associated with mutations in the slc a gene. the aims of the studies was to identify and characterize the causative gene for dominant kidd-null red blood cell phenotype (injk). jk-weak (bold)/ jk-neg expression n within aa from tm a-helix end v i, a t, w r, w r, g e, g e, v d* cytoplasmic n-terminal v m, g s, e k, l p in membrane tm and urea-pore a-helices r w, r q, g d, i t, a v, l r, a t ‡, a a §, l f, n s, s p, t m / * second nucleotide variant in this allele is synonymous (p.p p). ‡reported as jk-neg but considered jk-weak by isbt. §near splice point. study design/method: we identified several families with dominant inheritance of the kidd-null phenotype in multiple kindreds in spain. we performed whole-genome linkage analysis, exome sequencing, expression (rt-pcr and western) analyses, and urea lysis using patients' cells. in addition, two probands underwent urine concentration tests. results/finding: using molecular approaches, we mapped the affected locus to a mbp region in q . - . with an lod score of . . using deep sequencing, we identified a potential deleterious mutation in the znf gene, which deletes bp resulting in loss of an entire zing finger domain. the identical del -znf mutation is present in all affected individuals, and is absent from all controls tested (n> ). in addition, two adult individuals who are homozygous for the entire haplotype including the deletion within the znf locus, thus completely lacking the common allele, were identified. we also obtained dna from an unrelated injk individual reported from japan. in this individual, there was a similar, though not identical, znf del . none of the other potential genetic variants identified in the spanish kindreds was present in the dna from the injk individual from japan. consistent with the fact that the kidd antigen, encoded by the slc a gene, is a urea transporter that has been associated with renal function, we found that people with the znf del in spain had an inability to concentrate their urine. conclusion: a predicted zinc finger deletion at znf , prevalent in southern spain due to a founder mutation, leads to ut-b dysfunction and underlies the dominantly inherited kidd-null blood phenotype. the phenotype associates subnormal urine concentrating ability. in background/case studies: di-( -ethylhexyl) phthalate (dehp) makes pvc film flexible and useful for blood products. during storage, dehp can leach from the bag film into solution and be metabolized. studies in rodents have suggested that exposure to dehp may be associated with adverse health effects, albeit at high dosages. attempts to find dehp alternatives for blood bags have been difficult due to the rbc membrane-stabilizing effect of dehp. bis( -ethylhexyl) terephthalate (deht) a non-ortho-phthalate is structurally and functionally similar to dehp, but distinct from a metabolic and toxicological standpoint. deht can undergo complete hydrolysis and has an excellent safety profile; it is not classified as a carcinogen, mutagen, reproductive toxicant or endocrine disruptor. the study objective was to evaluate the quality of fresh frozen plasma (ffp) stored in deht containers versus ffp stored in dehp containers at days and year. study design/methods: thirty-six wb units were collected into cpd solution, leukoreduced, centrifuged, and separated into rbc and plasma. abo identical plasma units were pooled together in groups of three. the pools included group a, group o and group ab. each plasma pool was weighed, mixed, sampled, divided into dehp and deht pairs, and frozen at less than - c within hours of collection. in vitro plasma testing (pt, aptt, factor v, factor viii, fibrinogen, protein c, and protein s) was done on day (pool), day , and year of storage. dehp and deht paired plasmas were thawed and tested at the same time. plasticizer concentrations were determined on day , day , and year of ffp storage. dehp and deht and their monoesters were analyzed by liquid chromatography-mass spectrometry. internal standards were deuterated-dehp, mehp, deht and meht. the lower limits of quantification (lloq) were: dehp . ppm; mehp . ppm; deht . ppm; and meht . ppm. results/findings: mean and standard deviation (sd) for key clotting factors and plasticizer results are summarized in the table. there was no statistical difference in any plasma parameter between dehp and deht bags at the same time period. factor viii retained greater than % of its initial value. plasma stored in deht bags had an average plasticizer content % lower than that of the dehp bags. background/case studies: plasma prevents dilutional coagulopathy in trauma victims by replacing coagulation factors and substrates during resuscitation with red blood cells (rbcs) and/or crystalloid solutions. spray-dried plasma (spdp) is lightweight and can be reconstituted in minutes making it ideal for use in combat and pre-hospital settings to rapidly provide plasma in situations where it is impractical to administer fresh frozen plasma (ffp). the spray-drying process preserves coagulation proteins, but high molecular weight multimers (hmwm) of von willebrand factor (vwf) are decreased. the objective of this study was to compare spdp and ffp in reconstituted whole blood (rwb) to test the hypothesis that spdp is not inferior to ffp in facilitating platelet adhesion and thrombus formation. study design/method: under an irb-approved protocol, whole blood from healthy volunteers was collected into sodium citrate and centrifuged at g to separate rbcs from platelet-rich plasma (prp). prp was diluted -fold in pipes-saline with . mm pge and centrifuged at g. the platelet pellet was resuspended in either spdp or ffp and recombined with the packed rbcs to create rwb with hematocrit of - % and , - , platelets/ml. in addition, two rwb pairs were reconstituted with spdp diluted : (spdp %) with plasma from a patient with type vw disease (t vwd). samples were fluorescently labeled with a gpiibiiia-specific antibody and the sample was flowed through a type i collagen-coated microchannel at a shear rate of s - for seconds. still images of adherent platelets and thrombi were captured in order to calculate surface area coverage (sa) along the length of the channel. ratio paired t-test was used to compare sa in samples reconstituted with spdp vs. ffp. the margin of noninferiority was % (spdp/ffp > . ). results/finding: six batches of spdp/ffp were evaluated using subjects. there was no statistical difference between the spdp/ffp pairs (p . ). the mean ratio of spdp/ffp was . with a % ci of . - . . comparing spdp vs. spdp %, there was no difference (median ratio . , range: . - . ) in sa. two-way anova demonstrated that batch did not significantly affect ratio of sa in spdp vs. ffp. conclusion: spdp, despite a decrease of vwf hmwm, was not inferior to ffp in ability to support platelet adhesion and thrombus formation. on average, sa in samples reconstituted with spdp was % greater than in samples reconstituted with ffp. the lower limit of the th % ci is a difference of %, which is less than the a priori determined margin of noninferiority of %. even with % dilution with t vwd plasma, there was no reduction in platelet adhesion and thrombus formation in the spdp rwb samples. these data support the development of in-human studies to evaluate the efficacy and safety of spdp in preventing and reversing trauma-related coagulopathy. spray-dried plasma deficient in high molecular weight multimers of von willebrand factor retains hemostatic properties michael a. meledeo* , qiyong peter liu , grantham c. peltier , ryan c. carney , ashley s. taylor , colby s. mcintosh , james a. bynum and andrew p cap . u.s. army institute of surgical research, velico medical inc background/case studies: restoring coagulation factors is key in acute resuscitation after traumatic hemorrhage, but blood products are frequently unavailable in emergency response due to shelf-life restrictions and storage needs. a single unit spray dried plasma (spdp) process has been developed that produces a long-lived and readily stored product that has a reduction in high molecular weight multimers of von willebrand factor (vwf) and an increase in low molecular weight multimers. vwf is critical in platelet adhesion and thrombus formation. following work demonstrating enhanced function with use of glycine-based reconstitution solutions for spdp, this study examines two different spdp pretreatment conditions. study design/method: the samples were: ( ) ffp; ( ) ffp with mm glycine; ( ) regular spdp without pretreatment (rspdp), rehydrated with glycine-hcl:glycine; ( ) spdp pretreated with glycine-hcl ( mm); and ( ) spdp pretreated with glycine-hcl:glycine ( mm: mm; both pretreated were rehydrated in water). six donor-matched plasmas of each type were tested. vwf activity was measured by ristocetin cofactor assay. fibrin polymerization kinetics were analyzed by turbidimetry. thrombin generation (tg) was observed by thrombogram. chemistry was evaluated by i-stat. residual cell material was quantified by flow cytometry. coagulation properties were measured by thromboelastography (teg) in plasma and reconstructed whole blood ( % hct with platelets/nl from typematched donors). platelet adhesion to collagen under shear was measured by bioflux. results/finding: pretreated spdp showed enhanced vwf activity over rspdp (p < . ). fibrin polymerization density was slightly diminished in rspdp vs. ffp ( . vs. . o.d., p < . ), but tg was unchanged. bicarbonate/base excess were lower in spdp samples vs. ffp (p < . ). residual cellular material (especially platelet-derived) was reduced threefold in rspdp vs. ffp (p < . ) and an additional twofold in pretreated spdps vs. rspdp (p < . ). teg results were unchanged in plasma-only samples; in reconstructed wb there was a reduction in amplitude (clot strength) in all spdp samples vs. ; p < . ). platelet adhesion was equivalent in pretreated spdps and ffp, while rspdp was improved vs. all other samples ( . % surface coverage vs. . - . %, p < . ). conclusion: spdp has a longer shelf life and easier storage requirements than ffp and was equivalent or superior to ffp in most of these in vitro assays. spdp pretreated with glycine solutions was similar to ffp in most assays and showed superior vwf activity and fewer residual cellular materials but inferior support for platelet adhesion to collagen while under flow compared with untreated spdp. clinical significance of these findings is unclear, but overall in vitro outcomes suggest clinical studies are warranted. the interaction between red blood cell transfusion and lung injury: the influence of blood component manufacturing methods mathijs wirtz* , anita tuip-de boer , ruqayyah almizraq , jason p. acker , philip j. norris , jennifer a muszynski and nicole juffermans . academic medical center, university of alberta, canadian blood services, blood systems research institute, nationwide children's hospital background/case studies: red blood cell (rbc) transfusion is associated with acute lung injury, in particular in patients on mechanical ventilation. the causative factor is not known but may include residual cells or extracellular vesicles (evs) . in this study we investigated the functional effect of different manufacturing methods of rbc products on the response of pulmonary cells in an in vitro model of mechanical ventilation. study design/methods: groups of rbc products (whole blood filtered [wbf] , red cell filtered [rcf] , apheresis derived [ad] and whole blood derived [wbd]) were manufactured from donors (blood type a or b). supernatants were prepared after - (fresh) and - days of storage (stored) for measurement of thrombin generation and ev analysis. a type ii alveolar cells were seeded onto flexible membranes and incubated with rbc supernatant. cells were subjected to % stretch using a cellstretcher. control cells were not stretched. after hours, il- and il- production were measured. results/findings: both fresh and stored supernatants from ad products significantly increased pulmonary cell il- and il- production compared to incubation with other rbc products and non-incubated controls, which was further exacerbated by cell stretching. ad products also had significantly increased thrombin generating ability compared to other rbc products, as well as a significantly increased number of rbc-derived evs compared to rcf and wbd products (p< . ) . incubation of stretched cells with stored wbf products resulted in higher il- production compared to other blood products and stretched controls. rcf products did not activate pulmonary cells, had an absence of tg and had low levels of evs compared to other products. conclusion: manufacturing methods markedly influence the interaction of rbc products with lung cells. ad products activate lung cells, which is further aggravated by cell stretching. this may in part be mediated by rbc-background/case studies: investigators previously demonstrated immunosuppressive effects of rbc supernatant on monocytes in vitro, with greater effects seen in response to older units. recent clinical data suggest that rbc manufacturing method may influence immunomodulatory potential, but this has not been directly measured. we used in vitro models to test the hypothesis that rbc supernatants obtained by different manufacturing methods will have differential effects on monocyte function. study design/method: rbc products were manufactured by different methods from individual donors, each: (whole blood filtration [wbf] , red cell filtration [rcf] , apheresis, and whole blood derived [wbd] ). rbc products were stored in sagm (wbf and rcf) or adsol-containing preservative solution (apheresis and wbd). supernatants were obtained after - days (fresh) and - days (expiry). monocytes were co-cultured in media plus % rbc supernatant or media only (control) followed by lps stimulation. experiments were performed in replicates, each with a distinct monocyte donor. comparisons between groups by anova with dunnett's post-test for multiple comparisons. data are mean sd of % of control values. results/finding: exposure to apheresis or wbd rbc supernatants suppressed monocyte lps-induced tnfa production capacity compared to controls (table ) . this was true for fresh units and those at expiry. for monocytes exposed to rbc supernatant alone without lps, interleukin- production was higher after exposure to fresh wbf ( % control, p . ) or wbd at expiry ( % control, p . ). conclusion: manufacturing method and/or storage solution significantly alters immunomodulatory effects of rbc supernatant on monocytes in vitro and may confound analyses of clinical effects of rbc storage duration, particularly within international multi-center studies. a magnetic levitation system to study the impact of donor gender, age and blood storage conditions on red blood cell density profile gozde durmus* , alessandro tocchio , anita howell , kaushik sridhar , jason p. acker and utkan demirci . stanford university, canadian blood services, centre for innovation, background/case studies: the amount of hemolysis in red blood cell units increases as the product ages and has been shown to be lower in female blood donors than in males. it is hypothesized that female donors possess, on average, a younger population of red cells, which results in the lower hemolysis that is observed in the pre-menopausal population. it is also hypothesized that the differences between donor populations are mitigated by lysis of older cells when whole blood units undergo processing steps to produce red cell concentrate (rcc) units. as red blood cells (rbcs) age in circulation, they undergo characteristic changes in density and membrane composition that allows for them to be separated from younger cells. study design/method: our aim is to study the effect of donor factors and method of manufacturing and storing conditions on the average rbc age and density of red cell units. we have recently developed a powerful yet simple and inexpensive magnetic levitation-based platform, which allows realtime, high-resolution imaging and monitoring of various cell populations. this label-free system allows density profiling for individual red blood cells, with an unprecedented resolution of - g/ml. first, to determine the effect of rcc storage on the density profile of rbcs, levitation and single-cell density profiles were measured at , , , , and days. in addition, to determine the effect of donor age and sex on the rbc density profile, blood samples from volunteers with four different age and sex categories (male, - years; male, > years; female, - years; female, > years) were profiled. results/finding: first, we observed that the levitation and density profiles as well as morphology of rbcs within rcc units change significantly during storage. in addition, rbc density was significantly different between young ( . g/ml) and older female donors ( . g/ml) (p < . ). moreover, rbcs from young males ( . g/ml) were significantly less dense compared to rbcs profiled from older female donors ( . g/ml) (p < . ). conclusion: we have developed a magnetic levitation system for the point-of-care, real-time evaluation of rbc and red cell concentrate (rcc) quality. we envision our results might inform decision makers about impact that donor deferral criteria may be having on the quality of red cell concentrates available in the blood banks, for the optimal clinical outcomes. cytokine production of pulmonary cells il- (pg/ml) il- (pg/ml) background/case studies: oxidation reduction potential (orp) or redox is the ratio of activity between oxidizers and reducers. redox imbalance caused by a higher production of reactive oxygen species (ros) and reactive nitrogen species or a decrease in endogenous protective antioxidants results in oxidative stress (os). while os can cause cellular injury and death, it is also important in the regulation of a healthy immune response to injury or disease. in the present study we investigated changes in hemoglobin, free heme, and orp as red blood cells (rbc) age and the effects of red blood cell age on icu patient morbidity and mortality. study design/method: icu patients were enrolled in this prospective observational trial investigating the effect of transfused rbc age on icu patient morbidity and mortality. all rbcs were pre-storage leukoreduced and abo identical. citrated blood samples were collected from each rbc unit prior to issue. the rbc supernatants were tested for free hemoglobin/ heme and orp. the patients were followed prospectively. results/finding: a total of rbc units were transfused. patients and rbc characteristics are shown in the table. significant reductions were detected in orp values over storage duration (p< . ). substantial correlations were also found between orp and free hemoglobin (p< . ) and orp and free heme (p< . ). interestingly, there was a statistically significant difference between the average orp values of the transfused rbc in patients who developed infection with higher orp values measured in rbc units given to patients who developed post-transfusion infections vs (p< . ). no significant differences were observed between orp and patient mortality, hospital/icu days, or thrombosis. also, no correlations were detected between free heme/hemoglobin or rbc age and infection development. conclusion: these data demonstrate that older blood has lower orp values as well as increased free heme/hemoglobin. there were no differences in orp values between the different blood groups once rbc age was controlled for and there were no statistically significant differences in patient mortality associated with orp, free heme/hemoglobin, or rbc age. the decreased orp values observed in the older blood are likely attributable to the "storage lesion". higher transfused rbc orp values were associated with subsequent development of infection, and younger rbcs were found to have higher orp values. thus, this data supports that young/fresher blood may predispose to subsequent development of infection in critically ill patients. further studies are needed. background/case studies: no randomized trials in humans have addressed whether only exposure to red blood cells (rbcs) that have been stored for a long time is associated with harm. we explore the effect on inhospital mortality of transfusing rbcs stored for more than days compared to rbcs stored for days or less. study design/method: data from a multi-national randomized controlled trial were used for this exploratory analysis. the patients were hospitalized adults who required transfusions and were randomly allocated to receive the freshest rbcs in inventory or the oldest (standard issue) rbcs providing a large cohort of patients receiving rbcs with storage durations along the entire rbc storage continuum of to days. using a time dependent variable patient exposure was defined by the maximum storage duration of rbcs received. this was then used to classify individuals on each day of hospitalization into one of three mutually exclusive exposure categories: freshest (exclusively exposed to rbcs less than or equal to days storage duration -reference group), medium age (at least rbc of - days storage), and oldest (at least rbc greater than days storage). the primary outcome was all-cause in-hospital mortality. cause-specific cox regression models of in-hospital death assessed the effect of exposure of rbcs in each category to exclusive exposure to rbcs stored for days or less. the effects of fixed and time-dependent confounders were dealt with through stratification and regression. sensitivity analyses were conducted with a) weekly partition with cut-points every days, and b) a finer partition using cut-points every days. results/finding: , patients receiving , rbcs were included in the analysis. exposure to rbcs stored for more than days was not associated with increased risk of in-hospital death compared with exposure exclusively to the freshest rbc units (stored for days or less) after adjusting for several fixed and time-dependent potential confounders (hr . ; % ci: . , . ; p . ). exposure to blood stored for at most - days yielded a similar hazard ratio (hr . ; % ci: . , . ; p . ). in the sensitivity analyses using weekly partitions, exposure to rbcs stored for greater than days compared to exclusive exposure to rbcs stored days or less was not significant (hr . ; % ci . , . ; p . ). the confidence intervals around the hazard ratios for the other -day intervals all include . similar findings were obtained with partitioning exposure data into day intervals where exposure to rbcs stored for - days was not associated with increased risk of death compared with exclusive exposure to rbcs stored for - days (hr . ; % ci . , . ; p . ). the confidence intervals around the hazard ratios for the other -day intervals all include . conclusion: individuals exposed to rbcs stored for more than days were not at increased risk of in-hospital death compared to individuals exposed exclusively to rbcs stored for days or less. transfusion of anaerobically stored red blood cells improves recovery in experimental rat hemorrhagic shock model alexander williams* , cynthia walser , tatsuro yoshida , andrew dunham and pedro cabrales . university of california san diego, new health sciences inc. background/case studies: hemorrhagic shock (hs) severely decreases oxygen (o ) delivery and induces cardiovascular collapse. in parallel to controlling the hemorrhage, clinicians respond by infusing large volumes of red blood cells (rbcs) to restore blood volume, o carrying capacity, and hemodynamic stability. the quality of the transfused rbcs determines the recovery from hs, and extent of clinical sequelae prompted by the hs. this study compares the ability to recover from hs with conventionally stored rbcs, anaerobically (o saturation < %) stored rbcs, or anaerobic/hypercapnic (o saturation < % and pco (@ c) $ mmhg) stored rbcs. study design/method: packed red blood cells (prbcs) stored in as- after leukorfiltration were created from donor sprague-dawley rats. prbc units were randomly stored under either ) conventional; ) anaerobic; or ) anaerobic/hypercapnic conditions. rats ( - g) were hemorrhaged to % of blood volume, held in hypovolemia for minutes, and resuscitated to recover blood pressure to % pre-hemorrhage with prbc stored for either or weeks. systemic hemodynamics, cardiac function, and blood gas parameters were monitored during shock and resuscitation; and vital organ inflammation, oxygenation, and function were evaluated post resuscitation. data were analyzed using two-way anova, followed by the appropriate post hoc analyses. ( %) neg patient showed short term response and ( %) patients showed progressive disease. at the neg group standard eval ( %) patient showed response and ( %) had progressive disease. ( %) neg patient had long term response compared to ( %) pos patients. at the pos short term eval ( %) patients showed response and ( %) patients had progressive disease. at the pos group standard eval, ( %) patients showed response and ( %) patients had progressive disease. overall, ( %) pos patients responded compared to ( %) neg. conclusion: there is a trend in lower response rate in patients with negative antibody screens compared to positive controls. these findings suggest that an anti-cd neutralizing substance could play a role in treatment response. alternatively, reduced cd expression may also contribute. the low response rates seen in both groups may result from biased selection. the need for repeat t&s and presumed repeat transfusions may be preselecting patients with more aggressive disease. also, only a small number of patients were suitable for review. a larger prospective study that controls for such variables is needed. a review of blood utilized during provider-activated and critical administration threshold-triggered massive transfusion events patrick ramos* and john hess. division of transfusion medicine, harborview medical center background/case studies: traditional definitions of massive transfusions -e.g., the transfusion of ten or more units of red blood cells (rbcs) in a -hour period -are limited in prospectively identifying patients requiring massive transfusions, excluded patients who may not survive long enough to meet criteria, or ignored the acuity of the event. to address these issues, a level i trauma center adopted the critical administration threshold (cat) as an additional indication for activating its massive transfusion protocol (mtp). this study reviewed blood utilized during massive transfusion events based upon whether the mtp was provider-activated versus cat-triggered. study design/method: all massive transfusion events between january and april were reviewed to identify the start time, termination time, number of components transfused, and the start time of each component transfused. the transfusion of three or more blood components in an hour defined cat. a massive transfusion was any event in which the concern for hemorrhagic shock either necessitated a provider to activate the mtp or blood components were transfused at a rate that met cat criteria. the massive transfusion start time is based on either the time the provider activated the mtp or the time the first blood component was transfused, whichever came first. unless the patient expired first, the termination of the massive transfusion event was determined by identifying the point in time in which the patient went three or more hours without the transfusion of any additional blood components. this information was tabulated to determine the monthly number of provider-activated mtps, cat-triggered mtps, and average blood component transfused per massive transfusion. conclusion: blood utilization is lower within the cat-triggered mtps even though it outnumbered provider-activated mtps. however, the mode for both groups suggests that most massive transfusion require less blood components than the average rate. using the mode provides an approximate % replacement of blood volume. this should be enough to counter the early signs and symptoms of hemorrhagic shock. though this study did not review the appropriateness of provider-activated mtps, using cat as an indicator ensures clinicians are prepared for a potential massive transfusion. further investigation is needed to determine the factors contributing to the downward trend of the average blood components transfused. the mode would suggest optimistically that patients are being stabilized faster and resuscitated more efficiently. if this is the case, defining massive transfusion should include the rate of components transfused in addition to the total volume transfused. the long term storage effect of . m dithiothreitol on red cell antigen integrity in reagent red blood cells heike carrel* , laurie sutor , , germ an leparc , marjorie doty and william crews . carter bloodcare, ut southwestern medical center, oneblood background/case studies: anti-cd drugs, such as daratumumab, pose a problem for the transfusion service. they may cause a number of false positives, including positive direct antiglobulin tests (dat), indirect antiglobulin tests (iat), and panreactivity in eluates. such results can prolong compatibility testing and delay delivery of blood products for patients. treating reagent red cells (rrbcs) with . m dithiothreitol (dtt) removes drug interference due to daratumumab and allows for the detection of underlying alloantibodies. this study aimed to investigate the effect of dtt-treatment on rrbc antigen integrity over a day period. study design/method: twelve aliquots of human plasma, each containing an antibody of a single, known specificity (anti-d, -c, -e, -c, -e, -m, -s, -s, -fy a , -fy b , -jk a , and -jk b ), were tested against untreated and . m dtttreated rrbcs (immucor panoscreen i, ii, iii; dtt from acros organics). dtt treatment of rrbcs was performed using the methodology described in the aabb technical manual ( th edition). each of the plasma aliquots was further separated into aliquots and stored at - c until day of use. fresh aliquots were thawed each day to avoid unintended antibody integrity degradation. a polyethylene glycol (immucor) enhancement technique was used and reactions were read at the iat phase. hemolysis, if present, was observed in the diluent each day prior to mixing the cell suspension and given a grade based on the haemonetics color comparator chart. serological antibody reaction strengths were observed and documented each day. ( ) a monthly breakdown for both groups also displayed a downward trend in the average use of blood components. results/finding: there was noticeably more hemolysis with the dtttreated cells over time compared to the untreated cells. red cell antigens remained serologically detectable on the dtt-treated cells throughout the study, despite a greater degree of observed hemolysis. there was minimal difference in reactivity strength between untreated and dtt-treated cells for antigens not affected by dtt. in most instances, the dtt-treated cells reacted slightly more strongly. none of the antibodies produced reactivity strengths of less than with the untreated or dtt-treated cells during the study. conclusion: long term storage of . m dtt-treated rrbcs does not compromise antigen integrity. advance dtt-treatment and storage of a large aliquot of rrbcs may serve to increase efficiency in the transfusion service. background/case studies: monocyte monolayer assay (mma) is a cellular bioassay used to evaluate the hemolytic significance of blood group antibodies and aid in the selection of rbcs for alloimmunized patients. the requirement for fresh auto/allogenic monocytes for mma is highly restrictive due to tedious processing of fresh peripheral blood (pb). our previous study described processing and cryopreservation of buffy-coat (bc) derived and fresh pb-monocytes for mma assay. the aim was to evaluate the functional properties of cryopreserved bc-monocytes as substitute for fresh pbmonocytes in mma in evaluation of previously reported clinically significant rbc alloantibodies. study design/methods: peripheral blood mononuclear cells (pbmcs) were isolated from buffy-coats (histopaque- ), pooled, suspended in cryopreservation media ( % dmso; : ) and stored in liquid nitrogen. pbmc membrane integrity post-thaw was determined by trypan blue exclusion. pbmcs were cultured on poly-l-lysine-treated coverslips ( c, % co , h) and monocyte monolayers incubated with fresh or cryopreserved antigen positive (o ) rbcs sensitized with either anti-d (positive control), anti-scianna- (sc ) or anti-anwj or lipopolysaccharide stimulated for h. aliquots of the sensitized rbcs were tested for opsonization by indirect antiglobulin test (iat). phagocytosis index (pi) was determined microscopically as the number of fully phagocytosed rbcs/ monocytes. supernatants were analyzed for cytokines using luminex technique. results/findings: cryopreserved pbmcs showed . % viability postthaw. we report no significant difference in phagocytosis of anti-d sensitized rbcs by cryopreserved monocytes vs fresh monocytes. we show a significant increase in tnf-a, il- b, il- , il- , mip-a (p < . ), mip-b and gro (p < . ) secretion from cryopreserved bc monocytes vs both fresh bc and pb-monocytes. sc -and anwj-sensitised rbcs resulted in a pi of . % and . . % respectively vs anti-d sensitized rbcs (pi: . %). a weak ( ) reactivity by iat was observed for anti-anwj sensitized rbcs while anti-d sensitized rbcs resulted in iat reactivity. these results correlated with previously reported results for clinical significance and mma when using freshly obtained autologous or healthy donor monocytes. conclusion: this study shows that cryopreservation preserved monocyte viability and phagocytosis function for mma. as previously reported with fresh monocytes mma assay, the two alloantibodies tested with cryopreserved bc monocytes were shown to have a phagocytic index of clinical significance (pi> %). the use of cryopreserved bc-monocytes has the ability we describe antigen typing discrepancies in patients, involving antigens (c, jk a , s), revealed when serologic results differed from the phenotype predicted by dna testing. all patients had - positive dat with anti-igg and warm autoantibodies identified in the plasma. investigation of the antigen typing discrepancies showed both false negative and false positive results using monoclonal reagents. study design/method: standard tube hemagglutination methods were used for antigen typing. rbcs were treated with edta glycine-acid (ega) using gamma ega kit. genomic dna was isolated from wbcs and hea precisetype performed. results/finding: the rbcs of patients and typed c-on initial testing with immucor gamma-clone anti-c, but were predicted c by hea precise-type. ega-treated rbcs gave reactions with the same anti-c reagent. patient rbcs gave variable reactivity (vw- ) with bio-rad seraclone and ortho bioclone anti-c. patient rbcs gave reactivity with all anti-c reagents when incubated for the maximum incubation time allowed. patient rbcs were jk(a ) with immucor gammaclone anti-jk a , which the manufacturer states is suitable for testing dat rbcs, but predicted jk(a-) by hea. ega-treated rbcs tested jk(a-) with the same reagent. rbcs from patients and tested s with bio-rad seraclone anti-s ( - ), but predicted s-by hea. further testing with immucor gammaclone anti-s showed rbcs from both patients were s-. ega-treated rbcs from both were non-reactive with both anti-s reagents. conclusion: commercial monoclonal reagents are valuable resources, especially when phenotyping dat rbcs but not all manufacturers include reagent limitations regarding testing of dat rbcs. we describe cases of false negative tests with monoclonal anti-c due to antigen blocking by igg, and cases with false positive tests with anti-s (n ) and anti-jk a (n ) typing. false positive tests would potentially be anticipated, but false negative results due to antigen blocking are unexpected. extended incubation as indicated in the reagent insert may reveal weak reactivity when antigen blocking is involved. results concordant with dna testing were obtained with ega-treated rbcs, but it is generally accepted that this is not necessary when using a direct-agglutinating monoclonal reagent. these cases caution the potential for both false negative and false positive results for samples with - positive dat and supports testing to dissociate igg from rbcs strongly dat before antigen typing. in addition, this report highlights the benefits of dna testing as part of the routine reference laboratory workup. background/case studies: sensitization to antigens expressed on transfused cells, by triggering premature antibody-mediated clearance, diminishes the therapeutic effectiveness of transfusion and may also lead to serious delayed hemolytic transfusion reactions. accepted us clinical practice, while providing that sensitized patients receive only cells lacking "offending" antigens, nevertheless ensures continued alloexposure, and thus possible sensitization, to additional antigens, thereby complicating patient management. to mitigate sensitization risk, especially in an era of increasingly cost-conscious procurement, a quantitative assessment of the immunogenicity of specific antigens will be desirable. giblett, long ago, introduced a relative scale relating the rbc antigen immunogenicities to (an assumed) immunogenicity of "k" (http://bit.ly/ opqfew ). here, we show that an absolute estimate of immunogenicities may be extracted directly from observed antibody counts provided these are properly normalized to the fraction of recipients at risk (namely those lacking a specific antigen) and the expected fraction of donors expressing that antigen. study design/method: we define immunogenicity, or sensitization risk, r, for any antigen ("ag") of interest, as the conditional probability of alloantibody ("ab") formation, given allo-exposure to ag, i.e. r : prob(ab|al-loexp), so that prob(ab) prob(ab|alloexp)*prob(alloexp) and r ; rewriting prob(alloexp) prob(recipient, "r", lacks ag)*prob(donor , "d" has ag); and estimating prob(ab) nab/nr, nab denoting the number of ab in nr recipients, we obtain: nab/(nr*prob(r lacks ag)) r * prob(d has ag), the left-hand side representing the observed sensitized fraction, u, i.e. the number of observed ab in relation to the number of recipients at risk. conclusion: several antigens, though corresponding antibodies may be rare (e.g. "jsa", "e", "u"), nevertheless are highly immunogenic, requiring only a single exposure (on average) for sensitization; in contrast, others (on average) will require many exposures and thus pose a relatively low risk. in conjunction with patient genotypes, our r -scale will facilitate the selection of patient-specific cells so as to minimize the risk of (proliferating) alloimmunization even when perfectly matched cells are not available. our approach may be readily extended to additional rbc antigens and other antigen systems. background/case studies: aabb and fda require a month deferral of donors with a tattoo applied using non-sterile needles or reusable ink. we review state regulations to ascertain if tattoo establishments are licensed and required to use sterile or single-use needles and single-use ink. we recently added two large states in which we collect blood to the acceptable states list (asl). we compared the rates of donors deferred before and after the addition of these states to determine potential donor gain with changes in state tattoo licensing regulations. study design/method: we analyzed allogeneic interview responses to the screening question, "in the past months have you had a tattoo?" and if 'yes', whether the tattoo was applied by a state regulated entity. blood centers in states were selected for the analysis before and after state tattoo regulation. in state a, a comparison period of similar months before ( / - / ) and months after ( / - / ) was selected; for state b, a similar months before ( / - / ) and months after ( / - / ) was selected. frequency and rate of responses were compared in before and after periods. among those who responded to having a tattoo in a regulated state, donations were reviewed for presence of infectious disease markers including hiv, hbv and hcv. results/finding: a higher proportion of donors presenting to give blood admitted to having a recent (< months) tattoo in the post period in both states. this increase occurred immediately following the addition of states a and b to the asl (data not shown). among those who responded yes to having a tattoo, in states a and b respectively, there was a -and -fold increase in accepted donors (table) . the absolute number of accepted donors with tattoos increased from to (state a) and to , (state b), which annualized, represents a potential gain of , (state a) and , (state b) additional donations. all donors who had a tattoo in regulated states (asl) tested negative for hiv, hbv and hcv. conclusion: to counter rising numbers of ineligible donors resulting from recently added deferrals, we considered recovery of donors deferred for tattoos as a way to enhance our donor base. the immediate rise in the number of donors reporting a tattoo following the addition of the states may reflect a decline in self-deferrals based on having had a recent tattoo. we demonstrated an increase in the potential number of donations without compromising safety. background/case studies: transgender donors represent a small fraction of blood donors. determining their eligibility to donate has been challenging for blood centers. to assess behavioral risk, the donor is required to answer gender specific questions. the same is true when assessing trali risk where the donor is asked about a history of prior pregnancies. prior to the implementation of the fda's final rule, blood centers asked donors for their birth gender and determined eligibility based on that gender. if the donor changed their gender they were asked to answer both the male and female questions. the final rule now allows blood centers to accept the donor's stated gender and to determine eligibility based on that gender. in order to assess the risk of failing to ask a transgender male donor (birth gender female) the pregnancy question, a review was done to determine the number of transgender males who were actively donating with a large blood center. and tracked. donors were contacted to resolve any descrepancies. donors who had changed their gender from female to male and who had answered yes to prior pregnancies were identified. hla antibody test results were reviewed for these donors to see if they had been tested and whether they had tested positive or negative. results/finding: from - , there were donors identified who had changed their gender from their birth gender; female donors changed their gender to male and male donors changed their gender to female. there were ( %) transgender male donors, birth gender female, who had answered yes to the pregnancy question at one of their donations. three of these donors were apheresis donors who had been tested for hla antibodies. one tested positive and the other two tested negative for hla antibodies. the four other donors were whole blood donors and had not been tested. an hla test was added to these donors' records so that the test could be performed the next time they presented to donate. conclusion: transgender male donors may have had prior pregnancies and are also choosing to become pregnant after having transitioned from female to male. six percent of transgender males that we identified reported a prior history of pregnancy. at our center, when a donor requests a gender change from female to male, an hla test is requested for the next donation. first time donors are qualified based on their stated gender so transgender donors with a history of pregnancy will not be identified unless they volunteer this information. consideration should be given to using educational materials to prompt the donor to reveal a history of pregnancy at the time of donation so that hla antibody testing can be performed. effect of variable volume scale introduction in a large multi-site blood center ralph r vassallo*, marjorie d bravo and hany kamel. blood systems, inc. background/case studies: regulations allow whole blood donation [wbd] of up to . ml/kg or % of estimated blood volume [ebv] . traditional measuring/mixing devices are set to halt blood flow at fixed volumes which, with testing samples, are consistently below the % limit. variable volume scales [vvs] can be programmed to vary unit volume (up to ml) by donor ebv. this maximizes transfusable rbcs and plasma and recovered plasma [rp] volume. rp from wbds is a small but important source of derivatives and blood center cost recovery. we report the effect of introducing the hemoflow vvs on donor reaction rates and rp volume in a large blood center. compared to previous fixed settings, variable collection volumes were expected to decrease by ml at ebvs < . l in donors ! yo, but increase by - ml for all others. study design/method: donor vasovagal reaction [vvr] rates (prefaints, prolonged/offsite reactions, and loss of consciousness [loc]) for successful wbds were obtained from the center's hemovigilance database for the mos. before a mo. phased implementation of the vvs, and the subsequent mos. multivariable analysis [mva] by -mo. periods was performed in a model incorporating donor sex, age, first-time [ftd] vs. repeat status, ebv and donation site. both the volume and number of units of plasma sent for fractionation were available for the same time periods from the blood center's data warehouse. results/finding: compared to the baseline period, a significant increase in prefaint reaction rates were noted in pre-implementation (impl) periods & , continued during impl and post-impl periods & , returning to the baseline rate in post-impl periods & (table) . more severe reactions showed an increasing trend that only became significant in post-impl periods & . the mva showed the vvs as independent factor contributing to the increased prefaint and more severe reactions. however, its contribution, as measured by odds ratios, was consistently lower than those exerted by known donor determinants of reaction rates: young age, low ebv, ftd status and collection site (not shown). plasma unit volume increased an average of . ml during post-impl periods & from the temporally matched baseline & pre-impl period . conclusion: following an initial increase in mild vvrs during and immediately after implementation of the vvs, vvr rates fell back to baseline, suggestive of transient staff distraction from usual donor care, or a minor effect of increased blood loss with a superimposed improvement trend. the subsequent increase in prolonged/offsite reactions and loc after prefaint reactions had already returned to baseline suggests that staff training, work load, donor compliance with mitigation strategies and other determinants of donor reactions have a far greater effect than the small additional blood loss due to the vvs. small but significant increments in rp volume improve derivative availability and offset the cost of the vvs. comparison of vasovagal and citrate reaction rates in donors according to type of apheresis procedure pierre robillard* and yves gr egoire . hema-quebec, h ema-qu ebec background/case studies: apheresis procedures expose donors to various volumes of citrate depending upon type and length of procedure and type of machine used. citrate reaction (cr) results from various degrees of hypocalcemia in donors. blood volumes taken from donors vary according to type of procedure and use of volume replacement. loss of blood volume is in part responsible for the occurrence of vasovagal reactions (vvr). this analysis was conducted to estimate the incidence of cr and vvr according to various types of apheresis procedures performed at our blood center. (yfv) were reported in some brazilian states -rio de janeiro, sao paulo, minas gerais and espírito santo, mainly. the vectors of those cases were mosquitoes from the haemagogus and sabethes genders, whose habitat is the tropical forests. since many brazilian urban areas are very close to rain forests, there is an outbreak risk in those areas, where the infection is transmitted by the aedes mosquitoes. in order to minimize this risk, rio de janeiro health authorities decided to promote a mass vaccination in late march, . the vaccine is produced with live and attenuated yfv, which can circulate for at least weeks after vaccination. in some individuals, the vaccine can elicit viscerotropic effects and sometimes severe diseases. due to that, brazilian blood regulation authority established a week deferral period after yfv vaccination. this action could dramatically affect the availability of blood donors. this study shows the measures taken by rio de janeiro blood center to circumvent this risk and attract more donors. study design/method: the strategy consisted in offering the population, at a single place -the blood center -the possibility to donate blood and, immediately after donation, to get vaccinated against yfv. there were no financial advantages to the donors, since yfv vaccine is completely free of charge for any brazilian citizen. the vaccine was administrated by trained nurses, in an office close to the donors session. if, for any reason, the prospective donors were not able to donate, the vaccine was also offered to them, provide there were no contraindications. the blood center annnounced just before the mass vacination campaign launching that it would vaccinate people who came to the blood center to donate blood. if, for any reason, the prospective donors were not able to donate, the vaccine was also offered to them, provide there were no contraindications. results/finding: during the five days of campaign, we received , blood donors candidates; from those, , were accepted as a blood donor, after medical interview. the deferral rate was . %. at the same period of the year , there were , prospective donors, and blood donations. the deferral rate was . %. the "get vaccinated against yfv . . .but give blood before" campaign was able to attract, in a five day period, , additional donors, compared to same dates. that represents a . % increase in the number of blood donations, without deferral rate increment. there was a slight increase in the proportion of first-time donors, from . % in to . % in . conclusion: the strategy was more than successful, and it allowed the blood center to build a blood inventory large enough to avoid risks of shortage due to mass vaccination against yfv. dose loss which must be accommodated when collecting plt donations to ensure the us plt dose of ! . x is met. currently, triple set kits for pr are only approved in europe. plt loss, and adjusted apheresis targeting parameters may impact split rate (sr) or products per apheresis procedure. inventory suitable for pr without impacting us blood center srs warrants evaluation and optimization. study design/method: , apheresis collections from centers with different srs were analyzed. a baseline sr for conventional pc was calculated assuming i) a minimum dose (allowing for production loss) of . x for single (s), . x for double (d), and . x for triple (t) conventional pcs, ii) concentration and volume requirements from apheresis device manufacturer were used. for each collection, dose, volume, and concentration were assessed for pr kit compatibility, based on storage medium (pas or % plasma) assuming i) a minimum dose (allowing for production loss) of . x for s and . x for d for pr units, ii) removing small quantities from units with excess volume or dose to meet pr specs., iii) if all or part of an out of parameter d or t collection could be divided into one or more kits for pr, eligible parts undergo pr, and the remainder treated conventionally, iv) collections unsuitable for pr specs. or would decrease sr if treated would be counted as conventional pcs. results/finding: conclusion: blood centers today can adopt pr for a significant percent of their current supply (as high as %) without affecting their sr. compatibly increases further by dividing t and large d donations. percent achievable depends on their current s, d, t proportion of collections and practices. changes to d and t collection parameters, optimized donation and counting accuracy, and volume reduction will improve pr compatibility further. individual analysis is warranted for each blood center. rbc rbc plt/p plt plt/rbc/p plt/rbc plt plt/rbc plt/p #donations citrate exposure (mls) - study design/method: a randomized ( : ), placebo-controlled, single blind, subject, single-site study of ascending microdoses of autologous (apheresis-derived) thrombosomes was conducted. subjects were divided into cohorts, receiving increasing doses, ranging from / , - / of the lowest effective dose found in the above rabbit model. cohorts and received the / th dose, but cohort received two / th doses one hour apart. the primary end points were safety and tolerability. subjects were monitored in-hospital for hrs post infusion and followed for up to days for adverse events, global neurological assessments, abbreviated physical exams, and laboratory tests. results/findings: there were no serious adverse events (saes) or subject discontinuation post-infusion due to a significant decrease in platelet count from baseline. there were a total of aes: were treatment emergent (teae), of which were treatment-related ( thrombosomes and control). all teaes were mild or moderate in severity. in cohorts and , / thrombosomes subjects had treatment related adverse events. one cohort subject developed an upper respiratory infection and elevated wbcs within hours post infusion, which resolved by hours, and an elevated d-dimer at hours post infusion, which resolved by day . this subject also had an elevation of prothrombin fragment at baseline, which increased post transfusion and peaked at hours with resolution by day . one cohort subject developed non-specific t-wave changes at and hours following her nd infusion that resolved by day without clinical symptoms. troponin levels and echo stress tests were normal. ekgs were considered possibly a normal variant or related to placement of the ekg leads. another cohort subject developed an igg platelet autoantibody on days - , which was undetectable on days - ; there was no change in platelet counts. the thrombosomes autoantibody assay was positive at baseline, days - , and negative on days - . background/case studies: cryopreservation of platelets (plts) could extend the shelf life from - days to over two years. cryopreserved plts (cryoplts) appear to have a greater in vivo hemostatic effect than liquidstored plts. plts have been shown to require protein synthesis capabilities for certain functions such as clot signaling and immune responses. this study was designed to assess whether reconstituted cryo-plts carry out protein synthesis upon thawing and short term storage. study design/methods: apheresis plts were cryopreserved with % dmso and stored at c. after thawing, the unit was reconstituted in thawed ffp spiked with either lm puromycin (pm) or nm biotinlabeled pm. plts were stored at room-temperature with agitation. samples were drawn immediately after reconstitution as well as after , and hours to assess pm incorporation as a measure of protein synthesis, and for in vitro assays to determine platelet activation by cd p binding, phosphatidylserine exposure by annexin-v binding and microvesicle count in the supernatant. plt microvesicles (pmv) were prepared from the supernatant by ultracentrifugation. plts and pmv were lysed in a triton x- containing buffer and qualitative proteomics was performed on samples following affinity-purification with streptavidin beads. results/findings: in vitro parameters of reconstituted and subsequently stored platelets were in line with previously published results, with high surface levels of cd p and phosphatidylserine. pmvs were generated during cryopreservation and the count increased by -fold during hour storage. immunoblot analyses of the plts showed a -and -fold increase in pm incorporation after and hours of storage, respectively. massspectrometry revealed unique proteins that were synthesized after hours of storage, which was confirmed for gtpase and gtpase-regulatory proteins rac , rap and rhogdi by immunoblot analyses. analyses of the pmv translatome also revealed the presence of synthesized proteins; however, these did not change throughout storage. this finding suggests that a defined panel of proteins is packaged into pmvs upon freezing and thawing. additionally, the pmv translatome profile comprised a smaller subset of synthesized proteins compared to the cryo-plt translatome, including the proteins rac , rap and rhogdi. conclusion: this study has demonstrated that cryo-plts can synthesize proteins upon reconstitution in ffp and subsequent storage. discovery of a subset of these proteins in the pmv suggests their encapsulation, possibly in a selective manner. this observation provides novel insights into the capacity for protein synthesis in cryo-plts and the potential regulation of protein packaging into pmv. background/case studies: in , the authors' hospital-based blood bank received variances from the fda and aabb for the use of cold stored platelets (csps) with a shelf life of days. these group a csps, stored in a refrigerator in the emergency department, were used to support the trauma program for use in massively bleeding patients. the placement of the csps on the air ambulances, stored in coolers, was the next logical step in providing platelet therapy sooner to these patients. study design/methods: eight double unit csps were collected using the trima accelv r . two double csps were pathogen reduced using the inter-ceptv r pathogen reduction system. half of the csp pairs were subjected to flat storage in a refrigerator; the other half were loaded into a credov r - cooler with units of ffp, units of rbcs, and unit of whole blood. three to ml of platelets were collected via syringe from each unit at min (before storage in cooler or refrigerator) and after . , , , , and hours of storage for functional validation of platelets. the platelet count, agonists (thrombin receptor agonist peptide (trap), adenosine diphosphate (adp) and collagen stimulated platelets aggregation), non-activated and agonists activated platelet surface expression of phosphatidylserine (ps, annexin-v binding), p-selectin, fibrinogen receptor (pac- binding) were measured by coulter counter, channel aggregometer, and digital flow cytometer. paired wilcoxon rank sum tests were used to analyze differences in degradation rates with p< . deemed significant. conclusion: platelets, including pathogen reduced, stored in an oxygendeprived environment, (cooler), do not lose functional capabilities when compared to those platelets stored in a refrigerator with adequate oxygen for hours. therefore, cold stored platelets transported in a cooler are a viable option for providing timelier platelet intervention for severely injured patients prior to hospital arrival. c -a h molecular sieving: beyond genotyping ghazala hashmi , reinhard klemm and michael seul* , . biomolecular analytics, immunoinformatica background/case studies: more than a decade after its commercial introduction (hashmi http://bit.ly/ ohlehe), blood group genotyping, though available in several formats, has remained a tool for special tasks, e.g. the profiling of difficult patient samples or the identification of rare antigen combinations, while serology has remained the tool of choice for routine antigen typing. here, we introduce molecular sieving as an alternative to the current approach of managing special donor unit inventories. this novel process for dna analysis combines the "multiplexing" of markers offered by existing genotyping methods with the pooling of multiple samples in manner permitting the step -wise refinement of candidate sets by molecular attribute patterns. study design/method: molecular sieving is a special format of leansequencing, a proprietary process that permits the simultaneous analysis of up to four samples for alleles encoding rbc antigens in mns,rhce, lu,kel,fy,jk, di,yt,do and co, including the identification of rhce alleles. molecular sieving extends these capabilities to the analysis of pools to attain large scale. thus, in one format of the process, * * samples are accommodated in a single run. following the completion of the sieving step, candidates may be directly assigned to requests, or may be selected to enrich a subsequent profiling step for samples with rare or otherwise desirable attributes. here, molecularsieving was used to identify suitable donor units for sensitized sickle cell anemia ("sca") patients (tb in cas-tro , http://bit.ly/ oplxhr, excluding le and e(variant) and assuming request per patient), presenting with up to allo-antibodies ("ab") in multiple combinations. proprietary "greedy" algorithms were invoked to optimally pair candidate units with requests. results/finding: sieving of only = plate holding * candidate units from actual black donors, followed by profiling of samples selected to enrich for "e neg" and "c neg" and "c-e-k-fya neg", produced assignments for of requests ( . %), as indicated by colors, and shown in the row "assigned" below: thus, the number of assignments substantially exceeded the number of wells processed. moreover, the remaining pooled samples produced additional assignments to a second set of requests, for a total of assignments from only wells. in another scenario, sieving of a full plate of * samples, produced $ assignments for two successive batches of requests from sca patients, a yield exceeding . x. sieving alone typically fills - % of requests of moderate complexity ( ab). conclusion: molecularsieving, by widening the "funnel" while focusing the search for candidate donor units, attains a new level of efficiency in procuring suitable units for patients with hemoglobinopathies. molecular sieving for identifying red blood cells with special phenotype attributes kristopher fernandez , monica kalvelage , ghazala hashmi* and michael seul . biomolecular analytics, lifeshare blood centers background/case studies: providing transfusion support to patients with sickle cell anemia and other hemoglobinopathies remains a challenging logistical task that must accommodate pre-existing allo-antibodies in multiple combinations preferably while minimizing the risk of (continued) transfusion-related sensitization. the allelic diversity of the predominantly black patient population, especially at the rh locus which encodes a variety of "partial" phenotypes further complicates the problem (chou http://bit. ly/ ppvfeq ). study design/method: molecular sieving is a proprietary new process that, in order to rapidly probe candidate donor units in large numbers for multiple phenotype patterns, permits the analysis of pools and pools of pools of samples for a multiplicity of alleles (including at the rhce locus) that encode mns, rh, lu, kel, fy, jk, di, yt, do and co antigens. based on sieving, samples may be grouped by molecular attribute patterns ranging from single "ag " (e.g. e ,c ,e ,c ) to specific combinations of "ag " (e.g. c e k fya and c e jsa ) or combinations of alleles such as those encoding partial rh phenotypes. sieving, optionally, may be followed by profiling of samples selected for desirable attribute patterns. genomic dna from (predominantly) black donors, independently genotyped by one of two commercial methods were provided by lbc. at bmx, pools were prepared prior to amplification, and analyzed by a novel leansequencing method. results/finding: all pool genotypes were consistent with available individual sample genotypes. antigen patterns of particular interest included two groups, namely: several for which pools were homozygous and certain others t for which pools were heterozygous. illustrative of the former pattern type are these: appropriate pool queries revealed that sieving alone identified, among the c samples, that were also v and vs and, among the e samples, that were also negative for any partial_e phenotype. illustrative of the latter pattern type are pools identified as heterozygous ("het") for alleles encoding antigens of high or low prevalence. by segregating het pools into subpopulations, we were able to select specific "ee" pools of which were demonstrated (in subsequent profiling) to contain an e-sample. we also identified pools "het" for alleles indicating the possible presence of a rare donor, for example yta|b ( pools), co a|b ( ) and others. conclusion: molecularsieving of a single -well plate identified many desirable "antigen-negative" phenotypes and permitted selection of pools for combinations of "ag-neg" patterns including "partial:" rh phenotypes and combinations of c , e and jsa . these samples are thus confirmed "ag neg" and available for assignment. sieving also facilitated the enrichment of subsequent refinement of molecular attribute profiles in accordance with pending or anticipated demand. "antigen-neg" pattern partial_c partial _c, _e samples available after sieving background/case studies: sequence information generated from next generation sequencing (ngs) is often computationally phased using haplotype-phasing algorithms. utilizing experimentally derived haplotype information improves this prediction, as routinely used in hla typing. among the blood group systems, however, experimentally derived haplotypes are known for short genes only, such as icam (landsteiner-wiener) and ackr (duffy). for longer genes, such as abo of > kb, most haplotypes are only statistically derived. we recently established a large dataset of long ermap haplotypes, which code for the scianna blood group system. study design/methods: the nucleotide sequence of > kb each was used for all physically confirmed ermap alleles that we previously published. full-length sequences were aligned and variant sites were extracted manually. the bayesian coalescent algorithm implemented in beast v . . was used to estimate a coalescent phylogeny for these variants and the allelic ancestral states at the internal nodes of the phylogeny. results/findings: we found at least clades representing clusters of to alleles. for each clade, one observed allele was identified as the ancestral allele for its cluster of alleles. using the alleles, we were able to predict alleles with high posterior probability, which were ancestral to the observed alleles and, while not yet observed, may be extant. conclusion: we explored the phylogenetic structure and evolutionary events underlying the origin of different ermap alleles and predict ancestral alleles. in the present study, we show means to predict alleles and to calculate the distinct probabilities of correctness for such predicted alleles. the probabilities can be instrumental in defining a cut-off value to determine which computationally predicted alleles are worth confirming by physical evidence. the alleles identified by studies like ours may be utilized in designing of microarray technologies, imputing of genotypes and mapping of ngs data. the new alleles with nucleotide insertions would be predicted to cause complete loss of expression of the corresponding antigen from a bioinformatics perspective and to encode group o. rather very weak expression of the respective antigen and lack of the corresponding antibody in the plasma was found, confirming these represent subgroups of a and b and suggesting that transcriptional slippage, which has been observed before, is responsible for low level antigen expression. abo genotyping is powerful when both serology and molecular results are evaluated together, and these studies are needed to inform development of bioinformatics tools to accurately associate abo genotypes with phenotypes. background/case studies: evolutionarily related abo and gbgt genes encode a and b glycosyltransferases (at and bt) and forssman glycolipid synthase (fs), which catalyze the biosynthesis of a and b, and forssman (fors ) oligosaccharide antigens responsible for the abo and fors blood group systems, respectively. human at and bt possess leuglygly and metglyala, respectively, at codons - , and these tripeptides are important in determining the sugar specificity of enzymes, n-acetyl-d-galactosamine (galnac) for at and galactose for bt. functional fss possess gly-glyala at the corresponding codons, and exhibit galnac specificity. it has been recently shown that human at gained weak fs activity when the leu-glygly was substituted by glyglyala, suggesting that the tripeptide is involved in the recognition/binding of acceptor substrates, in addition to donor nucleotide-sugar substrates. study design/methods: we have searched for additional mechanisms that might enable human at to express fors . a variety of amino acid substitution constructs of human at were prepared. additionally, exon deletion constructs of at mrna transcripts were also prepared. dna from those expression constructs was transfected into cos (b galnt ) cells, and cell-surface expression of fors antigen was immunologically monitored with a monoclonal anti-fors antibody. results/findings: we found that met thr/ser substitutions also conferred human at with weak fs activity. we also found that the deletion of exon or of human at transcripts bestowed weak fs activity. because altered rna splicing is frequent in cancer, this mechanism may explain, at least partially, the appearance of fors antigen on certain cancer cells and tumors in forssman antigen-negative human species. furthermore, the co-introduction of one of those changes together with the glyglyala substitution synergistically conferred strong fs activity, in addition to strong at and bt activities. conclusion: the substitution of the glyglyala tripeptide codon in the catalytic domain may modify the acceptor specificity of the enzyme. met thr/ ser or exon / deletion may alter the intra-glogi localization of the enzyme. and those mechanisms function in synergy. the overlapping usage of acceptors by glycosyltransferases encoded by abo and gbgt genes is reminiscent of common ancestral origin of alpha , -gal(nac) transferase genes. the finding that at can synthesize fors implicates that the boundary between abo and fors systems may not be as strict as was previously delineated due to the crosstalk in-between. rh typing is required by the fda and fact/aabb for identity testing. since most antibodies in cb plasma are maternal in origin, the abo/rh phenotype relies only on the red cell typing. a and b antigens are not fully developed at birth, presenting about one third of a or b antigen expression levels compared to adult cells. this can result in indeterminate abo results for some cb products. we evaluated the use of dna-based methods for abo typing to aid the resolution of inconclusive ("indeterminate") or discrepant serologic typing results. study design/methods: a total of , cb units (cbu) were typed for abo/rh (beckman coulter pk system blood grouping and phenotyping) during the period / / - / / . abo genotyping targeting specific snps for groups a, a , b, o , and o and, if needed, gene sequencing was conducted in cases with indeterminate results, and in cbu that were provided for transplantation with abo discrepancy found at the transplant center. results/findings: sixty-two ( . %) cb samples had no reportable abo/ rh phenotype on initial testing, and therefore the cbu could not be used clinically. molecular abo/rh typing resolved all but one. all cases were heterozygous (a/o, b/o, or a/b); in % the predicted abo phenotype was a rh neg (table a ). the predominant donor race was caucasian ( %). four cbu with abo discrepancy were also evaluated by genotyping (table b) . in of those, abo typing performed at the hospital on the day of transplant differed from that reported by the cb bank; the fourth was identified by posttransplant abo typing of the recipient. molecular genotyping resolved the discrepancies. cbu identity was always verified by confirmatory hla typing. conclusion: there is currently no fda approved dna-based abo assay. however, abo genotyping is a useful method for samples where antibody tests alone cannot be conclusive, and can "rescue" cbu that could not be used otherwise. further, genotyping can help resolve abo discrepancies. abstract cobas v r hev for use on the cobasv r / systems is a qualitative pcr test for the detection of hev rna in human plasma. the purpose of this study was to evaluate the prevalence of hev rna among us blood donations collected in the midwest, a region reported to have a higher prevalence of hev infection, and the eastern us. study design/methods: , fresh and , frozen edta plasma samples from american red cross donors, collected from february - , were de-identified and screened by individual donation testing (id-nat) using cobasv r hev for use on the cobasv r system under a research protocol. samples were primarily from midwestern and eastern regions of the us. samples reactive on cobasv r hev were further tested by an alternate hev nat, hev rna quantitation, hev genotyping, and for hev antibodies. results/findings: of , valid results, a total of donations were reactive on cobasv r hev and all were confirmed positive. the confirmed donations were from a -year old male in indiana, a -year old male in california, and a -year old female in kentucky. all donations were positive by hemi-nested pcr and alternative hev nat; however, only the kentucky donation had a high level of hev rna ( iu/ml), and was strongly positive for both igm and igg hev antibodies. the indiana donation was genotyped as a, the california donation genotype b, and no genotype determined for the kentucky donation (see table) . the clinical specificity for the cobasv r hev test in id-nat was % ( % exact ci: . % to %). conclusion: based on the confirmed-positive donations of , tested, the hev prevalence was . % ( % exact ci: . % to . %) with a detection rate of : , ( % ci, : - : , ). to date, no cases of tt-hev have been documented in the us. however, based on the prevalence observed, immunosuppressed transfusion recipients may be at increased risk for transfusion-transmitted hev. background/case studies: monitoring the epidemiology of ttis within the donor population is critical to provide an ongoing assessment of infection risks associated with fda policy changes such as the msm deferral criteria. ttims is a multi-center, federally-funded program intended to derive hbv, hcv and hiv prevalence, incidence, viral genotypes, and donor risk factors for greater than % of blood collected in the us. ttims is supported by two distinct coordinating centers (laboratory and risk factor, lrcc, and donation database, ddcc). here we report months of prevalence along with demographic trends from the ddcc. study design/methods: four blood providers and their respective testing laboratories participated. standardized consensus-positive (cp) monitoring definitions were established for donor test results for hbv, hcv and hiv. these results, along with demographics for each donor and donation status (first-time vs repeat) were assembled into a single data set. rates of nucleic acid test (nat) yield (seronegative) and concordant positives (serologic plus nat positives) were combined to comprise cps, were computed overall for donors and donations and by demographic, geographic and temporal characteristics. where appropriate, rates were compared for differences using % confidence intervals. this analysis contains data from / / - / / . results/findings: among , , donations reported ( . % from firsttime and . % from repeat donors), there were respectively , and cp results for hbv, hcv and hiv with corresponding rates of . , . and . per , (pht) donations. prevalence among firsttime donors was, as expected, higher than among donations from repeat donors with ratios of : , : and . : for hbv, hcv and hiv. rates (pht) among males were higher than among females for all markers (hbv . vs . ; hcv . vs . ; hiv . vs . ). in general, higher rates for all markers were seen among minority donors, those in the - -year age group (also - year for hiv), and those from the southeast (and south central for hiv and hcv, and southwest for hbv). no trends were noted over time when -month periods were compared. conclusion: data from major us blood systems were successfully combined and are a baseline for monitoring purposes. demographic trends are similar to those observed in other donor studies and generally agree with community trends. changes in rates will require analyses in the context of potential changes in the demographic structure of the donor population. screening donated blood from babesia endemic regions of the united states using a transcription-mediated amplification assay on a fully automated system vanessa bres* , melanie c proctor , deanna self , monique portugal , adrian gurrola , laura tonnetti , sonia bakkour , cheryl lobo , michael paul busch , susan l stramer and jeffrey m linnen . grifols diagnostic solutions inc., american red cross, blood systems research institute, new york blood center background/case studies: the procleix v r babesia assay on the procleix panther v r system is a qualitative in vitro nucleic acid test currently under development. the assay, which is based on transcription-mediated amplification (tma), detects four clinically relevant babesia species (b. microti, b. divergens, b. duncani, and b. venatorum) in human whole blood specimens. this test is intended to screen blood donations individually and in pools of up to donations. whole blood samples are lysed and then pooled on the automated procleix xpress v r system prior to testing on the procleix panther system. these studies evaluated the preliminary analytical and clinical performance of the procleix babesia assay on the panther system. study design/method: analytical sensitivity was determined by diluting in vitro synthesized rna transcripts for the four babesia species. fresh b. microti-infected hamster whole blood, cryopreserved b. duncani-infected hamster whole blood and fresh b. divergens-infected human erythrocytes were tested to determine the limit of detection (lod) of parasites/ml (p/ml) by probit analysis. clinical sensitivity and specificity were determined by screening , unlinked whole blood donations collected from august th to april th in the northeastern united states. initial reactive donations were confirmed by repeat testing, pcr, and/or igg immunofluorescence assay (ifa). reactive individual donor lysates were tested in pools of . results/finding: the procleix babesia assay detected all four babesia species with a % lod ranging from . - . copies/ml. the preliminary % lod in parasites/ml ranged from . - . p/ml for b. microti (n ), from . - . p/ml for b. duncani (n ), and from . - . p/ml for b. divergens (n ). of the , donations screened, initial reactive and confirmed positive donations were identified for specificity of . % ( %ci: . - . %). of the confirmed positive specimens, were reactive by both ifa and pcr, by ifa only and by pcr only. all confirmed positive samples were reactive in lysate pools of . donors of reactive donations resided in ct ( ), nj ( ), nh ( ) and me ( ) for an overall incidence of : , , and : , in ct. conclusion: the procleix babesia assay on the procleix panther system demonstrated high clinical specificity and sensitivity and detected all four babesia species with similar sensitivity. all confirmed positive donations were also detected in pools of thus demonstrating the effectiveness of pooled lysate screening. conclusion: use of the lag avidity assay shows that in both first-time and repeat hiv-positive us blood donors, newly-acquired (i.e., incident) hiv infections are more frequent in younger donors. the use of this approach provides an additional monitoring tool to assess changes in characteristics of donors whose risk exposure was proximate to the date of donation and will also complement traditional incidence methods by allowing derivation of incidence by donor type. epidemiology of hepatitis b virus, hepatitis c virus and human immunodeficiency virus in united states blood donors lauren a crowder* , whitney r steele , ed p notari , james haynes , roger y dodd and susan l stramer . american red cross, american red cross (retired) background/case studies: from - , the prevalence of hbv and hcv in us blood donors decreased, while hiv rates remained constant. however, incidence has not been recently calculated. here we report the prevalence, incidence and residual risk (rr) of hbv, hcv, and hiv in a large us blood system from - . study design/methods: prevalence was calculated in -year intervals. incidence was measured as the number of positives among repeat donors divided by the total time at risk, in person-years (py). rr was calculated using the window periods of . , . and . days for hbv, hcv and hiv, respectively. linear regressions were calculated with p< . (*) as significant. results/findings: from / / - / / , there were more than million donations from , , donors ( . % female, % first-time (ft), . % caucasian). there were significant decreases in donation prevalence for hbv and hcv (p . and . ), but no significant decrease in hiv during the years (see table for f and r values). a significant decrease was seen in ft donor prevalence for hbv and hcv (p . and . ). prevalent ft donors were significantly more likely to be male ( . % -hbv, . % -hcv, . % -hiv; p< . ). incidence for all agents declined (significant only for hbv; p . ). the decrease in hcv incidence was not significant, but there were fewer incident donors in the last -year period ( in - vs. in - ) . hcv incident donors in - were more likely to be male ( . % vs . % in - , p< . ) and were younger ( . % vs. . % in - < years, p . ). overall, incident donors were more likely to be caucasian males (p< . ). rrs for all agents decreased over time with rrs in - of in , , ; in , , ; and in , , for hbv, hcv and hiv, respectively. conclusion: prevalence, incidence and rr of hbv, hcv and hiv have generally decreased within this blood system over the -year time frame. as donor screening and deferral regulations evolve, it is important to monitor these risks. it is critical to note that even in a large population, small changes to the number of positives can have a significant impact on prevalence and incidence rates. furthermore, in , mayv was isolated from a patient in haiti, suggesting the virus is already circulating in the caribbean. the extent of mayv transmission could be underestimated due to limited surveillance and diagnostic capabilities; therefore, it is necessary to be prepared for mayv emergence and the potential risk for the blood supply in case it can be transmitted through blood transfusion. study design/method: platelet components (pc) prepared in pas were spiked with mayv and treated with amotosalen and uva illumination. samples were collected pre-uva and post-uva illumination for infectious titer determination. as- rbcs were spiked with mayv, mixed with glutathione (gsh)/processing solution, dosed with lm amustaline, and incubated for hrs at room temperature. samples were collected prior to the addition of amustaline (pre-treatment) and following the hr incubation (post-treatment) to determine infectious titers. infectious titers for all samples were determined by plaque assay on vero cells. the extent of inactivation was determined by comparing the infectious titers (plaque forming units (pfu)/ml) in pre-vs. post-treatment samples. results/finding: mayv was inactivated to the limit of detection in both pc and rbcs. in platelets, > . log , or > . log pfu/ml, inactivation of mayv was achieved. in rbcs, inactivation of mayv was > . log , or > . log pfu/ml. conclusion: this study demonstrates robust inactivation of mayv by both amotosalen/uva treatment in pc and amustaline/gsh treatment in rbcs. these systems are efficient at inactivating alphaviruses that have demonstrated or have the potential for transfusion-transmission, including mayv, chikv and rrv. prt offers potential as a mitigation strategy for maintaining blood component availability in areas where multiple alphaviruses are epidemic or endemic, and testing is not feasible. (data have not been submitted for fda review and intercept for red blood cell is not approved for commercial use). thrombotic thrombocytopenic purpura with high adamts- inhibitor may represent a distinct disease subset in response to therapy based on immature platelet count (a-ipc) dynamics hamza n gokozan* , , hollie m reeves , and robert w maitta , . case western reserve university school of medicine, university hospitals cleveland medical center background/case studies: thrombotic thrombocytopenic purpura is a lifethreating consumptive thrombocytopenia and microangiopathic hemolytic anemia causing diffuse ischemic damage to tissues. early therapeutic plasma exchange (tpe) initiation has improved survival. absolute immature platelet count (a-ipc) has been found to aid in diagnosis and follow-up of ttp patients. a-ipc changes in response to therapy in patients with low adamts activity and high inhibitor have not been analyzed in a patient cohort. we analyzed a-ipc response to therapy in five patients with adamts deficiency and high inhibitor at a large tertiary academic medical center. study design/method: patients had adamts activity of < % and high inhibitor ( . - ). mean age of cohort . years (range - ). four patients were female and one was male. patients presented with microangiopathic hemolytic anemia, thrombocytopenia (mean . x /l, range - x /l) and low a-ipc (mean . x /l, range . - . x /l). patients were initiated on daily tpe and prednisone; additional immunosuppression during hospital stay for cohort consisted of rituximab mg/m ( patients) and cyclophosphamide mg/m (one patient). tpe continued until platelet count reached x /l for at least two consecutive days. immature platelet fraction (%-ipf) and a-ipc (%-ipf x platelet count) were obtained with daily pre-tpe cbc. a-ipc ratio was calculated from baseline. results/finding: patients responded rapidly to daily tpe (mean of . days [range - days]) when they achieved a three-fold increase in a-ipc from baseline (mean . x /l, range . - . x /l) and a rapid improvement in platelet count. however, this improvement in platelet count was not accompanied by expected decreases in a-ipc, suggestive of recovery from disease. all patients experienced platelet (mean . x /l, range - x /l) and a-ipc (mean . x /l, range - . x /l) decreases that occurred concurrently while receiving daily tpe so that after a mean of . days (range - days) mean platelet count was . x /l (range x /l) and mean a-ipc . x /l (range . - . x /l). patients were initiated in either rituximab or cyclophosphamide therapy in conjunction with tpe after a mean of . days of a-ipc and platelet count instability. a-ipc trended to levels indicative of restoration of a negative feedback after this time. conclusion: rapid decreases in platelet counts after a good response in ttp patients may raise suspicion for presence of high adamts inhibitor. patients with a high inhibitor have similar a-ipc dynamics during which initial high a-ipc production is followed by unexpected decreases in a-ipc concurrent with platelet counts. recovery occurs once negative feedback between platelet and a-ipc production is re-established. patients with a high inhibitor may represent a distinct subset of ttp as suggested by a-ipc responses. benchmarking the centralized urgent plasma exchange service for patients admitted with a diagnosis of thrombotic thrombocytopenic purpura at a multi-hospital healthcare system jansen n seheult* , michelle n stram , joan sevcik , alesia kaplan , and joseph e. kiss , . department of pathology, university of pittsburgh medical center, blood systems inc., university of pittsburgh background/case studies: consensus guidelines recommend that therapeutic plasma exchange (tpe) must be started as early as possible and within - hours after the diagnosis of thrombotic thrombocytopenic purpura (ttp) has been made; however, there are limited data documenting actual practice. there are several operational facets of delivering a centralized urgent tpe program in a multi-hospital healthcare system, including: central venous (cv) access, ordering, release and delivery of thawed plasma, and transportation of personnel and equipment to perform the procedure. this study analyzes the time elapsed between major steps from diagnosis to initiation of tpe in patients admitted with ttp. study design/method: a retrospective review of the electronic medical record and laboratory information systems from january , to november , was conducted to identify all ttp patients undergoing urgent tpe. demographics, comorbidities, and other pertinent laboratory tests (such as adamts- activity levels, complete blood count, biochemical markers of hemolysis and coagulation studies) were reviewed on all identified patients. temporal data for tpe request, cv access placement, plasma product release (which usually happens after cv access), arrival of tpe team and initiation of the procedure were extracted from procedure notes and the blood bank information system. descriptive and summary statistics were generated using stata version (statacorp, tx). group comparisons were made based on hospital location, level of care and history of ttp using a wilcoxon rank-sum test. results/finding: of the ttp patients identified, were excluded due to missing temporal data for important variables. the majority ( %) of patients were treated at central academic centers, with the remainder being treated at peripheral sites. fifteen patients ( %) had a prior history of ttp and % had severe adamts deficiency on admission. the median time from tpe request to initiation was . hours (interquartile range: . - . hours). there were non-significant trends to shorter time intervals from request to cv access and request to tpe initiation in patients admitted to the intensive care unit (icu) versus non-icu patients (table ) . treatment was not started within an -hour window in patients; the median time to cv access was significantly longer in these patients ( . vs . hours, p< . ). two of these patients had a prior history of ttp and only four patients had severe adamts- deficiency. the majority (more than %) of the time interval between tpe request and tpe initiation was spent obtaining cv access and plasma products. there were no significant differences in time intervals comparing patients with a new diagnosis of ttp versus patients with recurrent/ relapsed disease (table ) or between patients treated at a central academic center versus a peripheral hospital. conclusion: the consensus - hour target window from tpe request to initiation appears feasible for a centralized tpe program servicing a multi- a transfusion vol. supplement s hospital healthcare system. addressing limitations in availability of cv access would likely yield the greatest improvement in timeliness of urgent tpe. cytoreductive therapy for cellular hyperviscosity: utility of cytapheresis treatment for chronic myelogenous leukemia and essential thrombocythemia. jan c hofmann* and dobri d kiprov. california pacific medical center background/case studies: several retrospective, case series have suggested that cytoreductive therapy to treat cellular hyperviscosity and prevent thrombotic events in patients (pts) with chronic myelogenous leukemia with accelerated transformation (cml-at) or essential thrombocythemia (et) may improve short-term outcomes. however, no randomized controlled trial (rct) assessing the efficacy of cytapheresis treatment in this group of pts has been performed. study design/method: from january, through january, , we performed cytapheresis (cy) treatments (txs) for pts with either cml-at or et, and clinical and/or laboratory evidence of cellular hyperviscosity. pts ( %) had cml-at and received leukapheresis (lp) txs; pts ( %) had et and received thrombocytapheresis (tc) txs. cml-at pts presented with median wbc x /l (range - x /l), of which % had blast percent > % or blast count > x /l. median age was years ( - years); % were male. cns symptoms (sxs) of leukostasis (lks) were defined as: headache, cognitive decline, confusion, somnolence, visual abnormalities, or seizure; pulmonary (pulm) sxs of lks were defined as: dyspnea, hypoxia, or bilateral chest infiltrates. % of cml-at pts had no sxs of lks; % pts had sxs of either cns or pulm lks ( sxs), and % pts had sxs of both cns and pulm lks ( sxs). et pts presented with median platelet (plt) count of: x /l ( - x /l)and % pts had sxs of thrombosis (evidence of cva or tia, mi, or dvt). median age was years ( - years); % pts were male. results/finding: all pts received a course of cy tx with following objectives: ) decreasing the risk of thrombotic/ hemorrhagic complications related to hyperviscosity, and ) stabilizing cml-at pts for induction chemotherapy (ind chemo). wbc (or plt ct) tx goals were: wbc count (ct) < x /l for cml-at pts, and plt ct < x /l for symptomatic et pts and < x /l for asymptomatic et pts. cml-at pts received median of lp txs (mean . txs/pt; range - txs). et pts underwent median of tc txs (mean . txs/pt; - txs). outcomes were evaluated by percentage of pts who: ) reached wbc (or plt ct) tx goal, and ) received ind chemo. "improved" outcome was defined as pts who reached their wbc (or plt ct) tx goal during cy tx; "stabilized" were pts who achieved > % reduction in wbc (or plt ct) without reaching goal; and "unchanged" were pts who achieved neither. in cml-at cohort, % pts improved, % pts stabilized; and % pts worsened. in et cohort, % improved, % stabilized, and % were unchanged. for cml-at pts, median final wbc ct x /l (range - x /l); % pts received ind chemo. for et pts, median final plt ct x /l ( - x /l); % pts had resolution of thrombotic a transfusion vol. supplement s symptoms. % of cml-at pts and % of et pts expired within - days after course of cy tx. of expired pts, pts had both blast crisis and sxs of cns/ pulm lks; pt had intracranial hemorrhage or cva; and pts were hypotensive, intubated, or unable to tolerate ind chemo. conclusion: pts with cml-at or et and evidence of impending thrombosis may benefit from cytoreductive therapy. a limited number of cytapheresis treatments (median - txs) can enable a high percentage of pts to receive definitive treatment and may improve short-term clinical outcomes. a rct to assess efficacy of cytapheresis treatment versus induction chemotherapy (or platelet inhibitor tx) alone in this subset of pts would be very useful. background/case studies: partial normal saline replacement during plasma exchange procedures is common practice. benefits of using normal saline as a replacement fluid include reduced procedure costs and possible reduction of the hypothetical hyper-oncotic effects of standard albumin formulations. however, the use of normal saline may increase the risk of undesired, and potentially costly, adverse events, such as hypotension and citrate reactions. the goal of this study was to compare the frequency of reported adverse outcomes for patients that received all albumin versus albumin/ saline as replacement fluid for plasma exchange at our institution. study design/method: a four year retrospective chart review was done of all therapeutic apheresis procedures performed by our apheresis service that used % albumin or % albumin- % normal saline ( / ) as replacement. patients who received plasma entirely or partially as replacement were excluded. the procedure type ordered ( % albumin vs / ), the percent of normal saline actually used during the procedure, age, gender, and any noted adverse events during the procedure were recorded in all cases. repeated procedures were modeled using a generalized linear mixed model to examine the risk of having hypotension and/or citrate toxicity where % albumin was used versus those that used / . covariates included were fluid types, age and gender. odds ratios (or) and % confidence intervals (ci) were used as a measure of risk. we used the term significant for a two-sided p-value < . . results/finding: during the study period, procedures were documented for subjects ( % female), age range - years, of which , ( . %) received / . the type of fluid used as replacement had a significant effect on the risk of having either hypotension or citrate toxicity. replacement with % albumin had a significantly lower risk of having either event than by using / , [p . , or (ci): . ( . , . )] , and also had a significantly lower risk of causing hypotension [p . , or (ci): . ( . , . )] in addition to a lower risk of causing citrate toxicity [p . , or (ci): . ( . , . )]. age had a significant effect on having a hypotensive event [p . , or (ci): . ( . , . )] but no effect on citrate toxicity or the combined outcome. gender had no effect on frequency of any event. conclusion: partial saline use as a replacement fluid with albumin during plasma exchange significantly increases the risk of hypotension and citrate toxicity during the procedure. age also increases the risk of hypotension. use of saline as replacement fluid during plasma exchanges should be minimized to maximize patient safety especially in older patients. background/case studies: therapeutic apheresis (ta) is a complex procedure that is mostly well-tolerated and rarely associated with adverse events (aes). there are few studies published on aes associated with ta but they lack uniformity of data. moreover, there is no common database in the united states (us) to report ta-associated aes. we evaluated the annual incidence rates of aes associated with ta at a large tertiary academic medical center over a year period and compared it to published literature. study design/method: we conducted a -year retrospective study of ta procedures performed and aes were classified according to criteria described in table . during the study period, ta were performed using cobe spectra (software versions . and . ) and since the spectra optia apheresis system (version . ). literature search was conducted for data published on aes associated with ta. four studies from us and non-us studies (canada, europe and japan) were analyzed. trend for ae rates from - was also analyzed. statistical analysis was performed using chi square and spearman rho tests. results/finding: the overall ae incidence was . % ( of , procedures) during year period. frequency of aes associated with therapeutic plasma exchange (tpe) was significantly higher ( . %, p< . ) compared to other ta procedures. we found significant correlation between number of tpe and aes (spearman rho . , p . ) over the years and significant down trend of moderate and severe aes with a spearman rho of - . (p . ) and - . (p . ) respectively. there were no fatalities during the study period. majority of aes were grade i ( %) and grade ii ( %): / ( . %) procedures were not completed due to aes. comparison of aes [ . % ( / , )] to both european [ . % (n , , / , ) ] and other us studies [ . % (n , / , )] showed a statistically significant difference (p< . ). conclusion: overall incidence of aes was significantly lower than current published literature. incidence of aes published in other countries is significantly lower than rates published in us. differences in incidence of aes in literature emphasizes need for uniform reporting and stratification of aes and development of a common database to report ta-associated aes. we propose a grading rationale in order to standardize reporting of ae (table ) . variations in biochemical markers of bone metabolism during plateletpheresis: impact of socio-demographic and lifestyle factors? markus dettke*. akh vienna university hospital background/case studies: plateletpheresis is associated with short-term variations in biochemical markers of bone turnover. socio-demographic factors and lifestyle behaviors are recognized factors which influence mineral metabolism and bone health. in the present study we analyzed the influence of demographic and lifestyle factors on the observed changes in bone markers in a large cohort of routine platelet donors. study design/method: altogether platelet donors with a donation activity of up to platelet donations participated in the study. after a detailed anamnesis all participants underwent a standardized questioner asking for several lifestyle factors known to affect bone metabolism. blood was sampled before and after plateletpheresis and was analyzed for the bone formation marker osteocalcin (oc) and the bone resorption marker cross-linked telopeptides of type i collagen (ctx), among other parameters. the effect of calcium supplementation on bone metabolism was tested in a placebocontrolled crossover study involving ten donors. results/finding: plateletpheresis resulted in an increase in the serum levels of the bone resorption marker ctx and the bone formation marker oc. both parameters returned to base levels within hours after the end of the collection. multiple regression analysis including the parameters sex, age , positive family history of bone disease, but also individual factors like hormonal contraception, smoking, regular alcohol consumption or sportive activity revealed no influence of socio-demographic or lifestyle factors on the observed variation in ctx or oc. there was no association between individual donor career or the number of previous donation and the observed increase in bone turnover. the only predictive parameter we could identify was the amount of citrate exposure during plateletpheresis. increase in serum ctx, showed an inverse correlation to changes of serum ionized calcium. continuous iv supplementation of calcium-gluconate throughout plateletpheresis reduced the variations in bone markers, although this effect was more pronounced for ctx compared to oc. conclusion: the amount of citrate infused during routine plateletpheresis is a predictive parameter for the transient increase in serum markers of bone metabolism. known risk factors for bone diseases, including sex, age, smoking or alcohol consumption, seems to have a low impact on the observed citrate-related variations in serological biomarkers of bone turnover. transfusion with optimized blood products versus transfusion with standard products in a trauma-transfusion rat model mathijs wirtz* , jordy jurgens , jacoline buchner-doeven , joris roelofs , philip spinella , jennifer a muszynski , carel goslings and nicole juffermans . academic medical center, washington university school of medicine, nationwide children's hospital background/case studies: transfusion is associated with nosocomial infection and organ dysfunction in trauma patients, which may be mediated by soluble bioactive substances in blood products. we hypothesized that removing these bioactive substances improves host immune response and reduces organ dysfunction. study design/methods: blood products were prepared from syngeneic rat blood according to blood bank standards. soluble mediators were removed from red blood cells ( days old) and platelets ( days old) by washing. plasma was filtered through a . um filter. rats ($ grams) were poly-traumatized by crush injury to the small intestines, the liver lobes, and by fracture of the right femur and hemorrhaged $ % of their estimated blood volume, which was calculated to be ml/kg. hemorrhage continued until a mean arterial pressure of mmhg was reached. rats were randomized to resuscitation with standard blood products, washed/filtered blood products or sham. blood samples were taken up to h after trauma to assess biochemistry and coagulation status. ex vivo whole blood stimulation tests with lps were performed after sacrifice, and organ damage was assessed by histopathology. blood products were sampled to assess for biochemical changes. comparisons between groups was done by anova and dunnett's post-test for multiple comparisons. results/findings: filtering or washing of blood products significantly stabilized ph, sodium and potassium concentrations and decreased lactate levels in the products compared to standard products. both resuscitation groups received an average of ml/kg of blood products in a : : ratio. however, use of washed/filtered products did not improve organ failure, as assessed by histopathologic score and levels of creatinine, asat and alat. the coagulation status as assessed by thromboelastometry was deranged in all groups and normalized during transfusion, showing no significant differences between washed/filtered products and standard care. immune response to lps was decreased following trauma compared to healthy controls but did not differ between groups. conclusion: filtering or washing of blood products reduces some aspects of storage lesion of blood products, without affecting the hemostatic capacity of the products, but does not improve organ injury in a rat trauma and transfusion model, nor does it improve the immunosuppressive host response. these results suggest that washing or filtering of blood products may have no relevant clinical effects in a rat polytrauma model. safety and efficacy of tranexamic acid during cardiovascular surgery: a single center before-and-after study takuma maeda* and shigeki miyata. national cerebral and cardiovascular center background/case studies: tranexamic acid (txa), an antifibrinolytic agent, has been widely used in cardiovascular surgery, since several studies have shown that prophylactic use of txa is effective in reducing blood loss after cardiovascular surgery. however, there is concern about the risk of thromboembolic events and adverse neurological effects such as seizures, which might worsen patient outcomes. consequently, we stopped using txa in april , which enabled us to conduct a before-and-after study. the present study aimed to examine the association between txa and adverse effects (seizures, thromboembolism, and renal dysfunction) in patients undergoing cardiovascular surgery using a propensity score matching model. we also assessed the association between txa and other clinical outcomes (reoperation for bleeding, transfusion volume, blood loss, ventilation time, intensive care unit stay, and -day mortality). study design/method: this single center retrospective cohort study involved patients who underwent cardiovascular surgery with cardiopulmonary bypass or offpump coronary artery bypass grafting between january and july (n ). because of missing data on patient characteristics, patients were excluded. the incidence of adverse effects associated with txa and other clinical outcomes were evaluated before (january to march , n ) and after (april to july , n ) using a propensity score model. we estimated propensity scores using a logistic regression model for txa use as a function of baseline variable, generating pairs of patients who received or did not receive txa. we also evaluated the adverse effects of txa using segmental regression analysis. results/finding: propensity-matched analysis showed that seizures were more common ( . % vs . %, p< . ) and ventilation time was longer ( h vs h, p . ) significantly in the txa group than in the non-txa group. in contrast, transfusion volume and blood loss were significantly lower in the txa group than in the non-txa group ( ml vs ml, p . ; and ml vs ml, p< . , respectively). however, -day mortality was not statistically different between the groups ( . % vs . %, p . ). none of the other outcomes were significantly different. segmental regression analysis yielded similar results. conclusion: even though txa may be associated with an increased rate of seizures and longer ventilator time, it does not increase mortality. the use of txa is significantly associated with decreased blood loss and transfusion volume, providing social benefit by reducing the need for blood transfusion because the supply of blood components will be limited with the aging of japanese society. it seems to be advantageous to use txa because decreased blood loss and transfusion volume and the associated social benefit outweigh the disadvantages of an increased rate of seizures and longer ventilator time. sustained impact of blood management strategies in orthopedics: continuous quality improvement linda levinus* and michele deeney. new england baptist hospital background/case studies: transfusions are one of the most over-utilized treatments performed in any hospital setting (choosing wisely campaign, april , www.choosingwisely.org/societies/american-association-of-bloodbanks). costs and risks associated with transfusions are high and may have a significant impact on patient safety. in our institution we perform over , joint replacements and spine surgeries per year, making transfusion-associated costs very high. since our last formal evaluation of the metrics used post implementation of patient blood management (pbm) strategies, questions regarding the feasibility of continued transfusion reduction and sustainability of the program were raised by administration and key stakeholder physicians. the objective of this study is to determine what, if any, sustainable improvement to our blood utilization dashboard table ). the data collected show that there has continued to be a reduction in transfusion rate, and blood expenditures through fy . length of stay has also shown a continued reduction, which is an indicator that the pbm strategies implemented have not compromised quality outcomes. further, continued review and monitoring of the chosen metrics, evaluating changes to policy and practice related to transfusion medicine, and communication of findings to providers/administration upon immediate restrospective analysis, are integral to the continued success and sustainability of our pbm program. going forward, these practices, along with investigating use of additional pbm strategies, will provide the basis for an effective continuous quality improvement program in transfusion medicine for orthopedics. safety and efficacy of -factor prothrombin complex concentrate: a retrospective review of outcomes at an academic hospital stephanie jalaba*, hollie benson, nan zhang, jill adamski and theresa kinard. mayo clinic arizona background/case studies: -factor prothrombin complex concentrate (pcc) contains factors ii, vii, ix, x, proteins c and s and is used for reversal of vitamin k antagonists in acute major bleeding or urgent, invasive procedures. occasionally, it is used off-label when plasma is not optimal for achieving hemostasis. this study compares the efficacy of on-label and off-label use of pcc in correcting coagulation parameters and reducing allogeneic blood transfusion. study design/methods: a retrospective chart review was performed for pcc use at our institution in . marginal modeling (gee method) was used to account for within patient correlation and assess changes in lab values and products transfused. logistic regression (gee method) was used to evaluate potential risk factors for unsuccessful hemostasis (uh rate of transfusion after pcc ! rate before pcc) or thrombotic complications. results/findings: the reduction in pt (p . ) and ptt (p . ) was significantly greater in on-label than off-label use. interestingly, transfusion reduction in rbc (p . ) and plasma (p . ) after off-label use was significantly greater than on-label use. cases, both on-label and off-label, with uh were associated with cell saver, acute normovolemic hemodilution (anh), or cardiopulmonary bypass (cpb). the odds of having uh were . times (p . ) more with cell saver or anh, and . (p . ) times more with cpb. post-pcc thromboses were identified in cases, but no association was found with potential risk factors: use of antifibrinolytics, vitamin k, factor viia, or extracorporeal support. background/case studies: when a pregnant woman with high risk pregnancy (diagnoses such as abnormal placentation, multiple gestation) is admitted to inpatient bedrest the obstetrical team would like to assure ability to crossmatch red blood cells (rbc) at all times by always having an in-date type and screen specimen. per current aabb standards, this necessitates a new sample every days. this can lead to excessive iatrogenic blood loss and increasing difficulty with obtaining intravenous access in the patient, to the point that an invasive catheter such as a picc line may be placed. in order to mitigate these issues, we chose to extend the type and screen specimen to expire after days in patients without rbc alloantibodies other than passively acquired anti-d due to rh immune globulin administration. study design/method: patients expected to have an antenatal hospitalization of at least days with high risk for transfusion need are identified by the obstetrical service, which submits a request to the transfusion service for extension of pre-transfusion specimens to days. the transfusion service medical director reviews the case and gives final approval. we observed only patient did not have an in-date specimen when the extended out-dating was requested. thirty-eight ( ) patients were in-patients continuously until delivery. five patients were discharged prior to delivery- moved to another state, was admitted later at another local hospital, and three were readmitted for later deliveries. the mean interval from approval to delivery was days (range - ). six ( ) patients delivered within days of approval. after approval, the mean number of additional specimens per patient was . (range, - ). no patient required transfusion prior to delivery. five patients received transfusion of at least rbc at the time of delivery, and none had evidence of transfusion reaction. conclusion: since no new antibodies were identified prior to discharge or delivery and no transfusion reactions were observed, the process appears safe. with only patients delivering within days of approval for extended specimens, patients avoided collection of at least specimen each, and patients avoided at least collections each. since new antibodies are not detectable for at least days after immunization, even longer extension of pre-transfusion specimen out-date may be considered. although this requires further study, we believe our practice of extending the pre-transfusion testing sample expiration date to days is safe and is justified, when weighed against the risk of excess iatrogenic blood loss and placing an invasive line for blood sampling in a pregnant patient. iron metabolism in critically ill patients developing anemia of inflammation margit boshuizen* , , jan m. binnekade , benjamin nota , pieter r tuinman , kirsten van de groep , olaf l cremer , janneke horn , marcus j schultz , robin van bruggen and nicole p juffermans . academic medical center, sanquin research and landsteiner laboratory, vu university medical center, university medical center utrecht background/case studies: anemia due to inflammatory processes (anemia of inflammation, ai) frequently occurs in critically ill patients. in ai, inflammation-induced hepcidin decreases iron availability, a process that is thought to be regulated by erythroferrone, which impact erythropoiesis. knowledge on changes in iron metabolism during the course of ai is limited, hampering the development of strategies to counteract ai. this study aimed to investigate the dynamics of parameters of iron metabolism during the development of ai in critically ill patients. study design/methods: a case control study was performed in tertiary icus in the netherlands comparing patients who developed ai during icu stay with control groups: non-anemic patients with sepsis, non-anemic patients without sepsis, and patients with anemia due to acute blood loss. patients were matched on age and sex. a linear mixed model was used to assess differences in parameters of iron metabolism between groups and over time. results/findings: in patients with ai, levels of iron, transferrin and transferrin saturation decreased already prior to the development of anemia, with lower levels compared to controls (table) . ferritin and hepcidin were increased in ai compared to controls. in the course of ai development, erythroferrone decreased. differences in iron metabolism between groups were not influenced by disease severity. patients with ai differed from patients with anemia due to acute blood loss, the latter was characterized by high iron ( . vs. . mmol/l, p< . ) and transferrin saturation ( vs. %, p< . ), and low ferritin ( vs. mg/l, p< . ). conclusion: in critically ill patients with ai, iron metabolism is already altered prior to the development of anemia, suggesting a potential window of opportunity for therapy. iron metabolism in ai is more disturbed than in non-anemic septic controls, irrespective of disease severity, indicating that ai is not solely determined by severity of inflammation. iron metabolism in ai patients differs from patients with acute blood loss, suggesting that efforts to modulate iron metabolism in anemic icu patients should take the cause of anemia into account. clinical oral abstract session: novel approaches to processing and assessing cell therapy products a paradigm shift in stem cell isolation and storage jeffrey drew*. cells life group llp background/case studies: widespread use of umbilical cord blood is limited by processing yield and post-thaw recovery of viable nucleated cells. the recommended therapeutic cell dose is approximately . x cells per kg body weight indicating that a single cord unit may be insufficent to treat larger individuals. cell isolation methods were developed to remove erythrocytes whilst recovering the white cell fraction (wcf). however, all current methods result in significant loss of the wcf, some up to %, whilst leaving % of the starting volume of erythrocytes. additionally, there is an almost total loss of potentially important, low abundance cellular subsets. the use of cord blood for hematopoietic reconsititution and in regenerative medicine would be widened if processing methods improved postprocessing and post-thaw viable cell recovery. study design/methods: we have developed a solution consisting of a defined concentration of reagents routinely used in blood therapy. on combination with blood, this solution results in the selective sedimentation of erythrocytes by gravity within minutes. the wcf remains in solution and can be easily separated from the erythrocyte sediment. the wcf can then be concentrated by gentle centrifugation into a small volume containing less than % of the original erythrocyte content. the addition of dmso for cryogenic storage and controlled freezing using standard procedures then completes this simple process. results/findings: we have clearly demonstrated that this method allows almost the entire wcf to be isolated and/or concentrated with only modest loss of any of the cellular sub-sets thus far examined. in addition to improving pre-freeze yields, post-thaw recoveries of viable cells are markedly increased, with a yield of approximately % of the cd fraction post separation and freeze thaw (table ) . possibly more important, the cfu assay results reproducibly yield higher counts of cfu-gm, cfu-gemm and bfu colonies (table ) which is a strong indicator that this method will improve patient outcomes. in addition, our separation method isolates and preserves the megakaryocyte-like cells (cd cd ) and early projenitor cells expressing oct and nanog (markers for vsels) which are two examples of cellular subsets usually lost using current separation techniques. conclusion: these results demonstrate that our method achieves: . routine recovery of the wcf at levels higher than current methods, independent of volume. . higher percentage recoveries of all cell types tested than can be achieved with existing methods. . markedly higher post-thaw recovery of viable nucleated cells than any current methodology. . almost complete removal of hematocrit. as a result units of cord blood separated using this new method will contain cell yields that could only otherwise be achieved through pooling multiple separate units. therefore, this new method has the potential to increase the demand for cord blood in therapy, expanding to larger individuals and adults, where up until now, it has been suppressed due to limited cell yields delivered by existing methods. effects of implementation of an absolute lymphocyte count target, in addition to cd target, for hematopoietic progenitor cell collection edwin a burgstaler*, luis f porrata, dennis a gastineau, eapen k jacob and jeffrey l winters. mayo clinic background/case studies: lymphoma patients receiving > . x lymphocytes(lymph)/kg during peripheral blood stem cell transplant have superior survival. in addition to a cd cell target of . x /kg, a lymph target was also implemented. fifty patients before (no alc) and after (alc) implementation were retrospectively evaluated. study design/method: lymph and cd yields, number of collections, lymph target reached, and days to engraftment were examined. mobilization was g-csf (g) or g-csf plerixafor (g pl). consecutive no alc and alc procedures were examined. the mann-whitney and chi square tests were used for statistical comparison, p< . considered significant. results/finding: no alc and alc collections occurred among the patients. fenwal amicus was used for % of the no alc and % of the alc collections (terumobct spectra optia cmnc used for remaining). diagnosis was hodgkin's and non-hodgkin's lymphoma (no alc); hodgkin's and non-hodgkin's lymphoma (alc). pre procedure wbc and lymph counts were significantly higher for no alc (wbc . , lymph . x /l) than alc (wbc . , lymph . x /l). equivalent whole blood (corrected for ac) was processed for no alc ( . l) and alc ( . l). for alc group, extra collections beyond cd target were: days: %, day: %, days: %, days: %, and days: %. significantly more patients were mobilized with g pl in no alc group (n ) than alc group (n ) and collections in alc group had mobilization discontinued after cd cell target reached. there was no significant difference in g ( . x lymph) compared to g pl mobilized collections ( . x lymph); both were significantly higher than the collections where mobilization had been discontinued ( . x lymph). days to wbc engraftment ( . no alc vs . alc) and platelet engraftment ( . no alc vs . alc) were not significantly different. median number of collections for no alc ( ) and alc ( ) were not significantly different. data (medians) in the table. conclusion: not all patients achieved the . x lymph/kg or even the . x lymph/kg targets. implementation of a lymph target increased patients obtaining . x lymph/kg from % to %. only % had < . x lymph/ kg. discontinuation of mobilization once cd cell target was reached significantly reduced lymph yield. the median increase of one collection per patient following implementation was less than had been expected. extended preprocessing storage impairs cord blood hematopoietic stem cell activity suria jahan* , and nicolas pineault , . canadian blood services, university ottawa, canadian blood services, centre for innovation background/case studies: large distances between collection and processing sites combined with staff availability can result in long processing delays of umbilical cord blood (ucb) unit. current net-cord-fact standards specify that units can be stored for almost hours at room temperature (rt) as long as units are cryopreserved by -hours post-collection. the impact of such delay on hematopoietic stem cell (hsc) function is unclear since most studies have not used transplantation assays that measure hsc key properties and activities. we hypothesized that such processing delay reduces the engraftment activities of ucb units. we set out to measure the loss in engraftment activities associated with preprocessing storage. study design/method: ucb units (n ) were split with one half processed immediately (baseline - hours) and the second after hours storage at rt. ucb were then processed with hetastarch and buffy coat maintained cryopreserved in liquid nitrogen until use. viability was assessed post-thaw, and thawed ucb buffy coat cells were transplanted into nsg mice. serial transplantation was used to test the self-renewal and differentiation activities of hsc, while limiting dilution (ld) assay and poisson statistic were used to estimate the frequency of scid repopulating cells (src) in thawed units. results/finding: storage before processing had no significant impact on the recovery of viable post-thaw cd cells and cd cell (n ). primary nsg mice were transplanted with a ucb cell dose that contained a total of , annexinv neg viable cd cells. the latter was done to avoid any bias towards one group or another. short term platelets ( vs. hplt/ml, p . ) and leucocytes ( . % vs. . % hcd , p< . ) engraftment at -weeks were significantly reduced in stored mice vs. baseline (n ), and similar results were observed long-term at -weeks. long-term human bone marrow (bm) engraftment was also reduced in primary transplants from stored samples ( myeloid engraftment was however confirmed in both groups. bm cells from primary mice were transplanted into secondary recipients and human engraftment investigated months post-transplant. strikingly, the frequency of human cd bm cells was -fold greater in baseline vs. stored mice (p< . , n ). hence, storage at rt of ucb units is associated with a deficit in engraftment activity likely due to a loss in hsc activity and/or numbers. to distinct between both possibilities, the net number of src in baseline and stored samples for two units were calculated by ld transplantation assay. the net number of src measured -weeks post-transplants were reduced by % in unit , and by % in unit . conclusion: prolonged preprocessing rt storage significantly impairs the engraftment activities of ucb units. the reduced engraftment in secondary transplants coupled with the results from the ld assays suggest that this engraftment deficit origins from loss of hsc numbers. our results stress the importance of rapid ucb processing to avoid loss of engraftment activity. acoustic microfluidic separation of blood components charles lissandrello, ryan dubay, kenneth kotz and jason fiering*. draper background/case studies: new cell therapies require efficient and automated methods for purification of target cells prior to subsequent processing. while apheresis, density gradient centrifugation, and magnetic separation achieve some of the requirements, no method is currently available that fully meets clinical needs for a closed, automated, and scalable process. continuous acoustic separation in microchannels is emerging as a versatile method for sorting, separating, and concentrating cells from blood. it has advantages over centrifugation because it is scalable to small or large quantities and can discriminate cells by size as well as density. meanwhile, unlike magnetic methods, acoustophoresis is "label free" and adds no reagents to the therapeutic cells. it has been shown previously that acoustic separation can separate blood components including purification of lymphocytes. however, these studies used devices that were constructed from silicon or glass and have limited potential for scale-up or production as disposable cartridges. in contrast, we report the first ever demonstration of acoustic lymphocyte enrichment along with rbc and platelet depletion in a disposable plastic chip, and we present a cartridge concept that enables clinical scale throughput by linking microchannels in parallel. study design/method: acoustophoresis uses ultrasonic waves to oscillate a rectangular microchannel having a cross section on the scale of the ultrasonic wavelength ($ mm). this results in an acoustic force across the channel that drives cells toward the axial center stream. because the force increases with a cell's size and density, lymphocytes experience a weaker force than rbcs and other classes of wbcs. thus, as blood product flows through the device, the lymphocyte population is enriched at the sides of the channel and can be captured in a branching outlet. likewise, platelets can by separated from lymphocytes. initial and output cell counts are measured by a standard hematology analyzer. results/finding: in our acoustic system, lymphocyte purity (% of total wbcs) was enriched up to %, using leukapheresis product as the starting material. this enrichment was achieved in a single pass through the device (residence time of sec). total lymphocyte recovery was % and monocyte concentration was reduced %. furthermore, in a two-pass process platelets were reduced by %. in a -fold parallel system we tested rbc separation from plasma and achieved % separation at ml/hr. conclusion: acoustic lymphocyte enrichment along with platelet depletion from standard blood product was demonstrated for the first time in plastic microchannels. such disposable devices are suitable for scale up to clinical bioprocessing systems. lymphocyte purity is comparable to existing methods with the advantage of monocyte and platelet depletion and potential for an automated instrument. background/case studies: the use of natural killer (nk) cells as a cellular immunotherapy has increased over the past several years, specifically their use in patients with hematologic malignancies. nk cells have been used at our institution for the past years. most patients have a reaction with nk cell infusion with some reactions being quite severe. we retrospectively analyzed the reactions associated with nk cell infusions to help address why some patients have more severe reactions than others. study design/method: retrospective chart review of nk cell infusions performed at our institution from clinical protocols from - . an infusion reaction was defined as any symptom from the time of nk cell infusion up to hours afterwards. a severe reaction was defined as any symptom with grade or higher severity (graded on common terminology criteria for adverse events-ctcae). preliminary data was analyzed using r . . . two major endpoints of interest were: ) infusion reaction with any symptom and ) severe infusion reaction. to numerically summarize the association of continuous variables with our endpoints, the median, (range) and interquartile range (iqr) were used. a wilcoxon test was performed to test the association between the continuous variables and our end points. a chi-square test was used to test the association between categorical variables and our endpoints of interest. results/finding: there were a total of nk cell infusions. there were ( %) patients with an infusion reaction of any symptom and there were ( %) patients with a severe reaction. infusion rate (ml/min) was similar among those with any reaction (median . , p . ) and those with severe reaction (median . , p . ). infusion rate (ml/min/kg) was also similar among those with any reaction (median . , p . ) and those with severe reaction (median . , p . respectively). incubation of nk cell product overnight in il- vs il- had similar reaction rates for those with any symptom ( % had reaction with il- , % had reaction with il- , p . ) and those with severe reaction ( % had severe reaction with il- , % had severe reaction with il- , p . ). patients with severe reaction had a higher calculated monocyte dose (monocytes/kg) in the nk cell product (median . x ) versus those without (median . x , p . ). conclusion: our preliminary data analysis reveals that a higher number of monocytes in the nk cell product may contribute to severe infusion reactions, causing patients to have a grade or higher symptom. limitations to this study include this was a retrospective review at a single institution. a streamlined mixed lymphocyte reaction (mlr) assay for evaluation of human mesenchymal stem cell immunomodulation activity christopher p delavan , maryanne c herzig* , barbara a christy , james a. bynum and andrew p cap . us army institute of surgical research, u.s. army institute of surgical research background/case studies: mesenchymal stem cells (msc) have been investigated for treatment of acute respiratory distress syndrome (ards), graft versus host disease (gvhd), wound healing and trauma. a consensus is building that the immunomodulation by mscs is key to their therapeutic potential. mscs suppress peripheral blood mononuclear cells (pbmc) proliferation in vitro, suggesting a correlation for suppressing pbmc inflammatory responses in vivo. current mixed lymphocyte reaction (mlr) assays generally rely on either direct co-culture or indirect culture using transwell systems for monitoring the proliferation of isolated pbmcs in the presence of mitotically inactive mscs. in the study detailed here, mscs are analyzed in a direct co-culture with pbmcs using a luminescent atp assay. study design/method: blood was obtained from an in house blood bank and pbmcs were separated by centrifugation over ficoll-paque in leuco-sep tubes as specified by the manufacturer. the pooled donor pbmcs were stored at - . mscs derived from bone marrow, adipose tissue or umbilical cord (bm-msc, ad-msc, uc-msc, respectively) or human umbilical cord endothelial cells (huvec) were serially diluted starting at - , cells/ well and cultured in well plates for - h in their respective medias. on day , mscs were washed, resuspended in pbmc media and incubated with or without , freshly thawed pbmcs/well, in the presence or absence of phytohemagglutinin a (pha, - lg/ml). proliferation of both mscs and pbmcs was assessed in triplicate wells by quantitation of atp levels using the bioluminescent reagent cell titer-glo (promega). results/finding: pbmc proliferation in response to pha gave a robust atp signal by h, with > fold increase over control pbmcs. no increase in atp response or proliferation was seen in the absence of pha. co-culture with mscs inhibited pbmc proliferation dependent upon msc passage, source, msc media additive. intra-assay variance of triplicate samples was . %. inter-assay variation of msc preps run under identical conditions was . %. inhibition of pbmc proliferation was graded from - % over the range msc concentrations therefore an ec of msc cell number resulting in % suppression of pbmc could be determined for each msc prep. this ec however was dependent upon pbmc donor pool. conclusion: direct co-culture of live mscs with freshly thawed pbmcs give a robust determination of immunosuppression by mscs. graded responses can be determined, allowing comparison of potency between msc preparations. this streamlined assay can be performed within h, without irradiating cells and with minimal equipment outlay. background/case studies: a high prevalence of iron depletion (id) in blood donors has been documented by recent studies, but none targeted high school aged donors, who consistently contribute % or more of the us blood supply. differences between donors - years old (yo) and adults in baseline and donation-altered iron status are important to understand because teenagers need increased iron for physiological growth and development and may be more susceptible to harm from iron depletion. study design/method: donors aged - were eligible for ferritin testing if they donated at a high school (hs) blood drive at the start of the / academic year at two blood centers. samples from return donations over the remainder of the school year were also tested. the prevalence of absent iron stores (ais, ferritin < ng/ml) and low ferritin (lf, ferritin < ng/ml) were estimated for , , and - yo groups separately for both genders. linkage to operational databases established first-time (ft) vs repeat (rpt) donor status. linear regression analysis tested for differences in natural log of enrollment ferritin values by age. multiple logistic regression assessed whether young age independently predicts iron depletion controlling for donation frequency and other factors. results/finding: a total of donors contributed donations. donors were evenly split by gender, % were ft donors, and % were - yo. ft and rpt - yo donors had on average lower ferritin values at enrollment (p<. ), and a greater percentage were iron-depleted than donors - yo (table) . in repeated measures logistic regression analysis using data from all visits, female sex, greater number of previous donations, shorter interval since last donation, and lower body weight were risk factors for both ais and lf. controlling for these covariates, donors aged - have sharply higher risk for iron depletion than donors - yo. odds for lf were to times greater in the younger donors, and for ais were -to fold higher. preliminary statistical models indicate yo donors may have greater risk for lf than or yo by to percentage points, controlling for other factors (p . ). conclusion: the prevalence of iron depletion varies markedly by age, sex, and donation frequency, but was considerably higher in - yo donors than in adult controls. logistic regression analysis confirms lower age as an independent risk factor for iron depletion. blood centers should implement measures to mitigate higher risk for iron depletion and the potential adverse consequences for this population of vulnerable donors. mitigation of iron deficiency in young donors -a preliminary report ralph r vassallo*, marjorie d bravo, mary townsend and hany kamel. blood systems, inc. background/case studies: iron deficiency is observed in blood donors who meet regulatory hemoglobin (hb) requirements for blood donation. frequent donations result in negative iron balance and eventually lead to anemia. young donors may be at risk for adverse health consequences (cognitive dysfunction, pregnancy-related complications, fatigue, decreased exercise endurance and pica) even before anemia occurs. study design/method: serum ferritin testing was implemented on / / by a large blood collector. testing was performed on successful - y/o whole blood and apheresis donations. low ferritin (lf) was defined as a value < ng/ml in females (f) and < ng/ml in males (m). donors with low ferritin were notified of deferral from red blood cell (rbc) donations ( months for f and months for m) and counseled to take - mg of elemental iron daily for days. for m and f, a ferritin < ng/ml indicated absent iron stores (ais) and < ng/ml indicated iron deficient erythropoiesis (ide). ferritin levels ! ng/ml in f and ! ng/ml in m were considered as indicating an iron-replete state. conclusion: ferritin testing of young donors identified individuals with lf who would benefit from risk mitigation, e.g., delaying subsequent rbc donations and/or taking iron supplements. lf is more common in f than in m donors. lf is more prevalent in m and f donors with any rbc donations in the prior months. an appreciable number of donors with no rbc donations in the prior months presented with lf. these data may be useful in conducting a riskbased decision making exercise to establish recommendations for risk mitigation which could be different for m than for f, e.g., universal iron replacement in teen male donors may not be warranted above a certain hb value. ferritin blood screening in minor or young adult donors jennifer l ritter* , joan williams , michelle humphries , nancy haubert , ben reynolds , michael phillips , randall spizman , ralph r vassallo , hany kamel , sally caglioti , german leparc , and phillip c williamson . abstract completely investigated. the adolescent growth spurt, poor nutrition and onset of menses increase the risks of iron depletion in young donors. new studies show that teenage donors who give blood frequently may be more susceptible to becoming iron deficient than older repeat donors. study design/methods: over , serum samples from donors aged , and years were analyzed for ferritin levels using the beckman coulter au instrument and reagent kit. the anti-ferritin reagent is a suspension of polystyrene latex particles, of uniform size, coated with polyclonal rabbit anti-ferritin antibody. immune complexes formed in solution scatter light in proportion to their size, shape and concentration. the decrease in light intensity is measured spectrophotometrically. results/findings: background/case studies: the risk of cardiovascular (cv) disease in adults can often be identified during adolescent years. the presence of even borderline levels of multiple risk factors increases the likelihood of a cv event. our blood program routinely provides a total non-fasting cholesterol (tc) and blood pressure (bp) measurement for all blood donors. we added glycated hemoglobin (hba c) determination and performed analyses of the prevalence of abnormal (borderline or elevated) levels of multiple risk factors among , adolescents (ages - ; . % female) who donated blood from to . study design/method: abnormal risk factor levels were defined as hba c ! . %, sbp/dbp ! / mm hg and tc ! mg/dl, as suggested by the american heart association for adolescents. the presence of isolated risk factors was defined as one single abnormal risk factor per individual. clustering of risk factors was defined as the presence of or more abnormal risk factors in the same individual. donor sex was recorded at the time of donation. results/finding: table shows the prevalence of isolated abnormal risk factors and the prevalence of abnormal risk factor clustering in the study cohort. overall, , ( . %) adolescents had at least one abnormal risk factor ( . % of males, . % of females). of these, , adolescents had isolated abnormal risk factors, and , adolescents had clustering risk factors. higher proportions of males were in the abnormal bp alone, background/case studies: pre-donation determination of hemoglobin (hb) level in candidate blood donors is a pre-requisite in the majority of blood services and is used to ensure donor safety and blood product quality. however, a variety of hb testing strategies are used across blood services to satisfy this selection criterion. this study aimed to identify how hb screening practices vary across blood donation services and to what extent they influence deferral rates for low hb. study design/method: an online survey was performed among members of the biomedical excellence for safer transfusion (best) collaborative. additionally, data from literature were used to extend the dataset. the survey involved a detailed assessment of hb screening practices, numbers of donations and low hb deferrals for male and female donors separately. multivariable negative-binomial regression models were built to estimate the adjusted effects of minimum donation intervals, hb cutoffs (high/low with high defined as ! . g/dl for men and ! . g/dl for women), iron monitoring (y/n), iron supplements (y/n providing or prescribing), and geographical location on deferral rates due to low hb. results/finding: data were included from blood services worldwide and complete data were available for blood services. deferral percentages for low hb varied from . % to . % among male donors and . % to . % among female donors. hb deferral rates were notably higher in asian blood services. overall, iron monitoring was associated with % lower hb deferral rates in men ( % confidence interval [ci] % to %) and % lower rates in women ( %ci % to %). iron supplementation was associated to % lower hb deferral rates among women ( %ci % to %) but there was no evidence of such an effect among men (p . ). each one-week increase in minimum donation intervals resulted in % lower hb deferral rates among women ( %ci % to %) but not among men (p . ). at the % level of significance, higher hb cutoffs do not appear to have an effect among men or women. conclusion: the variation in hb deferral rates across blood donation services can be, particularly in female donors, explained by differences in hb screening and deferral practices. mitigation strategies should consider the variable response among men and women. these insights can help improve both blood service efficiency and donor care. were: characteristics of donors (age, sex, size, weight, region); hb levels, date and volume of donation for index application and previous donation; and number of previous donations (in the previous years and the lifetime). data were analyzed using logistic regression stratified by sex. results/finding: . % of all candidates for wb donation were deferred in continental france in . deferral was significantly more frequent in women ( . %) than in men ( . %), due to anemia in . % of deferred women and . % of deferred men. plotting mean hb recovery against time showed mean recovery times ranging from to weeks. analysis (table) identified main factors associated with a higher likelihood of hb recovery: higher logarithm of time since previous donation, lower levels of hb at previous donation, higher number of blood donations in the previous years. conclusion: the main factors associated with higher likelihood of hb recovery after wb donation are probably linked with hematopoiesis stimulation and selection bias among high-frequency donors. mean times required for hb recovery were long enough to require further studies to assess interdonation intervals in france. background/case studies: red blood cell (rbc) transfusion has been related to thrombo-embolic events. microvesicles in the rbc product may support coagulation, which in part may depend on storage time because microvesicles have procoagulant effects in vitro and the amount of microvesicles increase with storage duration. study design/method: we investigated whether transfusion of rbcs containing microvesicles promotes coagulation in human recipients. as transfusion is mostly administered to ill patients, we used a model of mild endotoxemia. eighteen healthy volunteers were randomized to receive either saline, days stored or days stored autologous rbc transfusion two hours after infusion of lipopolysaccharide (lps, from e.coli, ng/kg). blood was sampled every hours up to hours after lps infusion. results/finding: lps resulted in a mild increase in thrombin generation. during storage, the total number of microvesicles increased from . e (iqr . e - . e ) /ml in the fresh product to . e (iqr . e - . e /ml; p< . ) in the stored product (p < . ), which were mostly rbc derived vesicles. after transfusion, microvesicles from stored rbc products, but not from fresh products, could be detected in the circulation of healthy volunteers and were cleared within hours. however, infusion of stored rbc microvesicles did not augment thrombin generation. levels of d-dimer and thrombin-antithrombin complex were also unaffected. conclusion: transfusion of autologous rbcs containing high levels of microvesicles does not enhance coagulation in human volunteers with mild endotoxemia. background/case studies: transfusion-associated circulatory overload (taco) is characterized by hydrostatic pulmonary edema related to blood transfusion. we sought to examine contemporary risk factors and outcomes for taco during a period where patient blood manaement has led to declines in blood utilization. study design/methods: at four academic hospitals, cases of taco were detected by active surveillance of all adult hospitalized patients who received a blood transfusion, and transfused controls were matched to cases by transfusion intensity. taco incidence was calculated, and clinical characteristics were compared with control patients. odds ratios (or) were calculated using multivariable logistic regression. hospital mortality and length of stay were modeled using cumulative incidence functions in proportional hazards regression. results/findings: cases of taco and matched controls were enrolled from , transfused patients who received , blood components from may until july . taco incidence was case per patients transfused. in addition to well described cardiac and renal comorbidities, multivariable analysis identified the following independent predictors of taco: number of plasma units, emergency surgery, pre-transfusion diuretic use, and higher post-transfusion hemoglobin levels (see table) . compared to controls, taco cases were more likely to require mechanical ventilation ( % vs. %; p < . ), experienced longer intensive care ( vs. days; p . ) and hospital length of stay following transfusion ( vs. days; p< . ), and had higher mortality ( % vs. %; p . ). conclusion: the incidence of taco was lower than what has been reported by prior active surveillance studies. despite declines in its incidence and the number of blood components transfused per case, taco remains a complication of transfusion with significant associated morbidity and mortality. in addition to risk factors for cardiovascular and kidney disease, plasma transfusion and higher post-transfusion hemoglobin levels were associated with taco after controlling for other covariates in the model. additional research is needed to examine the utility of these risk factors in the development of real-time predictive algorithms and the benefit of reduced erythrocyte or plasma exposure in patients at high risk for taco. background/case studies: the residual risk of bacterial contamination of single-donor apheresis platelets (ap) was recently addressed by the march fda draft guidance to enhance the safety of platelet transfusion. this document also describes an existing pathway for ap outdate extension from to days using an fda cleared rapid test (rt). our hospital based transfusion service has used this rt to enhance the safety of ap transfusion since july and to routinely extend ap outdate to day since february . this study reports a month experience of secondary screening of ap using a rt. study design/methods: all ap were obtained from our hospital-based donor center or one of four external suppliers. ap were screened by culture based methods post-collection and prior to entry into our inventory. from july -january , ap underwent rt on day . day and units were transfused with physician approval when deemed medically necessary. any units remaining in inventory on day had a second rt performed. from february -january , ap underwent rt on day with routine outdate extension to days by performing a second rt on day and a third rt on day , as per manufacturer instructions. any positive rts were repeated in triplicate. repeat rt positive units were quarantined and cultured to identify true positives. false positives (fp) were defined as repeat rt negative (type ) or repeat rt positive with negative confirmatory culture (type ). all rt results were reviewed during both study periods. ap transfusion and outdate rates were also summarized. results/findings: since july , , ap were entered into inventory. of these, , ( %) were transfused prior to rt testing. the remaining ( %) underwent rt on day or day . of these ( . %) were rt positive ( type fp, returned to inventory; type fp, discarded), leaving a total available inventory of units tested by rt. of these, ( % of original inventory) were transfused before the end of day and the remaining ( % of original inventory) reached a day outdate. a total of ( % of original inventory) were transfused on day or day . of these, underwent a second rt on day ( rt positives; fp type one and fp type ) and underwent a third rt on day (no positive results). a total of ( % of original inventory) outdated on day . of these, underwent a second rt on day (no positive results). conclusion: to date we have performed rts on ap at our hospital. no true positives have been identified. use of rt over the study period decreased our outdate rate from a predicted % to only %. a total of ap have been tested twice by rt ( on day and ; on day and ) with ( . %) positive results, both of which were deemed fp by repeat testing or culture. a total of units have been tested times (day , day and day ) with no additional positives identified. we have not yet identified any units with an initial negative rt result that subsequently converted to a true positive. there is a low fp rate which should also be expected when performing repeat testing on the same unit. these data suggest that the yield for repeating the rt every hours, as currently specified by the manufacturer instructions, is quite low. additional studies are needed to clarify how rt can optimally be used to enhance detection of ap bacterial contamination. survival of trypanosoma cruzi in human blood components laura tonnetti*, aaron thorp and susan l stramer. american red cross background/case studies: trypanosoma cruzi, the agent of chagas disease, is associated with to million infections worldwide, mostly in latin america. despite the extensive immigration from endemic areas, only cases of transfusion-transmission (tt) t. cruzi have been reported in the us, before blood donor screening was implemented in . contributing factors to the low number of tt cases are a possible association between parasite lineage and tt, and high numbers of unreported cases. platelets are almost exclusively involved in t. cruzi tt cases; however, during preparation of components a large fraction of the parasites can be found in red blood cells (rbcs). we investigated if blood component preparation and storage time affect the survival of the parasite and thus play a role in tt of t. cruzi. study design/method: whole blood (wb) units were spiked with t. cruzi trypomastigotes to a final concentration between - , parasites/ml. each parasite concentration in wb was tested x . an aliquot of contaminated wb was used to prepare hemocultures to detect live parasites before preparation of components. rbcs were separated and half of the components leukoreduced (lr) by filtration. platelets and plasma were separated, along with one aliquot of plasma collected before lr. rbcs were stored at c for up to days; platelets were stored at c (rt) under agitation for days and plasma was frozen at - c. aliquots for culture were removed weekly from rbcs, daily from platelets and after days from frozen plasma. all samples were cultured in liver infusion tryptose (lit) media at c for detection of live parasites for up to weeks. results/finding: hemocultures from spiked-wb were positive at all concentration of parasites. lr'd and non-lr'd rbcs cultured before storage were positive at all concentrations. after storage at c, rbcs from all units spiked with , parasites/ml were positive for up to days; all further times yielded negative results. at lower concentrations, only non-lr'd rbcs spiked with parasites/ml were positive for up to days. plasma samples cultured before freezing were positive at the highest concentration in one non-lr'd sample, while all others were negative. platelets obtained from wb spiked with , and parasites/ml were positive up to days at rt. no parasites were observed in plasma or platelets prior to storage at lower concentrations. molecular analysis to determine the presence of parasite dna in each component is on-going. conclusion: platelet storage conditions offer a suitable environment for t. cruzi survival; however, high concentrations of parasites also survived in rbcs at c for up to weeks. leukoreduction offers partial protection, while freezing conditions appears unsuitable for t. cruzi survival. hemovigilance monitoring of platelet septic transfusion reactions (str) after treatment with intercept tm pathogen reduction or large volume, delayed bact/alert tm bacterial culture screening richard benjamin* , marion lanteri and larry corash . cerus corporation, scientific affairs department, cerus corporation background/case studies: amotosalen/ultraviolet a (uva) light (inter-cept tm blood system, cerus corporation) pathogen reduction (pr) and delayed, large volume, bacterial culture with the bact/alert tm system (dlvbc) (biomerieux, inc) represent respective best-in-class systems to reduce the risk of str associated with platelet concentrates (pc). where implemented, hemoviligance (hv) programs continue to receive reports of suspected str, most of which have low imputability as other causes are more likely or insufficient information is available to impute system failure. study design/methods: united kingdom ( - ), french ( - , swiss ( - ), and belgium( - hv reports, and cerus corporation's adverse event records were reviewed to assess the residual risk and imputability of str with amotosalen/uva-treated or dlvbc-screened pc. results/findings: approximately . million dlvbc-screened were issued with a day outdate after release into inventory days after collection, and $ . million amotosalen/uva-treated pc were released into inventory on day or , with a to day shelf-life. no septic fatalities were reported with either technology. the french, belgium and swiss hv programs monitored > . million conventional, non-dlvbc-screened pc and recorded str and fatalities. concurrently, zero definite and possible str were reported with , amotosalen/uva-treated pc, significantly fewer than with conventional pc (table ) ( . str per million vs. . per million, p< . ). one definite, possible, undetermined/indeterminate non-fatal str and contaminated "near miss" pc were reported with . million dlvbc-screened pc between and , for a reduced falsenegative rate compared with the prior five years ( . str per million vs. . per million, p < . ). hv programs highlight a major weakness when reporting str. stringent criteria are used to determine definite imputability, including evidence of patient infection, pc contamination and irrefutable evidence of a donor source, with confirmation of strain identity. reports with incomplete investigations are considered undetermined or indeterminate, or possible sepsis. some of these cases are almost certainly due to bacterial contamination of pc, suggesting that the actual rates of sepsis are considerably higher than that reported by hv programs. conclusion: best-in-class pathogen reduction and bacterial culture systems reduce str risk, although underreporting and inadequate clinical data may result in underestimation of the true rates. pathogen reduction of background/case studies: despite extant mitigation measures (e.g. diversion pouches and primary platelet culture at the collection facility), bacterial contamination of platelets and associated septic transfusion reactions remains a leading cause of transfusion-associated fatalities in the united states (us). consequently, the us food and drug administration has recommended adoption of additional measures such as point of release testing (port) and/or pathogen reduction to safeguard against transfusionassociated sepsis. however, port poses logistical challenges, particularly in institutions with high-volume platelet utilization, while pathogen reduction is a high cost intervention. we evaluated a second bacterial culture to contend with residual risk. study design/method: phased implementation of secondary bacterial culture testing (bact/alert tm ,biomerieux, inc., durham, nc) was initiated in october for all platelets received at our institution. at time of receipt at the blood bank (day post collection), products were sampled using a sterile connection device (tscd tm , terumo, elkton, md) and a sampling kit (sam-plok tm sampling kit, ml, itl biomedical, malaysia). five mls of product was transferred aseptically to bact/alert bpa (aerobic) culture bottles using the same sampling device. inoculated culture bottles were loaded into the bact/alert incubator modules and incubated at c for three days. results/finding: a total of / , ( . %) platelet products were successfully cultured ( / [ . %] and / [ . %] in october and march respectively). over the -month period, two true positive cultures were obtained (incidence of in platelet products). the cultures grew acinetobacter species (case a) and coagulase negative staphylococcus species (case b); both positive results were obtained four days following collection. repeat testing of cases a and b grew the same organisms identified in the initial cultures. there was a co-component in our inventory (case a) with negative initial and repeat cultures. none of the products were released for transfusion. the initial post-collection product cultures remained negative at the collection facility. over the same time period, no false positives were detected. implementation required hiring one additional dedicated fte; the total cost (technologist time, equipment and related supplies) was calculated to be $us . per product tested. the cost per averted case was $us , . conclusion: we demonstrate the feasibility of implementation of a secondary bacterial culture test of apheresis platelets to interdict bacterially contaminated units and prevent septic transfusion reactions. this presents a low-cost strategy (as compared to pathogen reduction) to mitigate risk of septic transfusion reactions. importantly, it offers a viable alternative to port in high volume institutions where logistic (e.g. time and personnel) constraints impede practical adoption of port. an increase in cases of blood culture positive transfusion reactions (bcptr) was noted at our hospital; bcptr was defined as bacterial culture positivity in the transfusion recipient and/or associated transfused blood product during investigation of a transfusion reaction. we sought to characterize the risk and clinical presentation of bcptr at our institution. study design/method: an analysis was conducted of all reported transfusion reactions at johns hopkins hospital (jhh) between january and december . the data, extracted from hemovigilance records, were evaluated to determine the incidence of bcptr; the severity and symptoms were evaluated in concordance with recipient data, including patient diagnosis, medications and clinical manifestations of the reaction. bacterial culture results were evaluated for both patients and associated blood products (i.e. partially transfused or residual product in blood bag). results/finding: in the -year study period, a total of transfusions reactions were reported, of which were bcptr ( . % of transfusion reactions). of the bcptr, ( %) were associated with apheresis platelets, ( %) with red blood cells, and ( %) with plasma. recipient diagnoses spanned hematologic/oncology (n ), renal (n ), cardiac (n ), autoimmune (n ), and obstetrics (n ). an organism was identified in both the blood product and recipient in ( %) cases; in ( %) cases an organism was grown in the blood product but not the recipient; and in ( %) cases an organism was isolated from the recipient only, due to inability to culture the product. the transfusion recipients in of the cases that did not isolate organisms in the recipients were on broad-spectrum antibiotics at the time of transfusion. symptoms of bcptrs included fever ( %), chills ( %), nausea and vomiting ( %), pain ( %) and dyspnea ( %). blood pressure (bp) decreased in %, increased in %; % of reported bcptrs had no change in bp. conclusion: the signs and symptoms of bcptrs are not specific and overlap both with underlying disease as well as other types of adverse transfusion associated events, thus contributing to delayed diagnosis and under-reporting. furthermore, high rates of antibiotic use in transfusion recipients can mask symptoms of true septic transfusion reactions. hospitals should consider expanding the clinical indications for culturing blood components that are implicated in transfusion reactions. furthermore, excessively stringent criteria (cdc/nhsn blood safety surveillance) for transfusion-transmitted infection, may contribute to misclassification of septic events in some recipients, particularly if on antibiotics. clinical oral abstract session: immonohematology and genetics --sickle cell disease and beyond blindspots and cross-reactivities of anti-human globulin specific for igg subtypes heather howie , jenna lebedev , linda kapp , xiaohong wang , meghan delaney , lay see er and james c zimring* . bloodworksnw research institute, bloodworks nw, university of washington school of medicine background/case studies: there are four different subclasses of human igg (igg -igg ), each with different effector function. essentially all existing data on the effect of igg subclass on hemolytic transfusion reactions and hdfn, were generated using ahg specific for igg subclasses. in recent decades, it has become appreciated that there are at least natural human variants of igg. in this study, the reactivity of igg specific ahg was tested against all known variants. study design/methods: the heavy and light chain variable regions of an anti-k monoclonal antibody were sequenced and cloned into expression plasmids that fused variable regions (in frame) with each of the known igg variants. plasmids were expressed by co-transfection into cho cells. the resulting panel of antibodies were pre-incubated with k rbcs and were then subjected to testing with currently available igg subtype specific ahg (monoclonal ahgs from southern biotech and sanquin, polyclonal ahgs from sanquin and the bindingsite). all testing was carried out by flow cytometry. results/findings: polyclonal reagents against igg , igg , and igg had cross-reactivity with variants found in other igg subclasses, and specific amino acids responsible were identified by site directed mutagenesis (table ). titrations of the ahgs did not identify a dilution at which crossreactivities were lost, but authentic targets were still detected. however, cross-reactivity could be neutralized by pre-incubating ahg with the crossrecognized igg forms (against a third party antigen); the remaining reactivity recognized the intended igg subtype without detectable cross-reactivity. no cross-reactivity was detected for polyclonal anti-igg or for any of the monoclonal ahgs tested. monoclonal anti-igg had a blindspot for igg - , due to the shorter hinge region on igg - . no blindspots were detected in other monoclonal or polyclonal ahg. conclusion: the relative quantitation of different igg subtypes has been studied in multiple immune settings, and plays important roles in diagnosis and research of human disease, including immunohematology. herein, we demonstrate that the reagents used to generate this body of knowledge suffer problems of cross-reactivities and blindspots. as such, the existing data regarding igg subtype biology may have some inaccuracies as a result of these defects in igg specific ahg. genotype matching for pediatric sickle cell disease patients nancy robitaille* , yves dominique pastore and maryse st-louis . chu sainte-justine, hema-quebec background/case studies: among the different treatment modalities available for sickle cell disease (scd), blood transfusion is frequently used. however, alloimmunisation remains a significant problem, even if prophylactic antigen matching is performed for c, e and kell antigens. this is partly explained by different antigen frequency among caucasian blood donors and african-american recipients, and by variants in the rh blood group of people of african-descent. blood group genotyping has been proposed as a potential way to alleviate this problem. the scd cohort of a pediatric academic hospital was genotyped for rhd, rhce and fy genes. the primary objective of our study was to evaluate whether compatible genotyped blood donors presenting similar rh variants could be identified. study design/methods: since , our local blood provider intensified recruitment of african-descent blood donors. these donors were phenotyped and genotyped for clinically relevant antigens by different means: genomelab snp stream, laboratory-developped assays and idcorext. as of , scd children were genotyped by sequencing rhd, rhce and fy cdnas after obtaining informed consent. extended red blood cell phenotypes were done at diagnosis at the hospital. patients' genotypes were compared to h ema-qu ebec's donor database to attribute blood donors to specific patients. results/findings: from diagnosis until september , ( %) patients had been transfused and had antibodies with known blood group antigen specificity: anti-c, anti-e ( ), anti-hrb, anti-fya, anti-jka, anti-jkb ( ), anti-s, anti-m, anti-sc , anti-leb ( ). seventeen patients ( . %) were either d or partial d. rhce results showed that patients expressed a normal c antigen and expressed partial c. as for e antigen, had a normal antigen, bore a partial antigen and were weakly expressed. fy(a b ) phenotype was found in ( %) patients. a total of genotyped blood donors of african-descent were available. the table below indicates the compatibility with these donors. conclusion: this study shows that several patients have rhce variants difficult to match, even with available genotyped blood donors from their community. although this measure is probably beneficial to decrease alloimmunisation, a larger donor pool is still needed to fulfill the patients' needs. the continued effort put towards recruitment and pheno/genotyping should improve the situation. using genetic markers to select responders and non-responders sickle cell disease (scd) patients for transfusion with rh haplotype matching red blood cell (rbc) units tamires delfino dos santos , emilia sippert , mayra dorigan de macedo , sheila fatima perecin menegati and lilian castilho* , . hemocentro unicamp, university of campinas background/case studies: rbc alloimmunization has been associated with several factors and with individual characteristics of each patient. we recently found that tnfa- a, il b- t cytokine polymorphisms, rhag g>a and hla-drb * alleles may predict a good responder phenotype (sippert et al, transfusion ) and that rhag a and hla-drb* alleles are closely linked to rh alloimmunization. based on this and considering the challenge to fulfill the transfusion needs of the patients with rh variants, we used these genetic markers to select responders and nonresponders scd patients for transfusion with rh haplotype matching rbc units and evaluated the risk of alloimmunization. study design/method: our study included non-alloimmunized patients with scd, homozygous for hbs, receiving a range of - rbc units. rbc antigen phenotypes of each patient and history of rbc antibodies were obtained from the medical records and transfusion service computerized database. rbc genotyping was performed using whea, wrhd and wrhce beadchip arrays (bioarray solutions, immucor) in accordance with the manufacturer's instructions. cytokine gene polymorphisms (tnfa- g>a, il b- c>t) and the rhag g>a gene polymorphism were analysed by pcr-rflp and taqman assays. hla class ii genotyping was performed using pcr-sso. results/finding: among non-alloimmunized patients, were homozygous or compound heterozygous for rh variant alleles. from those, had rhag a and/or hla-drb* alleles and at least one cytokine polymorphism (tnfa- a or ilb - t) associated with risk of alloimmunization and were transfused with extended and rh haplotype matching rbc units. the other patients with no risk factors associated with rbc alloimmunization were considered non-responders and were not transfused with extended and rh matching units. all patients were followed for one year and did not develop rbc antibodies. conclusion: these findings contributed to the development of a transfusion strategy for non-alloimmunized scd patients as typing for these polymorphisms could potentially help in the classification of responder and nonresponder scd patients, allowing blood with high level of compatibility to be five discrepant samples required sequencing. id core xt identified three rhce*cear samples encoding a partial c, and a partial e (predicted phenotype: vweak, vs-) and were confirmed by sequencing. the third sample was found to be rhce*cevs. ,rhce*cebi on sequencing (predicted phenotype v ,vs ). the samples were typed as v (or ce s ) and vs (or e s ) by hea. in addition, id core xt accurately identified rhce*ce[ g]in samples. this snp has been linked to various allelic variants affecting c and e antigenic expression. both samples were predicted to be c by hea. conclusion: blood group genotyping platforms vary depending on the specific snps that are included in each assay. such variations may be clinically significant when genotyping is used as a tool for providing matched blood. discrepancies leading to differences in the predicted phenotype could affect unit selection. despite the discrepancies between the methods, the high concordance rate and the limitations of serology warrant further reconsideration for the need for serologic confirmation of extended phenotypes. background/case studies: over three decades ago, two independent groups published work suggesting a novel categorization of warm autoimmune hemolytic anemia (waiha) on the basis of dat scores of agefractionated rbcs: type i waiha, comprising % of patients, showed increased binding of autoantibodies to aged rbc, whereas type ii waiha autoantibodies ( % of patients) bound young and old rbcs with no apparent prejudice. band- is a ubiquitously expressed rbc transmembrane protein which plays a vital role in maintenance of rbc structural integrity, cellular hemostasis, and regulation of senescence; and, has been suggested to be targeted by autoantibodies from patients with waiha. band- is regulated through phosphorylation of key residues; its hyperphosphorylation is a hallmark of normal rbc senescence, which causes band- to disengage from the cytoskeleton, increasing its lateral diffusion, thereby permitting the formation of band- aggregates forming new epitopes which are recognized by natural igg autoantibodies causing phagocytosis and destruction of senescent rbcs. type i waiha has been postulated to be caused by an exacerbation of normal rbc senescence. study design/methods: in an effort to confirm and characterize the two waiha subtypes we age-fractionated whole blood samples from patients with waiha on discontinuous percollv r gradients and looked for differences in dat results between less (young rbcs) and more dense (aged rbcs) fractions, fractionation patterns and band- tyrosine phosphorylation. results/findings: we confirm that two distinct types of waiha can be identified based on autoantibody reactivity with the youngest and oldest autologous rbcs. further, comparing type i and type ii patients, we found that type i is characterized by percollv r fractions (similar to healthy storage-matched controls) but increased band- tyrosine phosphorylation compared to healthy storage-matched controls, with phosphorylation occurring during younger stages of rbc development. type ii patients were characterized by - percollv r fractions, lacking the fraction containing the oldest rbcs, and showed a complete lack of, or dramatic decrease in, band- tyrosine phosphorylation compared to healthy storage-matched controls. conclusion: these results confirm the two distinct types of waiha. in type i waiha, the increased binding of autoantibodies to older rbcs coupled with increased tyrosine phosphorylation of band- suggests that rbcs from type i patients are aging faster than rbcs from normal healthy controls; this may represent an accelerated and pathogenic form of normal rbc senescence. in contrast, type ii waiha where autoantibodies bind strongly to either young or old rbcs coupled with a lack of fractionated bands that represent the oldest rbcs and a dramatic diminution in tyrosine phosphorylation of band suggests faster destruction of rbcs, consistent with the early published data, and metabolic changes that could affect rbc function. microbial pathogen primary sequence correlates with blood group antigen immunogenicity ian baine* , burak bahar , jeanne hendrickson , krystalyn e hudson and christopher a tormey . yale-new haven hospital, yale university, background/case studies: it is known that specific groups of patients immunologically respond more readily than others to rbc antigens. while rbc antigenic differences between donors and recipients are required for humoral immune responsiveness, other variables are also involved. studies have shown that there is significant primary sequence identity between common rbc antigens and microbes, and that cross-reactivity is possible between antigens in experimental models. we hypothesize that responder populations may be immunologically primed to form rbc alloantibodies via environmental exposure to cross-reactive microbial antigens, and that such a correlation may be linked to observed blood group antigen immunogenicity. study design/method: we performed peptide homology searches of the most immunogenic rbc antigens, based on previously published antigenicity findings. thirteen amino acid peptides containing the polymorphic residues of k, jk a , lu a , e, c, m, c, fy a , and s antigens were queried for identity with microbial peptides using the blast database (blastp, pam abstract algorithm, e value x - , word size , gap costs: existence exten-sion ). search results were restricted to bacteria and fungi, with a selective threshold of > % identity set for inclusion criteria. to corroborate with observed patient data, we also examined preceding cultures from alloimmunized patients to explore agreement between specific pathogens and rbc alloantibodies. results/finding: significant peptide identity was found between rbc antigens and pathogenic organisms including b. fragilis, p. aeruginosa, candida spp. among others. linear regression analysis of the number of genuses in microbial kingdoms meeting inclusion criteria showed a statistically significant inverse trend in predicting the degree of immunogenicity when fy a (an outlier) was removed (b - . , r . & p . ); that is, lower immunogenicity antigens were associated with larger number of kingdoms. k-medoids cluster analysis comparing immunogenicity and kingdoms showed that antigens clustered to low (c), moderate (e, c, s, m) and high (k, jk a , lu a , fy a ) immunogenicity groups, suggesting that an antibody response is inversely associated with environmental antigenic prevalence. of alloimmunized patients reviewed, were culture-positive. of these, % of the anti-c/c group ( of patients) and % of the anti-k group ( of patients) had microbe-antibody agreement. remaining microbe-rbc antibody agreements ranged from - . %. overall, . % ( of patients) demonstrated agreement. interestingly, we observed a particularly strong agreement between infection with klebsiella species and anti-k, despite the lack of > % sequence identity. while . % ( of ) patients reviewed had positive cultures for klebsiella species, . % of these ( of patients) demonstrated an anti-k. conclusion: our study highlights the potential connection between microbial infection and rbc alloimmunization, based on shared epitopes. we speculate that low-level antigenic exposure to highly prevalent microbial antigens such as commensals may promote immunotolerance, providing a model for the inverse relationship between rbc antigen immunogenicity and prevalence of microorganisms. longitudinal studies of microbial carriage (or acute microbial infection) and rbc alloimmune responses in larger patient cohorts may be informative. background/case studies: thromboelastogram (teg) has been incorporated into many hospital armories to manage transfusions during cardiovascular (cv) surgeries. some institutions use well-defined protocols for teg utilization at different stages of surgery (baseline, rewarming, postprotamine, and post-operative). on the other hand, at some institutions teg utilization is driven mainly by clinical judgment. when teg is ordered based on clinical judgment (clinical bleeding in most cases), some patients receive blood transfusions before teg is performed. there is no published literature on how pre-teg transfusions impact teg results and guide further transfusion requirements during cv surgeries. in this study, we have tried to address this issue. study design/method: we retrospectively reviewed tegs performed on patients undergoing cv surgeries at our institution from jan to dec , . no specific teg protocol was used to direct transfusions (plasma, platelets, and cryoprecipitate) during that period. only the first teg performed during surgery was included in the analysis. we excluded the patients that received only red blood cell (rbc) transfusions during the surgery because rbc transfusions are usually not based on teg results. for the tegs analyzed, teg results were divided into three categories: "normal" (reaction time (r), kinetics (k), angle (a), maximum amplitude (ma), and lysis at minutes (min) all within reference range), "hypocoagulable" (r> min, k> min, a< degrees, ma< mm) and "hypercoagulable" (r< min, k< min, a> degrees, ma> mm). fisher's exact tests and z-scores for two population proportions were used to identify statistically significant differences in teg results and blood product utilization. results/finding: out of tegs analyzed, patients ( %) received pre-teg transfusions. we found significantly fewer hypocoagulable teg results in pre-teg transfused patients than nontransfused patients ( % vs. %, p . ). the data also reflected a trend suggesting that there may be more normal teg results in pre-teg transfused patients compared with nontransfused ( % vs. %, p . ). there was no statistically significant difference in transfusions after obtaining teg results in both groups. however, there was a trend suggesting that hypocoagulable state was more likely to be corrected by transfusion in patients who were already transfused pre-teg compared to nontransfused ( % versus %, p . ). conclusion: pre-teg transfusions impact teg results (transfusions correct/normalize coagulopathy) but do not significantly impact further blood product utilization during cv surgeries. the decreased threshold (more transfusions) for correcting hypocoagulable state in patients who already received pre-teg transfusions may be due to more clinical significant bleeding in these patients to begin with. background/case studies: orthotopic liver transplantation (olt) is associated with significant blood loss, due to the complexity of the procedure and extensive liver vascularity, demanding blood transfusion. in this setting, cell salvage autotransfusion (cs) is been used as an alternative to decrease allogeneic red blood cell transfusion. however, as long as some studies have shown that cs in olt decreases allogeneic blood transfusion, others reported that cs presented little benefit or might have been associated with increased blood loss through fibrinolysis. in this study, we evaluate cs efficacy in reducing allogeneic blood transfusion in the intraoperative period. study design/method: we retrospectively evaluated data from liver transplants, performed from to in a single-center. patients were divided in two groups: one with cell salvage (cs) and another without cs (ncs). study endpoint included the requirement of allogeneic blood components transfusion during intraoperative period in both groups. cs was used in all liver transplant recipients but patients with malignancy and sepsis. blood transfusions were indicated based on clinical and hemodynamic criteria. clinical data included age, gender, diagnosis, body weight, height, warm and cold ischemic time and model for end-stage liver disease (meld) score. statistical analyses were performed using t-test, chi-square test, mann whitney test. results/finding: in this study period, olts were performed. a total of patients was submitted to cs. the median age was years (range - yo). cirrhosis caused by chronic hepatitis c virus infection was the main etiology of liver disease. hepatocellular carcinoma (hcc) was found in , % of the patients. the average meld score was , , and it was slightly higher in the cs group ( , vs , , p< , ) . there was no statistically significant difference in other variables such as body weight, height and cold ischemic time. the mean salvaged blood volume was ml and mean reinfused blood volume was ml. allogeneic blood transfusion was required in , % patients in the cs group, compared to , % patients in the ncs group. however, average red blood cells (rbc) and fresh frozen plasma (ffp) units transfused were lower in the cs group. the threshold for rbc transfusion was significantly lower in the cs group ( , units vs , units, p< , background/case studies: hemorrhage is a leading cause of mortality in trauma patients and morbidity in non-trauma patientsaddin en.cite.data. massive transfusion protocols (mtp) reduce mortality in trauma and nontrauma settings; however, this may be at the cost of blood product wasta-geaddin en.cite.data. blood product wastage benchmarks are loosely established, and data on wastage associated with mtps especially sparse. with a redesign of mtp and obstetric massive transfusion protocols (obp) which have different blood product preparation schedules, we assessed wastage, delivery method, and product utilization to identify differences in wastage during these protocols. study design/method: following institutional review board approval, a retrospective study on blood product wastage associated with the mtp and obp between july -december was performed. data on numbers of products dispensed and wasted were manually collected from transfusion service paper and electronic records and an automated data report from the electronic medical record. results/finding: the mtp resulted in higher total number of wasted products than the obp ( and products, respectively) however, obp wastage occurred more frequently in the month period. this reflects automatic thawing of cryoprecipitate in the first round of deployed products in the opb. mtp-trauma activations contributed higher wastage than non-trauma activations ( versus products). this is skewed by one month when products were wasted due to expiration of product on the floor. cooler-related issues ( ) and products dwelling too long out of a controlled environment ( ) were common reasons reported for wastage. the overall product wastage rates for mtp: trauma, mtp: nontrauma, and obp were . %, . %, and . %, respectively, with a total exsanguination protocol waste rate of . %. the difference between the overall proportion of waste between the mtp and obp protocols was insignificant (p . ). conclusion: wastage associated with both protocols was low and there is no statistical difference between mtp versus obp wastage. coolerrelated issues accounted for most product wastage, allowing for targeted waste reduction strategies including educational outreach and improved product delivery methods. better documentation of waste events identifies wastage trends for further product utilization optimization during these protocols. a year old female with multiple gun shots was admitted to a level one trauma center and received uncrossmatched group o, rh negative (d-) red blood cells (rbcs) through a rapid infuser during resuscitation. transfusion of uncrossmatched products before sample collection can lead to errors and confusion in blood typing, as can the venipuncture site used for collecting the patient's blood sample. the current fda guidance and aabb standard of two samples for determination of blood type to prevent cases wrong blood in tube (wbit) or electronic identification systems do not always catch or clarify these errors. study design/methods: patient was tested by manual tube method. two different technologists using two different reagent racks performed initial testing with matching results. results/findings: two samples were collected during resuscitation from the patient and typed as o d-. patient was transfused with units of o d-rbcs before stabilizing. two days later another sample was collected and typed as o rh positive (d ) with mixed field being seen on the anti-d. a weak d testing was performed to see if the negative result with anti-d could be strengthened through incubation. both original samples still resulted as d- (table a) . after consulting the patient care team it was discovered the samples were collected above the iv site after one unit had been completed and while the second unit was being transfused. it was also discovered all other clinical laboratory samples were rejected due to possible line contamination when results for the sodium, potassium, and glucose appeared inaccurate. the transfusion service laboratory is in a different area of the hospital and was unaware those samples had been rejected. conclusion: the initial samples were collected above the iv site and were contaminated with the d-blood product being rapidly transfused during resuscitation. the samples collected during the initial trauma response should have been rejected and a request made for samples drawn below the iv site. because both samples were collected while the unit was being transfused, contamination was in both. use of a handheld barcode system would not have caught this error because the patient had been correctly identified. future prevention of the above anomaly would be the education of transfusion testing staff to recognize an abnormal high hematocrit: secondly reminding the staff collecting samples to be aware of the proper collection procedures for laboratory testing, which would include type and screen. facilities also should strive to perform collection of the confirmatory sample from a completely different venipuncture site. impact of cell saver usage during solid organ transplants at a major institution holly ross* , edward smith , thomas brown , foeks jeremy , metcalf suzanne , james johnson , peter davis , karafa sw badjie and abba zubair . department of laboratory medicine and pathology, transfusion medicine, mayo clinic, department of anesthesia, mayo clinic background/case studies: our institution performs an average of solid organ transplants (sots) yearly. transfusion support for transplants can be tremendous, accounting for a large percentage of red blood cell (rbc) transfusions annually. even the best practices for allogeneic transfusion are not without risk. transmission of pathogens is possible with even the strictest screening methods, and each transfusion increases the risk of alloimmunization. the advent of intraoperative blood recovery has reduced the need for allogeneic donor rbcs during surgeries expected to bleed heavily. with the cell saverv r (haemoneticsv r , braintree, ma), patients' own blood shed during surgery is collected, washed, concentrated, and reinfused, lessening the need for transfusion support. this study sought to examine the amount of allogeneic donor rbc units saved during sots through the use of the cell saver for intraoperative blood recovery. study design/methods: data was collected for sots which utilized the cell saver. these included liver, liver/kidney combination, lung, and heart transplants. data a y.o. female was admitted to the trauma department after a motor vehicle collision (mvc) and transfused o( ) rbc units from the kiosk. her blood type was determined as o(-) with a negative rbc antibody screen (as). she was transfused more units of o(-) rbc. two months later, a repeat as identified two new rbc alloantibodies, anti-d and anti-e. the anti-d formation resulted from the o( ) rbc transfused from the kiosk, but the source of the anti-e was undetermined since e antigen is expressed in % of rh(-) individuals. the trauma department staff was notified of delayed serologic transfusion reaction and asked to investigate further since a y.o. female patient should not have received o( ) rbcs. study design/method: an investigative plan was developed by the trauma staff involving a patient census, review of the chart and kiosk inventory, obtaining feedback from clinical providers, and review of information provided by emergency services (ems). results/finding: the trauma unit was busy with admissions during the hours preceding the patient's arrival. the chart review found the following physical attributes; patient was overweight ( kg) with obvious facial deformities from the mvc, that compromised age assessment. it was determined that the kiosk was fully stocked with both o(-) and o( ) rbc units. one clinical provider recalls that the patient identification (id) might have been unknown. review of the ems communication states "patient is a y.o. female." conclusion: use of visual examination to determine age was significant in the selection of o( ) rbc for this patient. the trauma staff proposed and implemented a change in policy to prevent future incidents. any female patient that arrives without id or written confirmation of age will be transfused o(-) uncrossmatched rbc until a blood type can be determined. after being notified of the incident, the trauma staff took the lead in investigating and providing a process improvement resolution. this is credited to the excellent collaborative relationship between the transfusion service and trauma department on ensuring patient safety during emergent, uncrossmatched rbc transfusions. rate of abo/rh confirmation in outpatient pelvic organ prolapse surgery alexis r peedin*, taylor brueseke, yara park and jay s raval. university of north carolina background/case studies: approximately , surgeries for urinary incontinence or pelvic organ prolapse (pop) are performed annually. for abdominal pelvic floor disorder (pfd) surgeries, transfusion rates historically range from - %, whereas transfusion rates for vaginal and robotic pfd surgeries range from . - . % and . - . %, respectively. since the implementation of college of american pathologists (cap) requirements for abo/ rh confirmation, approximately % of patients who receive a transfusion in our hospital required a second abo/rh specimen to be drawn; however, limited data are available regarding the impact of this new requirement on patients preparing to undergo outpatient surgery that currently require preoperative type & screen (t&s). the primary objective of our study was to assess the rate of abo/rh confirmation in women who underwent outpatient pop surgery. study design/method: this was a planned secondary analysis of a retrospective cohort study of consecutive patients undergoing pop surgical repair from may -may in our academic tertiary care institution. among this sample, patients were excluded if their first t&s was drawn before our institution implemented the abo/rh confirmation requirement. fisher's exact test was used, and statistical significance was defined as p< . . results/finding: we identified patients for analysis, of whom ( . %) had a preoperative t&s ordered. two ( . %) of these patients had positive antibody screens; one patient had an anti-k and one had a warm-reacting autoantibody. fifty-nine ( . %) of the patients required a second abo/rh specimen per hospital protocol; ( . %) of these actually had a second specimen drawn. in patients for whom abo/rh confirmation was indicated, there were no differences between those who did and did not have abo/rh confirmed when comparing age, body mass index (bmi), pre-operative hemoglobin (hgb), or surgical approach (table ) . no abo/rh discrepancies were identified. one patient received unit of red cells after abdominal pop surgery. conclusion: the rate of requiring abo/rh confirmation before pop surgery was markedly higher than that seen in all patients receiving transfusions at our institution ( . % vs. %, respectively). because the vast majority of women undergoing vaginal or robotic pop surgery are not transfused perioperatively, hospital transfusion services should consider eliminating routine pre-operative t&s for this low-risk population in the maximum surgical blood ordering schedule, avoiding this unneeded test and subsequent abo/rh confirmation. volume reduction of red cells to reduce transfusion-associated adverse events related to hyperkalemia maressa t pollen*, laura knicks, linda van tol and c. michael knudson. background/case studies: one attribute of older blood is an increase in supernatant potassium level which can contribute to transient hyperkalemia. this problem is exacerbated in conditions of massive transfusion and in patients with renal failure. washing rbcs can effectively remove free potassium but is time consuming and can often only be performed on one unit at a time. here, we estimate the amount of potassium that is removed by volume reduction of red cell units. we also examined whether this technique would be feasible in the setting of massive transfusion in a patient with hyperkalemia. study design/method: expired or over temperature units (n ) that had been removed from inventory were utilized for these studies. each unit was weighed and a volume reduction procedure was performed. the supernatant was weighed and the potassium of the supernatant was measured using routine laboratory assays. for all formulas, weight was converted to volume using a specific gravity of . g/ml. the hematocrit (hct) of the volume reduced rbc was measured using a sysmex xs- i instrument. the percentage of supernatant removed was calculated by dividing the residual supernatant in the volume reduced unit (rbc hct x rbc volume) by the total supernatant prior to the procedure (residual supernatant removed supernatant). the remaining free potassium (meq) was calculated as the (concentration of potassium in the supernatant (mmol/l) x the estimated red blood cell residual supernatant volume. to simulate the process that would occur in the setting of a massive transfusion protocol (mtp), units were subjected to the volume reduction while recording the time needed to process all units. this was performed twice for a total of units processed in this manner. results/finding: the volume reduction procedure reduced the supernatant volume by an average of % (range %- %). in units between and days (n ), the estimated mean residual k was . meq (range . to . ). in the two mock mtp trials, the time to complete the procedure was approximately minutes and we estimate an additional - minutes would be required to modify and issue the units in our lis/emr. conclusion: a manual volume reduction protocol in red cell units significantly reduces the amount of potassium administered in a unit of red cells. this procedure may be useful when only older red cell units are available for a patient at risk for hyperkalemia. the procedure can be performed in less than one hour and may be useful under the conditions of massive transfusion. processing, cryopreservation, and non-specialized hospital collection. preliminary studies of three shipping conditions after collection were tested using sterile containers with sterile normal saline (ns) alone, ns plus antibiotic/antimycotic (ab/am) and a dry container. prolonged exposure to ab/am solution retarded outgrowth of mscs, but control of microbial growth in cultured tissue samples was needed. these findings were used to construct a validation study. study design/methods: a validation study designed to test procedures to collect, transport, process, and store umbilical cord tissue was measured by post-thaw outgrowth. collected uc tissue from consenting mothers was transported to the distant lab in validated shipping containers in a dry, sterile cup from vaginal ( ) and caesarian ( ) births. uc collections were divided into segments to test conditions. segment explants were placed on . % gelatin-coated gridded tissue culture plates ( explants per plate) in enriched medium specified for msc outgrowth containing antibiotic only with an endpoint of days. growth was scored as the number of squares with explants exhibiting outgrowth compared to the total planted explants. one segment (fresh control) was dissected and planted without further processing. the remaining tissue segments were soaked in (ab/am) saline solution for hr and hrs at c, respectively. tissue segments were frozen in cryo bags with a proprietary % dmso/large molecular weight sugar solution. background/case studies: it has been the practice in our institution to process or times the total blood volume (bv) of the patient, up to a maximum of liters (l) per procedure, to obtain peripheral blood cd stem cells. as a consequence, a patient often would need to spend hours or more on the machine. it would be desirable to be able to specify the exact volume of blood to process to achieve the desired cd cell yield, thus minimizing the patient's time on the machine, the nurse's time performing the procedure, and the number of bags that have to be submitted for cryopreservation and storage. study design/methods: our institution recently implemented the new spectra optia cmnc collection protocol, a continuous flow and continuous collection procedure that uses the automated interface management (aim) system to precisely manage the separation interface. an analysis of our collection data suggested a highly reliable collection process, so a prediction algorithm (pa) based on the linear regression between the patient's cd pre-count and cd yield, normalized per liter of blood processed, was derived utilizing the patient's cd pre-count, the patient's weight in kilograms (kg), and the target cd dose/kg. this pa calculated the exact volume of whole blood to be processed to achieve the requested dose of peripheral cd stem cells. the initial equation was modified to add an additional % to the predicted volume, to account for the natural variability of the process. this pa was then tested prospectively in the clinical setting. results/findings: in patients, representing both allogeneic and autologous donors, the average blood volume processed was . l. the range was . l - . l. the target dose was achieved in all patients. our previous practice for these patients would have required, assuming a standard bv procedure, processing an average of up to l per patient, with a range of - l. to quantify how well the new pa works, it was decided to evaluate the ratio between actual and predicted volume vs. the ratio between the actual and expected cd yield. the result was a high correlation between these two ratios (r . ), indicating that the algorithm produces very consistent results. conclusion: the predictability of our collection process during the time period analyzed was a robust r . , confirming the findings in the first data analysis. the blood volumes processed and patient time on the machine decreased substantially, with some patients only needing hours or less to achieve their target dose. nurses and lab medical technologists have seen a dramatic change in their workflow. the number of bags to process has dropped for the lab, with the consequent freezer space savings and the shorter collection times allowing the lab medical technologists to finish with their work earlier in the day. all in all, implementation of this pa has produced huge increases in patient and provider satisfaction. important factors that likely contributed to the success of the protocol included the precision and consistency of the aim system of the apheresis device, as well as the small number of nurses ( - ) who performed the procedures, resulting in less variability. the economic impact of this pa has not been quantified, but might be an interesting area for future studies. background/case studies: zarziov r , a biosimilar granulocyte colonystimulating factor (g-csf) has recently been introduced into clinical practice. its use has stimulated a certain debate regarding their possible less efficacy and security on cd mobilization. the aim of this study is to evaluate if there are differences between good and bad mobilizers and assess the need for plerixafor when a biosimilar as g-csf is used. study design/method: we retrospectively evaluated autologous mobilization processes performed between june and march . patients (n ) evaluated were diagnosed with malignant lymphoma (n ), multiple myeloma (n ) and primary amyloidosis (n ) and were mobilized according to standard protocols. collection cd cellularity target was established ! x e /kg. two groups, good and bad mobilizers, have been determined. predictors of unsuccessful mobilization were defined by > years old, previous fludarabine, lenalidomide, or bendamustine treatments or ! previous regimens, present peripheral cytopenias, active disease and previous mobilization failure. mann-whitney u test was used to compare means and comparisons of medians were performed by the median test. cd count was performed according ishage protocol. adverse events (ae) were analysed according to ctcae v . . results/finding: the media (range) general collection parameters were: cd (day ) . /ml ( . - . /ml), blood volume processed ml ( - ml) and . ( - . ) exchanged volemias. seventeen patients were considered bad mobilizers, needed plerixafor and had to undergone a collection procedure twice. there were statistically significant differences between both groups on mobilization characteristics and product cellularity [mean (sd) ); p . ]. there were no significant differences on mobilization characteristics and product cellularity between both groups. five mobilization ae were observed [muscle pain (n ), fever (n ) and flu syndrome; all grade ]. two patients could not undergo hematopoietic stem cell transplantation due low cd cellularity. conclusion: there are differences between products collected from the good mobilizer (rich in gm and cd ) versus poor mobilizer (with plerixafor) rich in cn and cmn. the mobilization with zarziov r could be smaller than expected since there are no significant differences if we compare the good mobilizers versus the bad mobilizers although the number of cases studied can be limiting. background/case studies: mesenchymal stem cells (mscs) have been widely studied and have shown beneficial effects on tissue regeneration, immunomodulation, and improvement of multiple organ failure caused by infection, sepsis, and trauma. however, mscs express tissue factor, which may be a risk factor for thrombosis especially if administrated systemically following trauma when coagulopathies are common. before applying mscs in a preclinical animal model, we sought to determine the procoagulant properties of rat mscs in vitro. study design/methods: bone marrow and adipose derived mscs (bmsc and amsc) were isolated from bones (femur and tibia) and visceral fat tissue in normal young sprague dawley rats respectively. both bmscs and amscs were cultured and passaged using dmem medium with % fetal bovine serum. bmsc and amsc at passage - were used in this study. the tissue factor expression of mscs was determined by immunohistochemistry. citrated whole blood collected from normal rats was treated with rat bmscs and amscs at low, medium and high doses ( . /ml, /ml and . /ml respectively). the prothrombin time (pt), coagulation properties and platelet aggregation (response to adp, collagen and par ) were measured by hemostasis analyzer, rotational thromboelastometry (rotem) and impedance aggregometry (multiplate) respectively within min and hr after incubation. results/finding: tissue factor was significantly expressed among both bmsc and amsc at all passages in vitro. bmsc and amsc at any dose and time of treatment neither shortened nor elongated pt in whole blood. however, both bmsc and amsc significantly shortened the clotting time (ct) (none: seconds, versus low, medium and high doses of amsc ( , , and seconds), and bmsc ( , , . seconds), p< . ), clot formation time (cft, p< . ) and increased alpha angle (p< . ) by natem measurement, but did not significantly affect the ct, cft and alpha angle by extem. maximum clot firmness (mcf) and fibrinolytic index were not affected by mscs. there was no significant impact of both bmsc and amsc on platelet aggregation simulated by adp, collagen and par . no significant differences of hemostatic and platelet function were found between the treatments of bmsc and amsc. conclusion: consistent with reports from human derived msc, both rat bmsc and amsc significantly expressed tissue factor in both early and late passages, which led to a significant decrease in clotting time at various dose and time of treatment. however, mscs had no direct impact on platelet aggregation in vitro. as considering the procoagulant capability of mscs, future study will be necessary to determine the optimal dose and safety of using mscs for systemic application in vivo. comparison of the terumo bct mnc and cmnc protocols for peripheral blood stem cell collections lindsey westbrook* , neil bagamasbad , reynold dilag , melissa nasser , nicole bauer , jennifer wheeler and mary berg . department of pathology, university of colorado -anschutz medical campus, department of medicine, division of hematology, university of colorado hospital, scientific support, terumo bct background/case studies: terumo bct recently offered a new method of peripheral blood stem cell (pbsc) collection using the spectra optia, an apheresis instrument. the new protocol, continuous mononuclear cell collection (cmnc) collects cells continuously as opposed to the older protocol, the mononuclear cell collection (mnc) protocol, which is batch collection or dual stage collection, involving an additional step where platelets are separated from mnc within a cell separation chamber. our institution has used both protocols and the purpose of this study was to compare pbsc product characteristics and run times between the cmnc and the mnc protocols. study design/method: a retrospective review and comparison of parameters from collection procedures using the mnc protocol and collection procedures using the cmnc protocol was done using the t-test. data from patients/donors (including allogeneic donors) as well as procedure details including run time, flow cytometry marker for stem cells (cd )-positive (cd ) throughput, cd collection efficiency (ce%), platelet loss a transfusion per total blood volume processed (plt loss/tbv), and collection product characteristics were included in the analysis. results/finding: numerical results are summarized in the table. the mnc and cmnc donor groups included and allogeneic donors, respectively. donor weight was not significantly different between the two groups. pre-procedure wbc values were also similar between the two groups. run time was found to be significantly shorter using the cmnc protocol compared to the mnc protocol. product volume was also significantly lower in the cmnc group compared to the mnc group. although the volume was lower, the cmnc product had significantly higher percentages of mononuclear cells (mono%) and lymphocytes (lymph%) collected when compared to the mnc product. the cd throughput was significantly higher in the cmnc group than the mnc group. the cd ce% was found to be slightly increased in the cmnc group, though not significantly. the platelet loss was not significantly different between the protocols when normalized for total blood volume. product hematocrit (hct%) was significantly higher using the cmnc protocol; however, the red blood cell volume never exceeded ml due to the lower product volume with the cmnc protocol. the cmnc protocol collects a smaller volume of a purer product when compared to the mnc protocol with comparable platelet and red blood cell loss. staff members who perform apheresis procedures are pleased by the shorter run time. background/case studies: hematopoietic stem cell (hsc) donors and their recipients need not have a matching blood type. eventually, the hsc recipient will become the blood type of the hsc donor. this scenario can become quite a conundrum if the hsc recipient becomes a patient in need of an organ transplant. in order for a patient to receive a donor organ, the patient and donor's blood type and hla typing must be compatible. study design/methods: blood type was determined using gel test cards. hla typing was determined by using sequence-specific oligonucleotide (sso), sequence-specific primer (ssp), and sequence based typing (sbt) technologies. hsc sources were bone marrow and umbilical cord blood. results/findings: patient # , originally typed as an a , had bone marrow donor and cord blood transplants. one of the cord blood transplants successfully engrafted. the engrafted unit was from a type o donor. patient # is now typing as type o. patient # was originally typed as a and received a bone marrow transplant from a type b donor. patient # is now front-typing as a b and backtyping as an ab. since the patient's abo front and back-type do not match, a note must be made, that when confirming abo during crossmatch, the abo will not match. the patient now has an hla and abo identical kidney match (his father who is a type b). previously, the patient and his father were abo incompatible. the abo and hla results on both patient # and patient # indicate that the hsc transplants have engrafted. results also indicate that the abo and hla now match that of the donor and differ from the recipient's original abo and hla type. due to various reasons, for example, a side effect of the immunosuppression, both patients now need a kidney transplant. both patients will be entered into the unet system according to their "new" abo and hla types, as unos regulations require patients to be listed as per the results of two separate abo typing tests. the patients' antibodies will be monitored as per lab policy and communication with the transplant centers and blood banks is crucial. background/case studies: mesenchymal stem cells (msc) are beneficial for tissue regeneration, immunomodulation and improvement of multiple organ failure caused by infection, sepsis, and trauma. mscs express tissue factor (tf) that activate the clotting cascade and interfere hemostasis. hypoxia is a condition that occurs after trauma globally during shock or at the site of injury, and is known to change or influence the phenotypes of cells, including mscs. in this study, we want to determine if hypoxia changes the expression of tissue factor and the pro-coagulant properties of rat msc in vitro. study design/method: bone marrow and adipose derived mscs (bmsc and amsc) were isolated from bones (femur and tibia) and visceral fat tissue in normal young sprague dawley rats respectively. both bmscs and amscs were cultured using dmem medium with % fetal bovine serum under either normoxia ( % o ) or hypoxia ( . % o ). msc growth curves were measured by cell counter. the tf expression was determined by immunohistochemistry. cd /cd and cd were measured as positive and negative markers of msc respectively by flow cytometry. the citrated rat whole blood was treated with msc ( . /ml) either from normoxia or hypoxia. the coagulation properties were measured by hemostasis analyzer and rotational thromboelastometry (rotem). results/finding: hypoxia potentiated the growth of bmsc by %, but depressed the growth of amsc by % at day in comparison to normoxia. both bmsc and amsc equally expressed cd and cd but not cd under any culture condition. tissue factor was significantly expressed among bmscs and amscs from both normoxia and hypoxia. whole blood treated with bmscs and amscs from normoxia significantly shortened the clotting time (ct: (control), versus (bmsc), and (amsc) seconds) by natem. hypoxia also significantly shortened ct ( (bmsc), (amsc) seconds, p< . as compared to control), but the changes in ct were not significantly different between bmscs and amscs. maximum clot firmness (mcf) and fibrinolytic index did not change after treatment with bmsc and amsc regardless of the normoxia or hypoxia conditions. conclusion: tissue factor is constitutively expressed in rat bmscs and amscs. adjustment of the msc culture condition to hypoxia did not affect tissue factor expression or the procoagulant properties of msc (bmsc and amsc). this study also suggests that the procoagulant properties will not be affected if mscs are recruited into injured tissues with hypoxic environments. future study will be necessary to determine the optimal dose msc and whether it is safe to use mscs for systemic application in trauma. effect of double-end cryopreservation on gene-transduced human hematopoietic stem and progenitor cells sandeep k srivastava*, jiaqiang ren, steven highfill, narda theobald, suksee deravin, andre larochelle, david f stroncek and sandhya r panch. national institutes of health background/case studies: current early-phase clinical gene therapy trials use freshly collected or cryopreserved cd cells as the starting fraction prior to gene manipulation. following gene-transduction and culture, the end product is infused fresh into recipients. for wider applicability and scale-up, gene therapy manufacturing protocols would benefit from double-end cryopreservation (dec) of cd cells during manufacture (i.e. immediately post-collection and again, post-gene modification). dec helps delink patients' preparative conditioning phase from cell manufacture, eases logistics of inter-facility cell transportation, and ensures fulfillment of regulatory product release criteria before infusion. our objective was to study the effects of dec on gene transduced mobilized peripheral blood (mpb) cd cells. study design/method: cryopreserved cd cells from healthy adult donors were thawed and transduced (tr) in retronectin coated tissue culture bags with an ef -alpha-yfp lentivirus ( . % concentration) and media (x-vivo- , human serum albumin(hsa), ng/ml each of cytokines (scf, tpo and flt -l) over days. untransduced (utr) cells were cultured as controls. tr and utr fractions were re-cryopreserved. a standard freeze-mix of % dmso, % pentastarch, hsa, plasma-lyte a was used for cryopreservation. viability, hematopoietic stem cell (hsc) (cd cd -cd ra -cd cd f cells) phenotyping and cfu assays were done following first thaw (pt ), post-transduction (ptxn) and second cryopreservation-thaw (pt ). results/finding: tnc recovery decreased gradually in the donor samples at each step. transduction efficiency, cd %, cfus were similar before and after pt . hscs ranged from to cells/ cd cells in the pt -tr arm compared to a range of to / cd cells after pt . viability, % cd and cfus were lower in the tr compared to the utr arm. this difference was not altered after pt (table) . conclusion: dec of mpb human cd cells decreases tnc recovery, but has minimal effects on cd cell phenotype, transduction efficiency and cell function. hsc numbers were within acceptable range after recryopreservation. lower viability and cd % in the tr arm compared to the utr arm is likely due to vector toxicity. this was unaffected by recryopreservation. additional studies to assess dec mediated changes on cd cell early apoptotic markers, telomere lengths, gene expression and engraftment potential in nod/scid mice will inform clinical trials. background/case studies: autologous peripheral blood stem cell (pbsc) transplantation has been used as a powerful resource during the treatment of some hematological malignancies. cryopreservation of these cells is routinely performed to allow for patient adequate conditioning and chemotherapy. in some cases, pbsc are harvested as a backup option and remain stored for several years, although effect of storage lesion in this product is still controversial. our work presents retrospective data on pbsc infusion after long-term storage. study design/method: all products were harvested after patient mobilization with g-csf by apheresis with cobe spectra v r . flow cytometry analysis of cd cells was performed prior to cryopreservation. the cryoprotective solution was freshly prepared by addition of % hydroxyethyl starch, % human serum albumin and % dmso at final concentration. pbsc were cryopreserved by direct immersion on - c mechanical freezer (dump freeze) and stored until transplantation. post-thaw viability was determined from stored cryotube samples by trypan blue exclusion minutes prior to infusion. cells were thawed and infused on bedside. engraftment was defined as the first day of consecutive days of neutrophil count > . x /l and platelet count > x /l after days. with g-csf for four days and patients with g-csf for five days with use of mozobil when cd was below x cells/l on the fourth day. hpc collection was performed on the fourth day of mobilization for healthy donors and on the fifth day for patients. all procedures were realized based on a prediction algorithm using pre-cd on the day of the collection and estimating wbc liters to be processed to obtain sufficient stem cells for the transplant. this algorithm was designed using linear regression of peripheral blood cd on the day of the collection versus collected cd per liter of blood processed. there was no distinction between patients and donors, once the efficiency coefficient was used for both. collected material was sent to analysis and total cd was calculated. final laboratory count of cd per kilogram was compared with the number predicted by the algorithm with spearman's correlation to evaluate whether the formula is effective. calculations were made using ibm spss software. results/findings: among patients collecting hpc for autologous transplantation, , % needed only one day of hpc harvesting, while , % needed two days and , % needed three or more days. our collection efficiency (ce) and standard error of the mean (sem) was - , %. after comparing predicted values with cd collected in the final product, we found a very strong correlation of . (p< . ) for patients and a strong correlation . for healthy donors (p< . ). conclusion: the use of a mathematical model with a prediction algorithm is safe, has low cost and provides a good tool to estimate wb liters to process and avoid unnecessary procedures in both patients and healthy donors. this study evaluated the phenotypic characteristics of uc-mscs derived from fresh and cryopreserved cord tissues (ct), as described in isct's position paper on minimal characteristics of mesenchymal stem cells (plastic adherent; ! % cd , cd , cd and % cd , cd , cd , cd , hla-dr) study design/method: umbilical cord tissue (n ) was washed, blood vessels removed, cut into . - mm pieces, and washed twice in saline. fresh tissue was immersed in . % saline for same day culture, while frozen tissue was cryopreserved for at least hours prior to culture. for colony forming unit (cfu) testing tissue was plated directly in a cm tissue culture flask following a wash in pbs with antibiotic/antimycotic. the tissue was allowed to adhere for minutes prior to the addition of cell culture media. media was changed several times a week. cells were passed when robust colony growth was observed and in subsequent cultures > % confluence. all cells were tested on an msc flow panel at passage just prior to confluence. results/finding: both fresh and cryopreserved tissue showed excellent colony forming capabilities. average time for cellular emergence of days (fresh . , frozen . ), and days (total) for the msc's to reach passage (fresh . , frozen . ). all cells were ready for flow analysis in approximately weeks time. there was no statistical difference between fresh and frozen tissue in their colony emergence (p . ), or their growth rates (p > . for all). flow cytometry showed average ! % for positive markers and % negative markers. there was no statistical difference between fresh and frozen flow result (p > . ). conclusion: uc-msc's show excellent adherence to plastic in both fresh and frozen explant cultures, with a consistent fibroblast-like morphology. flow cytometry analysis showed strong msc phenotype in both fresh and frozen samples. the data show that the cryogenic process does not appear to have any detrimental effects on the ability to obtain msc colonies. studies have shown that hsct improves survival and disease-free survival rates when compared to conventional chemotherapy treatments. the increase in the number of hscts over the last years has demanded quality and safety improvements of cell processing and cryopreservation services. cell recovery and viability are crucial parameters to assess ucb quality as a viable hsct graft source. study design/method: twenty-five ucb units cryopreserved for periods of up to years ( to ) were analyzed. units underwent red cell and plasma depletion and then subjected to controlled rate freezing and subsequent cryopreservation using dmso (dimethylsulfoxide) cryoprotectant with % concentration. informed consent and the unit discard terms for all units were obtained. units were thawed in a c water bath and . ml aliquots were diluted at a : proportion with % human albumin solution and plasmin were prepared, enabling dmso stabilization and concentration reduction. the following analysis were performed: nucleated cell count (tnc) in an automated hematologic counter and cell viability using flow citometry. post-processing (pre-cryopreservation) cell viability was tested using trypan blue as exclusion dye, while post-thaw cell viability was assessed using -aad marker through flow cytometric analysis. results/finding: ucb storage period was . years (mean) and cell recovery was . % (mean). there was no statistically significant correlation between storage period and post-processing cell recovery (p . ). post-thaw cell viability of . % (mean) showed no statistically significant correlation with unit storage period (p . ). post thaw cell viability results are within parameters defined in other studies. background/case studies: umbilical cord (uc) tissue is a rich source of mesenchymal stem cells (mscs) that can be collected noninvasively at birth and stored for potential future use. as such, a growing number of stem cell banks have established uc storage programs based on mounting preclinical evidence of its therapeutic potential. however, little has been reported on the ability to isolate msc-like cells from uc tissue after extended periods of cryopreservation. this work describes and characterizes the isolation of mscs from uc tissue cryopreserved as a composite material at a family stem cell bank for years. study design/method: donated uc units from consenting mothers were evaluated. units had been cryopreserved as composite tissue pieces in ln vapor in a dmso-based cryoprotectant for yrs. ( . . ; n ). units were rapidly thawed and rinsed in dpbs, then pieces were excised from each using a biopsy punch. pieces from each unit were explanted in a x grid pattern in msc-supportive medium and incubated for days, after which the tissue was discarded and media exchanged. cells were isolated on the th day, counted, and subcultured for two passages. at the end of each passage, cells were collected, counted and population doubling time was calculated. isolated cells from each unit were also evaluated for msc immunomarkers. results/finding: small, proliferative cells with fibroblastic morphology were obtained from all explants, yielding a % success rate. cells were positive for the msc markers cd , cd , and cd ( . . %, . . %, and . . %, respectively) and negative for the hematopoietic markers cd / ( . . %). passage and passage doubling times were . . days and . . days, respectively, which are in line with values reported for mscs isolated from fresh uc tissue. conclusion: due to their immature status, ease of collection, and potential therapeutic value, uc mscs are an appealing candidate for future clinical a transfusion vol. supplement s research and treatment. the present work demonstrates that the long-term cryopreservation of uc tissue does not disrupt the ability to isolate functional mscs from the tissue at a later date. importantly, growth characteristics of isolated mscs appear to be comparable to those reported for mscs from fresh uc tissue. based on the consistent isolation and lack of apparent impact on proliferation kinetics, it is reasonable to expect cell yields in the range anticipated for therapeutic requirements and more than sufficient for moving to clinical grade bioreactors for expansion. these results support the feasibility of storage of uc as a composite material for future potential cell isolation and expansion to clinically relevant doses. large volume leukapheresis with spectra optia cmnc protocol in adult and pediatric patients: performance and determination of cd yield prediction algorithm ines bojanic* , nelly besson , ivana vidovic and branka golubic cepulic . department of transfusion medicine and transplantation biology, university hospital centre zagreb, terumo bct background/case studies: large volume leukapheresis (lvl) have shown to enhance cd cell yield collected. this study evaluated performance and safety of the spectra optia cmnc protocol (version ) in adult and pediatric lvl. a prediction algorithm for cd cell yield was also tested. study design/method: we evaluated retrospectively lvl performed in adult patients, and lvl in pediatric patients treated in uhc zagreb from march till september . mobilization regimen combined chemotherapy and filgrastim; poor mobilizes received plerixafor additionally. a combination of acd-a and heparin was used as anticoagulant (acd-a:whole blood ratio : ). in patients weighting kg (n ), a rbc prime was performed. cd , lymphocyte(ly) and monocyte(mo) collection efficiencies (ces) were calculated. a customized prediction algorithm was determined on linear regression between pre-cd cell count and cd cells collected / blood volume processed. prediction accuracy was evaluated by comparing predicted cd values to real cd yield. results are presented as median (iqr). results/finding: in both groups, cd , ly and mo ces were high. target cd dose was successfully reached in procedure in ( , %)adults and in ( . %) children. all procedures were well tolerated: adverse reactions were restricted to mild citrate toxicity symptoms in ( . %) adults, while all pediatric apheresis went uneventful. no bleeding episodes occurred, and no transfusion was needed. product and procedure characteristics* a high correlation between precd cells and cd cells collected/ blood volume was observed in both groups (r . and . in adults and children respectively, p< . ) suggesting cd yield could be predicted based on precd cells and blood volume to process. linear regression equations served as prediction algorithm. the high correlation between predicted cd yield and observed cd yield (r . and . in adults and children respectively, p< . ) showed accuracy of the algorithm. implementation of the algorithm could have allowed sparing a median of . ( . - . )l of blood in adult procedures, and . ( . - . )l in pediatric procedures. conclusion: lvl performed using spectra optia cmnc protocol is safe and efficient in adults and in low body weight children. high cd , ly and mo ce were observed in both groups. implementation of a predictive algorithm can reliably minimize blood volume processed, shorten procedure duration, reduce anticoagulant volumes infused, and improve patient comfort. mesenchymal stem cell therapy in steroid refractory graft-versus-host disease (gvhd) emese molnar* , aniko barta , arpad batai , zoltan csukly , zita farkas , laszlo gopcsa , gabor tatai background/case studies: steroid refractory acute graft-versus-host disease (gvhd) is a serious complication of allogeneic hematopoietic stem cell transplantation (hsct). more experience accumulates in the immunomodulatory effect of mesenchymal stem cell (msc) infusion in numerous immunopathological disorders -such as gvhd -and signals. mscs have a hlarestrictive and non-immunogenic nature. study design/method: we have evaluated the efficacy of msc transfusions in cases of acute gvhd refractory to conventional immunosuppressive treatment. the patients with steroid-resistant gvhd had received third-party mscs (derived from wharton's jelly and bone marrow) times per case weekly at a dose of million cells/kg. clinical response was assessed days after administering the first dose. complete remission was defined as the complete disappearance of symptoms. partial remission was assessed by the significant relief of symptomsand by the general improvement of the patient's condition. results/finding: in all patients had received cycles of msctreatment ( dose per cycle). the median age was years old ( - ) with a male/female ratio of : . distribution of the original malignancies (n): acute myeloid leukemia: ; acute lymphoblastic leukemia: ; myelofibrosis: ; myelodysplastic syndrome: ; multiple myeloma: ; t-cell lymphoma: . nine patients had undergone allogeneic hsct with matched unrelated donors, the other three had stem cells derived from hla-identic relatives. the first episode of gvhd after hsct was started on the median rd day ( - ). the involved organs were skin ( ), gut ( ), skin and gut combined ( ) and even lung in cases. the median time of msc's first infusion was days after the stem cell transplantation (hsct) and ( - ) days after the first episode of gvhd. of the cycles of msc-treatment led to complete remission ( . %) and resulted inpartial remission ( . %). conclusion: we have evaluated msc-therapy as an effective treatment of gvhd in the majority of the observed cases with % overall cumulative response rate. the application of third-party mscs offers a promising alternative in the therapy of gvhd and other gvhd-associated complications after hsct. further research is needed to determine the optimal start of the treatment, along with the issue of long-term safety. background/case studies: stem cell collection by leukapheresis for transplantation is a significant endeavor for the patient and the clinical team. whether the collection is allogenic or autologous, the patient undergoing the collection and the physicians caring for the patient are always concerned whether they will be able to harvest enough cells for transplantation and engraftment. a typical goal for most adult procedures is million cd cells/kg. if a patient does not reach this goal on the day of the procedure, they will likely have to return the following day to undergo a second procedure to reach the desired goal. given the logistical challenges in planning transplantation, it is reasonable to attempt to optimize the number of cells collected while minimizing the number of collections. measuring a patient's cd cells/ml in their peripheral blood before the leukapheresis procedure has been used to predict if the collection will successfully reach the million cells/kg goal. the ideal minimum cd cells/ml that will lead to successful harvest has not been conclusively identified. study design/methods: we analyzed the collection data from patients to evaluate the predictive value of the cd cells/ml level. data was collected over months from every patient who underwent a stem cell collection. four patients were allogenic donors and were autologous donors. the patients' weight, diagnosis, and pre-procedure cd cells/ml level were all collected. the run time, amount of volume processed, and the absolute viable cd cells collected were recorded. the collection efficiency and the cd cells/kg were calculated for each patient. results/findings: our data showed a strong linear correlation between pre-procedure cd cells/ml and post-procedure cd cells/kg (r . ). any patient who had a pre-procedure cd cells/ml count of or greater had a collection of at least million cells/kg. any patient who had a pre-procedure cd cells/ml count of or less collected less than conclusion: the pre-procedure cd cells/ml level in the peripheral blood has a very strong predictive value for the post-procedure cd cells/kg level. to confidently know that a patient will be able to produce the desired million cells/kg, a pre-procedure cd cells/ml count of at least should be obtained. for any patient with a count below , they should be counseled that their collection is likely to take at least a second day and a second procedure. further studies, including potentially lengthening the run time and the volume processed, to evaluate how to handle the patients who fall between and cd cells/ml should be conducted. heidi elmoazzen , antonio giulivi , michael halpenny* , lisa martin , donna perron , chris bredeson , lin yang , locksley mcgann , paul birch and jason p. acker . canadian blood services, ottawa hospital background/case studies: a critical aspect of hematopoietic progenitor cell processing is the cryopreservation method. our program uses a "dump" freeze method consisting of product placement directly into liquid nitrogen vapour after addition of a cryopreservation solution containing dmso ( % final concentration) and hes (hydroxyethyl starch). pentastarch (hes source) a critical component of the cryoprotectant formulation was discontinued by the commercial vendor. this required that an alternative cryoprotectant formulation be validated to minimize the risk to patient safety without compromising engraftment quality. study design/method: the validation study consisted of phases; firstevaluation of the efficacy of four different cryoprotectant formulations, second -evaluation of full scale production and crypreservation and third -a concurrent validation for clinical transplant. phase i -samples from four different cryoprotectant formulations were tested for tnc, cd , viability and cfu at three points during manufacturing (fresh, post processing and post thaw). phase ii -mock hpc, apheresis units were used for a side-by-side comparison of freezing curves for the control and replacement formulations. phase iii -five clinical transplants were performed with hpc, apheresis products cryopreserved using the recommended replacement (hetastarch). results/finding: phase i -results indicate that aliquots cryopreserved in % dmso and . % hes (hetastarch) did not behave significantly different than cells cryopreserved in the control in terms of cell recovery, viability or cell proliferation assay (cfu). phase ii -the majority of freezing profiles displayed typical or expected bulk freezing profiles for both formulations. phase iii -transplants performed resulted in a mean engraftment time of . days for anc with no adverse patient reactions observed. engraftment times using the new hetastarch formula were compared to the previous engraftment times with no significant difference. conclusion: a change in the formulation of a cryoprotectant solution represents a major change that could have a significant impact on quality. in addition, maintaining the current % dmso final concentration was critical as post thaw washing is not performed at the clinical site, history demonstrating a very low toxicity rate with the existing formulation. this study demonstrated the acceptability of the hetastarch formulation using % dmso and . % hetastarch to replace pentastarch in the cryoprotectant formulation used for cryopreservation of hpc, apheresis products. background/case studies: autologous stem cell transplantation is usually performed with mobilized peripheral blood stem cells (pbscs). traditional mobilization regimens include granulocyte colony stimulating factor (g-csf) with or without chemotherapy, but have failure rates ranging from % to %. plerixafor is an adjunct agent used to improve mobilization in many clinical settings. however, its high cost is a significant concern. the manufacturer-recommended dose is . mg/kg, therefore patients weighing > kg would require a second vial, thus doubling the drug cost. in we implemented a policy of capping plerixafor at mg for patients weighing > kg. this retrospective study compares the mobilization of patients > kg who received capped doses ( ) ( ) ( ) ( ) , with historical control patients ( - ) who received full or uncapped doses. study design/method: patients weighing > kg with crcl > ml/min who received capped and full doses of plerixafor were identified in the pharmacy database. electronic medical records were used to collect baseline characteristics and cell collection data. results/findings: a total of and consecutive patients were included in the capped and full dosing groups, respectively. they showed comparable baseline distributions of age, weight, gender and diagnoses. plerixafor was given upfront, or as a rescue agent due to suboptimal mobilization in both groups. in the capped dosing group, fewer patients received chemomobilization or plerixafor upfront. when compared to historical controls, they used half of the number of vials of plerixafor, but collected similar numbers of cd /cells kg and achieved a comparable collection success rate. the strategy dose capping plerixafor at mg for patients > kg is cost-effective and achieves comparable mobilization outcomes while decreasing the drug cost by half. mean and range of %cd in peripheral blood were calculated. the data show that in the non-hispanic group, the youngest donors (< yrs) have a higher pre-apheresis %cd level than any of the other groups, reaching statistical significance when comparing the %cd pre-apheresis between the youngest group (< yrs) and the oldest group (> yrs). hispanic donors show statistically similar %cd pre-apheresis levels over all age groups. moreover, the hispanic older age group (> yrs) had a statistically higher %cd pre-apheresis level than the non-hispanic older age group. conclusion: in this analysis of sequential unrelated pbsc donors, hispanic donors maintain a similar pre-apheresis %cd level even as the donor ages, while non-hispanic donors show a decreasing pre-apheresis %cd level as they age. if proven, this data would suggest there are genetic factors that modulate a person's ability to mobilize stem cells as they age and that these genetic factors differ between ethnic groups. this small data set would suggest that people of hispanic ethnicity maintain a more robust and quickly responsive stem cell pool, even as they age. further studies of larger cohorts are needed to validate this observation. if proven, this has far reaching implications within the stem cell research and therapy arena. background/case studies: an update in hpc apheresis collection software led to higher collection volume in the organization's human progenitor cell (hpc) products without a corresponding increase in total cellular counts. incorporation of a volume reduction step was therefore warranted as larger product volumes require additional time to transfuse and lead to a larger dmso load to the recipient, often resulting in the need to transfuse over several days. the objectives of this study were to develop suitable mock hpc (mhpc) products and evaluate the effectiveness of the biosafe pericell volume reduction technology on white blood cell (wbc) recovery and viability. study design/method: hpc products are not readily available for development. mhpc were created from whole blood buffy coats (bcs). fresh abo compatible bcs were pooled and concentrated using centrifugation and manual extraction of supernatant and red cells. the mhpc products were then diluted in plasma to produce an appropriate concentration and volume. hpc collection data from last years was analyzed to determine the th percentile, median and th percentile values for both hpc volume and wbc concentration. six mhpc products were tested; three high wbc ( x cell / ml) and three low wbc ( x cells / ml) concentrations, each at high ( ml), low ( ml) and median ( ml) volumes. each unit was processed sequentially from high, median and low volumes. hence, the highest mhpc volume was processed for volume reduction first with a sepax (pericell protocol, cs. . kits), analyzed and then reconstituted and volume adjusted to the next volume target before being volume reduced again, and so forth. one additional mock product was prepared for a reproducibility study and was volume reduced three times. wbc concentration and -aad viability was determined before and after each volume reduction. a control sample was removed from the product prior to processing and sat on the bench top until the end of the protocol to assess the change in cell concentration and viability over time. results/finding: mock hpc products had a mean starting -aad viability of % [range - ]% and a hematocrit of % [ - ] which is well below the maximum allowable limit of the pericell. no significant differences in wbc recovery or change in viability were seen between the mhpc products. aggregate data showed that the mean wbc recovery of the volume reduction process was % [ - ] with a % [- - ] change in viability. the recovery protocol used to salvage product after each volume reduction gave a recovery of [ , ] % and a change in -aad viability of [ , ] % from the input product. the method was found to have a cv of . %. the change in wbc concentration and wbc viability of the test products was not significantly different from the unprocessed control samples. conclusion: mock bc products are a suitable alternative where hpc products are not available for development and are a good use of product otherwise directed for rejection and disposal. the volume reduction protocol evaluated had minimal impact on the wbc concentration and wbc viability in the mock products and was found to be highly reproducible, giving confidence that it will be a valuable processing step with hpcs and will facilitate transfusion of hpc products into the recipient. the protocol is now in use with patient hpc products and engraftment kinetics will be tracked in a postimplementation study. validating a transfusion clinical assessment. in the first phase, cryopreserved pbsc products were tested. two aliquots were thawed simultaneously for each product: one was passed through a pre-set infusion pump and a second control aliquot was drained by gravity. each aliquot was tested for baseline total nucleated cell (tnc) count and viability, and for final tnc recovery, trypan blue (tb) viability, cd -aad viability, and potency (cfu). the effect of longterm exposure to dmso was assessed by visually inspecting the product for aggregates and measuring viability up to hours post thaw. the second in vivo phase included use of an infusion pump for consecutive autologous patients, with comparison of infusion and transplant outcomes to previous infusions by gravity drip. comparison variables included infusion rate, adverse events (ae), and engraftment time. results/finding: no significant differences were observed between infusion pump and drip for the products tested in vitro, including tnc recovery, cell viabilities, and potency. for both methods tnc tb viability decreased by more than % within hour, while cd cell viability remained stable up to hours post thaw. small aggregates appeared after hour for both methods and increased by a similar rate over time. comparison of infusion and transplant outcomes between drip and infusion pump patients showed no significant differences for all measured variables. engraftment time was similar for both groups. anc days to engraftment for pump and drip were . . and . . , respectively (p-value . ). platelet days to engraftment for pump and drip were . . and . . , respectively (p-value . ). infusion rates were slightly higher for the pump group. for control patients, required transfer of products to syringes due to slow infusion rate and others experienced allergic and hypotension infusion adverse events. conclusion: no significant in vitro or clinical differences were observed between thawed pbscs infused by gravity or an infusion pump. these results demonstrate that the use of a pump for pbsc infusion is safe, provides consistent infusion rates, eliminates the need to transfer products to syringes, and results in comparable engraftment times. donor racial distribution among the zikv ineligible cbus was: caucasian %, asian %, black/aa %, and multi-race %. racial distribution of all clinical cbu donors was caucasian %, asian %, black/aa %, and multi-race %, suggesting there is no race correlation for this risk factor driven by cultural habits such as family travel. there were no cases in which onlythe sexual partner's potential exposure determined donor's ineligibility. conclusion: our study indicates that currently the leading risk factor for ineligible cb donors is potential exposure to zikv: % of all ineligible cbus and % of all banked cbus in the study period. we anticipate the number of cases to decrease following maternal education and travel warnings. recognizing the importance of zikv in public health, and its potential transmission via hct/p products, an fda approved screening test for hct/ p donors becomes a timely necessity. acknowledgments: funded by zimmer biomet, a zimmer biomet company, ibgrl red cell reference and nhsbt reagents background/case studies: during storage, red blood cells (rbcs) become less deformable, deplete , -diphosphoglycerate ( , -dpg) and adenosine triphosphate (atp), release pro-coagulation phospholipids, accumulate pro-inflammatory molecules, free iron and haemoglobin and increase their potential for adhesion to a recipient's vascular endothelium. longer rbc storage may impair transfusion outcome due to impaired oxygen delivery, promotion of oxidative stress, increased pro-inflammatory state and coagulation. a sterile, non-pyrogenic rejuvenation solution, containing pyruvate, inosine, phosphate, and adenine (citra labs, llc, braintree, ma), is approved by the u.s. food and drug administration for the rejuvenation of stored rbcs. the solution acts by restoring , -dpg and atp in stored rbcs to levels equivalent to those in the circulation. the aim of the study was to investigate the effect treatment with this rejuvenation solution had on the crossmatch reaction profile and phenotypic state of stored rbcs. study design/method: a ml aliquot was removed from abo/rh grouped, leucocyte depleted rbc units (n ), which were stored in sagm for days, to act as untreated controls. the remainder of each unit ($ ml) underwent treatment with the rejuvenation solution ( ml, minutes at o c), followed by cell washing twice in sagm ('manual' centrifuge-based process). to represent current transfusion laboratory practice, units were crossmatched against plasma from random donors, using both diamed gel column and glass tube technique. phenotype investigation with commercial antisera was performed to identify the effect the rejuvenation solution treatment exerted on rbc surface antigens (a, b, d, c, c, e, e, k, m, n, s, s, p , lu a , k, kp a , kp b , le a , le b , fy a , fy b , jk a , and jk b ), including whether it exposed crypt antigens (t, tn, tk*, th, tx*, and cad). crossmatch and phenotype agglutination scores observed for the untreated and treated rbcs were then compared. results/finding: crossmatch findings were defined as compatible, suitable, and incompatible. the study identified no difference between the crossmatch reaction profiles of untreated and treated rbcs. furthermore, no difference was observed in the phenotypic state between untreated and treated rbcs. conclusion: treatment of day old stored rbcs with the rejuvenation solution had no effect on crossmatch reaction profiles or phenotypic state when compared to matched untreated samples. background/case studies: cryopreserved platelet production is burgeoning worldwide. currently, there are no automated platelet cryopreservation methods. by contrast, red blood cell cryopreservation using the acp (haemonetics corp., baintree, ma) has automated the processing within a closed system, increased labour productivity and provided high quality blood components. purpose: to automate platelet cryopreservation procedure. study design/method: apheresis platelet concentrates (pc) were collected on the trima accel system. platelet counts were performed using an abx micros . pc were centrifuged at g in a sorvall rc c centrifuge (sorvall, usa) for min. the combination cryoprotectant dmso dextran (cryosure dex , germany) was used for pc cryopreservation. cryopreserved pc (cpc) were frozen and stored in a kelvinator chest freezer. cpc were thawed at degrees c (barkey plasmatherm) for min. cpc osmolality was measured with an osmomat osmometer. results/finding: staged platelet cryopreservation technology has been developed. platelets were cryopreserved in a closed system (patent no.: ru u ). during the first stage, cpc were spun to separate a plateletrich plasma (prp) fraction from platelet-poor plasma (ppp). the second step was to resuspend the prp by adding a combination of dmso dextran (cryosure dex ) , as a cryoprotectant, to obtain a final concentration of % dmso in the platelet suspension. the injectomat mc agilia and npbi compomixer m were instrumental in automating that phase. pc to be frozen had an osmolality of no less than mosm/l. prp and ppp were frozen at a cooling rate of - c/min and stored at - in the chest freezer for up to months. pre-transfusion defrosted platelets were also processed in a closed system (patent no.: ru u ). our transfer set made it possible to automate platelet resuspension in plasma through the agency of the exadrop v r . post-thaw prp was resuspended in plasma, which lowered the osmolality to mosm/l. freeze-thaw recovery of platelets was % or more of the original population. defrosted pc were stored at - with continuous gentle stirring from a helmer platelet agitator for no longer than hours before transfusion. it took no more than min to cryopreserve pc and process pre-transfusion thawed platelets. the automated processing accounted for the bulk of the time (over min). conclusion: the automated technique developed reduced the workload while offering reproducibility of the procedure and high cpc quality. the use of closed systems ruled out bacterial contamination. employing the infusion pump, platelet stirrer and precision flow regulator enabled adequate osmolality monitoring. bacterial detection in leukoreduced apheresis platelets on day and day evelyn c. oyler*. suncoast blood bank background/case studies: the recently published fda draft guidance describing bacterial testing to enhance the safety and availability of platelets outlined the steps for blood collection establishments and transfusion services to extend apheresis platelets dating for up to days. this evaluation will compare culture based and rapid based test methods for detecting bacterial contamination in apheresis platelets. study design/method: a large community blood center and transfusion service collects leukoreduced apheresis platelets (lrap) using amicus separator system (fenwal, lake zurich, il) and trima accel system (terumo bct, lakewood, co). previously-cultured lrap units were sampled on day for secondary culture using bact/alert (biomerieux, durham, nc) and rapid bacterial tests using bactx (immunetics, boston, ma) and pgd (verax, marlborough, ma). if lrap unit is still available, it is also sampled and tested for rapid testing on day . a total of lrap units were tested over a -month period: were cultured and rapid tested on day ; were rapid tested on day . the rapid test methods were also evaluated based on cost, ease of use, incubation time and indication for use. results/finding: of the lrap units evaluated for this study, there were true negatives (tn) and false positive (fp) on day when tested by bact/alert, with tns on day . bactx testing results showed tns on day and tns on day . testing using the pgd kit showed tns on day ; and tns and fps on day . fp results were confirmed by performing a secondary culture, which were found to be negative. bactx requires a specific analyzer and minutes are required for result interpretation. there is no instrument requirement for pgd and reactions can be read within minutes. conclusion: the results of this evaluation makes pgd the best fit for this blood center based transfusion service. pgd offers a shorter time for reading of results, does not need an initial investment for an analyzer and is indicated for lrap in % plasma and lrap in pas/plasma. its ease of use allows for testing of lrap on day and day during the night shift to be accomplished without additional staffing and allows to extend outdate to day storage of lrap. change in growth factor content of human serum for use as eye drops during frozen storage for year jos lorinser , pieter f van der meer , hans van der heiden and dirk de korte* . department of product and process development, sanquin blood bank, mu-drop background/case studies: growth factors are thought to be among the active components in serum used for treatment of dry-eye syndrome. stability of growth factors during frozen storage in mini containers ( ml) is unknown. if these products can be stored at - c it will be feasible to store this product in -star household freezers, making the product available for patients in need of serum eye drops. the purpose of this study is to demonstrate stability of growth factor content in human serum during longtime storage at - c or <- to - c packed in a new micro dose device for single use as eye drops. study design/method: serum produced from ml whole blood donations from non-remunerated healthy donors was quickly frozen. after frozen storage at <- c for - months and controlled thawing, six different sera were used to fill a large number of mini ( ll) containers, which were refrozen and stored at either - c or <- c. during storage at months intervals, samples were tested for several growth factors, using magpixv r luminex multiplex assays and compared to control samples stored at <- c. growth factors tested were pdgf-aa&ab/bb, tgf-ß / / , vegf, a transfusion vol. supplement s egf, fgf . the study was a fact-finding study, without preset acceptance criteria. results/finding: pdgf-ab/bb and tgf-ß were the most abundant growth factors, on average , resp. ng/ml. also pdgf-aa was detected at relatively high concentration in human serum, on average ng/ml. tgf-ß , egf and vegf were detected at relatively low values, resp. ng/ml, . ng/ml and . ng/ml. average levels of fgf and tgf-ß were close to detection limit (< . ng/ml). the controls stored at <- c showed for all growth factors close to % of the initial values in samples at t (moment of filling mini containers). for serum stored at <- c for up to months, most factors showed less than % decrease, except for pdgf-aa and tgf-ß , showing % resp. % lower values. for serum stored at - c the values for tgf-ß , egf and vegf were stable, whereas pdgf-ab/bb, pdgf-aa and tgf-ß showed a decrease of resp. , and %. conclusion: human serum eye drops can be stored in the new micro dose device at - c ( -star household freezers) or <- c (professional freezers) for at least one year after preparation without large decreases in growth factor content. the maximum decrease was found for pdgf-aa in serum stored at - c. it is yet unknown if the tested components add to the in vivo effectiveness of serum eye drops and what the minimal concentration is to ensure in vivo effectiveness. further stability testing in combination with in vitro and in vivo application is required to extend the shelf-life beyond year. ruqayyah almizraq* , heather inglis , phillip norris , , jennifer a muszynski , nicole juffermans , jelena holovati and jason p. acker , . university of alberta, blood systems research institute, university of california, san francisco, nationwide children's hospital, academic medical center, canadian blood services background/case studies: different blood manufacturing methods can influence residual cell numbers and membrane vesiculation, which may affect quality and safety of blood components. the aim was to identify, quantify and characterize residual cells and extracellular vesicles (evs) in stored rbc products produced by different blood manufacturing methods. study design/methods: thirty-two rbc units produced using whole blood filtration (wbf), red cell filtration (rcf), apheresis, and whole blood derived (wbd) methods were examined (n per method). residual platelets and white blood cells (wbcs) were measured on day using flow cytometer (fc). on storage day and , number and cell of origin/surface markers of evs were assessed with fc, and concentration and size-profile of evs were examined using tunable resistive plus sensing (trps). results/findings: on day , apheresis and wbd units had significantly greater residual platelets in comparisons to rcf (vs: apheresis p< . , wbd p< . ) and wbf (vs: apheresis p< . , wbd p< . ) methods. while rcf units yielded the lowest count of platelet-evs (cd a ) on day and , the highest number of platelet-evs were in apheresis (day ) and in wbd (day ). similarly, there was significant difference among methods in the number of wbc-evs (cd , cd , cd , cd , cd b ) and rcf contained the smallest concentration. moreover, both trps and fc showed an increase in the total number of evs on day vs day in all of the processing methods. noteworthy, trps showed that the number of small evs/exosomes (< nm) was greater than large evs (! nm) in all of the products on day and , and the highest level of evs < nm were in apheresis units. trps results also showed a significant difference in the evs size-profile amongst all rbc products (p< . ). conclusion: this study shows that the method of manufacturing significantly affects rbc and non-rbcs evs characteristics throughout storage, which has the potential to impact quality and safety of rbc products. the differences in the evs cell-of-origin, concentration, and size-profile observed between manufacturing methods, warrants further examination of their potential immunomodulatory effects and clinical consequences. coagulation and complement assays in whole blood stored at centigrade maryanne c herzig* , crystal lafleur , chriselda g fedyk , sherrill j. slichter and andrew p cap . us army institute of surgical research, u.s. army institute of surgical research, university of washington background/case studies: whole blood has been demonstrated to retain hemostatic activity, including platelet aggregation function, over at least weeks of storage at c without agitation. it may be possible to extend the preservation of platelet function by agitating wb. in order to more fully characterize the quality of wb stored at c with or without agitation, we evaluated complement activation as a marker of inflammatory potential. study design/method: subjects donated one unit of wb collected in cpd-a (citrate phosphate dextrose anticoagulant with adenine). the wb was not leukoreduced nor was it separated into components. units were stored under refrigerated conditions for , , , or days after collection. units were stored for days without agitation. units stored for , or days were agitated during storage with a model hybridization incubator at c set for end over end rotation at - rpms. at the appropriate time point, platelet free plasma was obtained from the wb sample and stored at - c. the frozen plasma was analyzed by elisa assays to determine: thrombinantithrombin complex (tat) as a marker of coagulation; soluble cd l as a measure of platelet activation and granule release; plasmin anti-plasmin complex (pap) as a marker of fibrinolysis; plasminogen activator inhibitor (pai- ) as another fibrinolytic measure; and complement activation markers c a, c d, c a and c b- . data was analyzed by one way repeated measure anova. results/finding: only % of the platelets were recovered in units stored for days without agitation. these levels did not meet fda requirements of . x platelets per wb unit. subsequently, wb was agitated and platelet recovery was - %. no difference was seen in elisa analysis for agitated or non-agitated samples. no change was seen in tat or pap levels between t (day of collection) and t , , , or measurements. significant elevations of pai- and scd l indicate activation of platelets and inhibition of fibrinolysis (p< . ). activated complement peptides c a, c a, and c d were all elevated over time (p< . ) while sc d- was not. however, only c a and c d levels at t were above normal reference ranges at . and . times maximum reference, respectively. conclusion: whole blood agitation appeared necessary to recover platelets at or above fda requirements. whole blood stored at c for - days did show some activation of complement proteins. in contrast to studies in stored red blood cells with elevations of sc d- reported, wb showed elevation of c a, a and c d and not sc d- . complement was gradually and modestly activated with most levels remaining within reference ranges over whole blood shelf life. meredith lummer* and christian todd . cerus corporation, community blood serivces background/case studies: the interceptv r blood system for platelets (cerus, concord ca) is used for the pathogen reduction (pr) of platelet collections, and replaces irradiation, cmv testing, bacterial culture and point of issue bacterial testing. to better understand pr compatibility and impact to split rate, data were analyzed from a mid-size blood center with roughly . x . x . x . x rcf . x . x . x . x apheresis . x . x . x . x wbd . x . x . x . x platelet collections must meet specific volume, concentration, and dose ranges to qualify for intercept pr. changes made to apheresis devices included adding the following collection targets: . x in ml, . x in ml, . x in ml, and . x in ml. study design/methods: four months of collections were retrospectively analyzed. platelet collections were evaluated to determine eligibility for pr treatment, and all products meeting pr processing specifications (unless intended for an hla matched recipient at a hospital not able to accept pr products) underwent pr treatment regardless of potential impact to split rate. a minimum post-treatment dose of . x or . x was required to classify collections as singles or doubles respectively. volume/dose mitigation (removal of volume to increase the number of products eligible for pr) was not utilized during this study. thus units were treated conventionally if volume, dose, and/or concentration did not meet pr specifications without further manipulation. results/findings: % of all single and double collections were eligible for and underwent pr treatment. split rate for single and double collections was . . conclusion: it is possible to treat % of single and double platelet donations with intercept pr at the blood center's current state with only a slight impact to split rate if centers are willing to make alterations to their targeting practices. platelet collections that fall outside of the specifications for pr are processed and distributed as conventional products. strategies to increase eligibility toward % while minimizing impact to split rate are being investigated, including incorporating new collection settings, splitting triples, and volume/dose mitigation. further evaluation is needed to determine the additional quantity of pr eligible products resulting from such changes. monique p gelderman* , andrey skripchenko , fei xu , ying li , stephen j wagner , pamela h whitley and jaroslav g vostal . fda/cber/ obrr/dbcd/lch, american red cross holland laboratory, american red cross mid-atlantic research facility background/case studies: platelets (plts) stored at room temperature (rt) can support bacterial proliferation in contaminated units and therefore septic transfusion reactions may occur. storing plts at cold temperature ( - o c [ct]) limits bacterial growth but results in rapid clearance upon transfusion. the development of alternate storage conditions usually involves costly radiolabeling human studies but success in these studies is difficult to predict based on in vitro studies. thus, an animal model of plt circulation that could predict performance of human plts in human volunteers would positively impact the development of alternate storage conditions. study design/method: we designed an immunodeficient (scid) mouse model to evaluate recovery of human plts and compared this side by side to a radiolabeling study in human volunteers that was conducted for evaluating a new plt storage condition: thermocycling plts ( hrs ct: hr o c [tc]). autologous apheresis plts stored for -days at rt, tc and ct were radiolabeled and infused into healthy human volunteers (n ) and the same non-labeled plts were also infused into mice (n ). blood samples from humans and mice were collected over time to generate survival and clearance curves of the plts in circulation. flow cytometry was used to detect and analyze the human plts in the mouse samples to generate such curves; counts < % were considered background. results/finding: the mean recoveries of infused plts were . . % for rt, . . % for tc and . . % for ct in humans. in mice, mean recoveries of the same plts were . . % for rt, . . % for ct and . . for ct (mean sd). to compare performance of the plts in humans and mice we expressed all recoveries as a percentage of the rt recoveries. in humans tc was $ % and ct was $ % of rt. in mice tc was $ % and ct was $ % of rt. the area under the survival curve (auc) was calculated for the individual mouse study and human trial data sets. the results of both auc were normalized to % for rt plts. human tc plts had % auc while ct plts had % auc compared to rt plts in humans. in comparison, the same tc plts had % auc and ct plts had % auc of the rt auc in the mice. the calculated ratios of the auc between the tc plts and ct plts of the human data set and mouse model data set are . and . , respectively. conclusion: the scid mouse model differentiates between rt plts and ct plts similar to humans based on auc and plt recovery data. however, the mouse model cannot differentiate between ct plts and tc plts as occurs in humans. even though the mouse model cannot differentiate between ct plts and tc plts, it may still be a useful tool to screen other novel storage conditions for human plts. converting the component manufacturing from a manual process to automation nicole peters* and geeta paranjape , . coastal bend blood center, carter blood care background/case studies: initiatives focused on improvements to donor collection processes drove us to investigate opportunities in our component manufacturing processes. our goal was to maintain blood quality while streamlining manufacturing and automating the in-process documentation. the compomat g was evaluated using a multi-team approach including component manufacturing staff, equipment management, qa, regulatory affairs and it. study design/method: after a comprehensive evaluation, the team decided to purchase the compomat g with the compomaster net software for data management. implementation was planned for a november go-live. . to centralize processing, new work counters were installed. fresenius kabi installed the compomat g s and compomaster in june . training and validations were successfully completed and a full launch occurred mid-march . device and sop training was performed. training qualification checklists were completed for each technician with a required number of successful units processed and completed december . validation was completed and signed off in march of . manufacturing data was collected using the compomaster net data management system and our quality control software for platelet (plt) parameters, including plt count, plt weight, and plt yield from before implementation (bi) and after implementing (ai) of the compomat g system. data points were collected from units bi and units ai. results/finding: upon initial implementation, staff training and use, the compomat g was found to be easy. plt weight spread was reduced from an average of gm to an average of gm. actual plt weights were reduced from an average of gm to gm, resulting in an average increase in recovered plasma of . ml per unit. plt count on average increased from a count of to ( /mm ) with a negligible change in plt yield. conclusion: plt weight spread was reduced by . % after implementation of the compomat g and our plt concentrations increased on average by %. we were able to consistently produce a smaller volume plt (average gm), which gave us . ml more plasma per unit for recovered plasma. the team intends to review a dryer cryo as a next step for potential additional plasma yields for recovered plasma. deglycerolization of manually glycerolized, frozen rccs using a closed system cell processor anita howell , angela hill , brandie dennis and jason p. acker* , . canadian blood services, centre for innovation, canadian blood services, university of alberta background/case studies: upon implementation of a closed system cell processor for glycerolization and deglycerolization of red cell concentrates (rccs), many rare rccs frozen using the current manual, open system glycerolization method will remain in the organization's frozen inventory. a study was undertaken to assess the feasibility of deglycerolizing this existing inventory on the closed cell processor and to evaluate how the change may impact post-thaw red blood cell (rbc) in vitro quality. as the closed cell processor uses a fixed centrifuge bowl for deglycerolization and rbc resuspension, both large and small units were assessed to determine the impact of cellular loss and variability in hematocrit on the post-thaw product. study design/methods: abo/rh matched lr sagm rccs were pooled and split to produce large ( ml) and small ( ml) rccs. the rccs were stored to d and glycerolized manually by mixing ml of glycerol with the rcc in a ml freezing bag. units were frozen at - c for ! h before being removed from frozen storage and thawed in a c water bath. large rccs and small rccs were deglycerolized using the organization's current procedure on the cobe cell processor prior to re-suspension in . % saline, . % dextrose. the remaining rccs were transferred into a l bag, spun to allow removal of excess glycerol by manual extraction to achieve a hematocrit of %, and deglycerolized in a ml centrifuge bowl on the acp- with re-suspension in as- . rbc quality was tested at h post-deglycerolization. results/findings: large rccs had significantly higher hemoglobin per unit (cobe: p . , acp : p . ) and lower cell recovery (cobe: p . , acp : p< . ) post-deglycerolization than smaller rccs on both cell processors. large rccs deglycerolized on the cobe had higher hemolysis (p< . ) and supernatant potassium (p . ) than did small volume rccs. large cobe rccs had higher hematocrits (p . ), hemoglobin (p . ), and recovery (p . ) than did large acp- rccs. however, all cobe rccs had higher (p< . ) hemolysis ( . . %) levels than did acp- rccs ( . . %). cobe rccs failed to meet regulatory hemolysis standards of . %. conclusion: addition of a ml bolus dose of glycerol to rccs of different volumes results in different concentrations of glycerol in the frozen rcc product and may lead to differences in frozen rcc quality. additionally, the size of the rcc impacts quality for rccs processed on the closed cell processor due to centrifuge bowl volume limitations which result in lower recovery, hemoglobin, and hematocrits. use of the closed cell processor with resuspension in as- and storage for h, met in vitro quality standards for recovery, hemoglobin, and hematocrit, and drastically reduced hemolysis levels in rccs glycerolized manually. the acp- cell processor can therefore be used to deglycerolize rccs glycerolized using a manual, open system glycerolization method. background/case studies: washed platelets may be indicated for thrombocytopenic patients who experience severe allergic/anaphylactic or febrile reactions to conventional platelet transfusions. platelet washing process is time-consuming which may delay transfusion. this study was conducted to evaluate the manual platelet washing method (mm) using . % saline and centrifugation and the semi-automated washing method (sam) using the cobe blood cell processor. study design/method: in this study, units of single donor platelets were evaluated ( washed using the mm and washed using the sam. the collected data included product weights (pre-and post-wash), platelet counts (pre-and post-wash), total plasma protein (pre-and post-wash), presence/absence of platelet clumps, calculated % protein removal, and calculated % platelet recovery rate. the platelet counts were measured on the sysmex exn and the total plasma protein samples were measured on the roche cobas . results/finding: table shows that the average platelet recovery for the sam ( %) was significantly higher compared to the mm ( %). the mm had a slightly higher average protein removal compared to the sam. no platelet clumps were observed in either the mm or the sam. it was observed that the hands-on time for the mm took - minutes longer than the sam. background/case studies: the interceptv r blood system for platelets is currently licensed for pathogen reduction (pr) of amicus platelets in inter-sol (pas- ) for input platelet doses of . to . platelets in to ml of to % plasma and - % pas. a new platelet processing set was designed with three storage containers (ts) to process apheresis platelet components in pas- containing doses of . to . platelets in a volume of to ml. study design/methods: apheresis pcs (amicus v r ) were collected in % plasma and % pas- . one study was performed at the nominal dose ( . - . x platelets), volume ( - ml) in % pas/ % plasma using single donor apheresis collections. two studies were performed to evaluate the high dose and high volume condition ( . - . x platelets in - ml) using either single or pooled donations. input pcs (n ) were treated with the intercept ts set by the end of day post collection; the incubation time in the compound adsorption device (cad) container ranged from to hours and the intercept treated pcs were stored in containers (n ). day and post-donation pcs were evaluated using a panel of in vitro platelet function assays results/findings: in vitro function data for apheresis pcs in pas- treated in the intercept ts set demonstrated acceptable in vitro function (table ). all intercept treated pcs had ph( c) ! . . platelet dose and volume recovery post-treatment ranged from % to % and % to %, respectively. conclusion: pathogen reduced platelet components processed using the intercept ts set from either single or pooled apheresis donations maintained acceptable in vitro quality through days of storage. intercept blood system for platelets ts set is currently not approved for use in the us. background/case studies: the possibility of transmitting infectious organisms via blood products, plasma and their derivatives is a major public health concern. while current screening measures have considerably improved transfusion safety by reducing the risks associated with known pathogens, they cannot protect from emerging infectious threats. the pathogen reduction technology (prt) represents a proactive strategy to further reduce transfusion-transmitted infectious risk. however, the scientific community broadly agrees over the fact that prt has negative impacts on the product's quality markers. this study aims at evaluating the impacts of the mirasol prt on platelet (plt) quality and plt processing. study design/method: two abo-compatible platelet concentrates (pcs) containing % plasma obtained from either apheresis or sagm whole blood (wb)-derived processing were paired, pooled and then split into two equal units. one unit was used as a non-treated control (ctrl) (n ). riboflavin was added to the other pc unit and then exposed to uv light according to the manufacturer's instructions for the mirasol prt (teru-mobct) (test) (n ). numerous in-vitro quality markers (plt concentration, atp, po , pco , ph, glucose, lactate, sodium, and potassium) were measured for both mirasol-treated and non-treated pcs on days , , and for apheresis pcs, and on days , , and for wb-derived pcs. two flow cytometry assays were used to evaluate cd p expression with and without thrombin activation, and to measure the percent annexin vpositive plt. transfusion vol. supplement s results/finding: platelet recovery was % and % for apheresis and wb-derived pcs, respectively. mirasol-treated pcs showed higher levels of annexin v-positive cells ( % (test), vs. . % . (ctl) on day ) and a higher rate of cd p expression than control pc units ( % (test), vs. % (ctl)) on day ). the mirasol treatment generates changes in ph, glucose and lactate for pcs during storage. conclusion: the mirasol treatment induces a loss in the net number of plts/unit and elevated platelet activation. changes in ph, glucose and lactate suggest that prt affects plt metabolism. finally, prt has numerous impacts on logistic, storage and processing time constraints of blood bank operations. nevertheless, the mirasol prt is routinely used in europe with acceptable clinical outcomes. evaluation of a test method to detect bacterial contamination in platelets; bactx tm assay ji hye park sexton* , lorraine blagg , christi e marshall , herman woodson , sean erony , krishna patel and eric gehrie . the johns hopkins hospital, johns hopkins hospital transfusion medicine dept, johns hopkins university school of medicine background/case studies: bacterial contamination of platelets (plts) is the leading infectious risk of platelet transfusion therapy and it is the most significant infectious cause of transmission-associated morbidity and mortality. therefore, detecting various potential bacterial contaminants in platelets in a timely manner is critical. the bactx assay is a rapid colorimetric assay that detects peptidoglycan, a cell wall component of both gram-positive and gram-negative bacteria. here, we report an analysis of the bactx assay at our hospital. study design/method: we aimed to determine the sensitivity and specificity of the bactx assay. intact leukoreduced apheresis plt (lrap) units were tested by bactx at storage day . as a control, each intact lrap was also cultured by an automated bacterial detection system (bact culture) on storage day . the results of the bactx test were compared to the results of the bact culture system. results/finding: a total of lrap were tested. lraps initially tested negative by bactx, while lraps initially tested positive by bactx. all initial positive bactx tests were negative when subjected to repeat testing. in contrast, all lraps tested negative with the bact culture system. the specificity of the bactx test was . %. we did not have any true positive test results; therefore, the sensitivity of the bactx could not be determined. conclusion: this is a small study of only platelet units. the expected rate of bacterial contamination of platelets is less than per units. the . % initial positive rate was therefore higher than expected, but given the small sample size, it is clear that further study is needed to more rigorously assess the true sensitivity and specificity of the bactx assay. in vitro quality of rejuvenated and washed cpd/as- and cp d/as- rbc alan d. gray* , matt landrigan , pamela whitley , michael wellington , sherrie sawyer , shalene hanley , emily rondeau , louise herschel , neeta rugg , patricia a.r. brunker , shawnagay nestheide , jose cancelas-perez , larry dumont and zbigniew m. szczepiorkowski . and , -dpg to fresh levels. the objective was to demonstrate that in vitro quality measures are maintained for rbc when stored for > hours after treatment with an fda approved rejuvenation solution. study design/method: whole blood ( - ml) was collected and processed at sites into leukocyte-reduced rbc (a total of n cpd/ as- and n cp d/as- ). ml of rejuvenation solution (citra labs) was added to each rbc on day (d- ), incubated for minutes with agitation at c water bath (helmer dh ), washed (haemonetics acp ), and stored in as- at - oc for days (d- through d- ). in vitro recovery (%) was calculated and hemolysis, atp, and , -dpg were determined on day , d- , d- after rejuvenation and washing (postrjv), d- , d- , d- , and d- . all units were cultured on d- postrjv and on d- , and then concentrated by centrifugation on d- . results/finding: in vitro rbc recoveries were . % and . % (as- and as- , respectively) and no bacterial growth was observed. hemolysis on d- was maintained < % in / ( %) as- units and / ( . %) as- units. all as- and as- units ( %) had hemolysis < % following concentration by centrifugation. morphology score was reduced to % (as- ) and % (as- ) by d- , restored after rejuvenation ( %, %, respectively) and maintained through d- (> %). atp was restored and maintained above fresh levels after rejuvenation. , -dpg was restored above fresh levels and was maintained ! % of fresh levels through d- . all values were significantly different compared to d- except as noted (p< . , paired ttest) ( table ) . conclusion: rejuvenation of stored rbc restores atp and , -dpg above fresh values and morphology to near fresh levels while maintaining improved in vitro rbc quality measures through d- when compared to nonrejuvenated rbc on d- . this study is funded by zimmer biomet. storage > hours is not fda approved for use at the time of this publication. liposomes and rejuvenation: new approach for improving quality of stored red blood cells luciana da silveira cavalcante , jason p. acker* , and jelena holovati . background/case studies: liposomes have been shown to minimize rbc membrane damage occurring during -day hypothermic storage (hs), while rejuvenation solutions have been shown to restore rbc metabolism by maintaining atp and , -dpg levels. this study aimed to evaluate the effect of combining liposomes and rejuvenation on the quality of stored rbcs. study design/methods: five leukoreduced packed rbc units obtained were pooled and split. the units produced were segregated into four experimental groups: sham control (s), liposome-treated (l), rejuvesol-treated (r) and liposome rejuvesol-treated (l r). the prbcs were incubated for h at c with hepes-nacl (sham), liposomes (dopc:chol, : mol%, mm lipid), rejuvesol or liposomes plus rejuvesol. the in vitro quality was accessed by hemolysis, deformability, aggregation, atp and , -dpg at day hs. results/findings: hemolysis was significantly decreased in all treatments compared to sham control ( . . %): l ( . . %, p . ), r ( . . %, p . ), l r ( . . %, p . ). ektacytometry analysis showed an increase in maximum elongation (ei max ) in r ( . . , p . ) and l r ( . . , p . ) treatments compared to s ( . . ) but not l ( . . , p . ). rbc rigidity (kei) increased in all treatments compared to sham ( . . ): l ( . . , p . ), r ( . . , p . ) and r l ( . . , p . ). aggregation amplitude was significantly increased by r treatment only ( . . au vs. . . au, p . ). atp levels were significantly higher in all treatments compared to sham ( . . mmol/g hb): l ( . . mmol/g hb, p . ), r ( . . mmol/g hb, p . ), l r ( . . mmol/g hb, p . ). the levels of , -dpg were no longer detectable in s and l treatments at day . the combined treatment was comparable to r ( . . mmol/g hb vs. . . mmol/g hb, p . ). conclusion: both rejuvenation and liposome treatments improved the quality of stored rbcs compared to sham control. the combined treatment (l r) did not have a greater impact in improving in vitro quality of stored rbcs compared to rejuvenation alone. step toward a unique and adaptable thermoregulation system lucie boyer , eric ducas , patricia landry , nathalie dussault , jacques bernier , danny brouard* and anne maltais . h ema-qu ebec, institut de technologie des emballages et du g enie alimentaire background/case studies: h ema-quebec (hq) is facing major logistic challenges in the transportation and distribution of blood components over a large geographic area. in collaboration with the institut de technologie des emballages et du genie alimentaire, our applied research group is working on the development and optimization of a transport packaging for the -ml whole blood leukotrap rc system (haemonetics corp.). the objective is to design a packaging system for the rapid cooling (t < c) of one to six -ml whole blood units (wbu) within h from collection. moreover, the insulating and thermoregulation system must maintain the internal temperature of wbu between c and c for h under extreme external conditions (- c to c), including the initial blood cooling period. study design/method: the proposed packaging design is based on an external coroplast box containing six vacuum insulated panels (vip) for increased insulating efficiency. preservation of the initial cooling period and extended thermoregulation properties were ensured by an assembly of preconditioned c phase change material (pcm). the number of pcm, their position and conditioning were optimized and tested in order to meet the expected performance criteria. preconditioned pcm were stored into vip boxes for h at - c before each test to mimic a worst-case scenario for remote blood drives. for the experimental testing, -ml wb bags were filled with ml saline . % at t c to mimic freshly collected wb. probes were positioned inside the saline-filled bags to monitor temperature profiles of wbu under extreme winter (- c) and summer ( c) conditions. shipping boxes were filled with either one or six bags (n ). results/finding: the results showed that the thermoregulation box prototype is able to cool wbu bags under c in . . h and maintain their internal temperature between c and c for h with final values ranging between . c and . c for the extreme summer scenario. similar results were obtained for the extreme winter scenario; units reached the c threshold value in . . h and the bags' internal temperatures were within the acceptable range for h. conclusion: the insulating and thermoregulation system met hq performance criteria. preliminary results showed that pcm could be conditioned at temperatures higher than - c without any significant impact on the system performances. hq is currently validating the shipping box prototype performances. additionally, we are working on reducing the pcm conditioning time to optimize logistic operations. as this packaging has many advantages in terms of durability, price and convenience, hq intends to evaluate this system for the packaging and transport of other lines of blood products. stuart weisberg* , christopher c. c silliman , beth shaz , marguerite kelher and claudia s. cohn . new york blood center, bonfils blood center, department of laboratory medicine and pathology, university of minnesota background/case studies: platelets collected and stored in platelet additive solution (pas) reduce recipient exposure to donor plasma components. to better define the effects of pas on platelet supernatant composition, we compared total protein, isohemagglutinin titers, hla antibodies and in vitro neutrophil (pmn) priming activity in supernatants of pas-c platelets to plasma platelets. study design/methods: apheresis platelets from group o blood donors were collected into either % donor plasma (n ) or % pas- / % donor plasma (n ). within hours of collection, samples of the product supernatant were frozen, assayed for total protein concentration, anti-a and anti-b titer, and pmn priming activity within the total and lipid extractable fractions. all samples were screened for hla antibodies. screen-positive samples were tested using luminex single bead assays for antibody strength and specificity. soluble cd ligand (scd l) was measured using solid-phase elisa. results/findings: supernatants of pas-c platelets had significantly lower total protein concentration, anti-a and anti-b titers compared to plasma platelets. there was no significant difference in the number of hla-antibody screen positive pas-c ( / products) compared to plasma platelets ( / products); however, the hla-antibody screen-positive supernatants of pas- a transfusion vol. supplement s abstract c platelets had fewer hla specificities ( specificities) compared to those of the plasma platelets ( specificities). pmn priming activity was significantly increased in the supernatant of pas-c platelets. the lipid extractable fraction was not affected; however scd l levels were increased in the supernatant of pas-c compared to plasma platelets (table ) . conclusion: decreased plasma proteins likely underlie lower rates of allergic and febrile non-hemolytic transfusion reactions seen with use of pas-c platelets. decreased anti-a and anti-b titers may prevent hemolysis from minor abo mismatch. lower hla-antibody specificities may mitigate transfusion related acute lung injury (trali). increased pmn priming by pas-c platelets is likely due to platelet membrane release of scd l and not bioactive lipids. although scd l has been associated with trali, only pmn priming with lipid -not cytokine -agents has been causally linked with trali. the mechanism and clinical impact of increased scd l in pas-c platelets remain to be elucidated. background/case studies: current guidelines require a reduction of residual white blood cells (rwbc) below x wbc in us and x wbc in europe, per unit. the established reference method for testing rwbc in platelet (plt) and red blood cell (rbc) products is flow cytometry. alternative technologies have been developed including hemocytometry and microfluorometry. study design/methods: this study compared performance and workflow efficiency of the facsvia, a flow cytometer with a simplified workflow and automated loader to the adam automatic microscopic cell counter based on imaging technology. nonfiltered whole blood (wb) samples, apheresis platelet units (n ) and leukoreduced (lr) rbc units (n ) were used to generate spiked samples. apheresis platelets and lr rbc were filtered to deplete wbcs and were used as a diluent. nonfiltered wb samples were the source of wbcs to prepare a sample of wbc/ul. the spiked samples of , . , , , and wbc/ul were prepared from the source sample of wbc/ul and filtered platelet and rbc units. to evaluate linearity, wbc concentrations ( , . , , , , wbc/ul) were measured using adam and facsvia. samples were stained and run in triplicate on each analyzer. data was analyzed using linear regression. the results were proportional to the wbc concentration in the spiked samples. reproducibility of the two systems was measured by running spiked samples ( , , , wbc/ul). tubes of each sample were stained and run per system. the %cv and %diff were calculated. a batch of samples (plt and rbc) were run on both analyzers, repeated for days. workflow efficiency was assessed observationally by measuring the time of tasks performed. tasks recorded were instrument qc, assay controls and sample testing and analysis. results/findings: the wbc concentration results for plt and rbc samples on facsvia correlated well with adam (r-plt . , slope . ), (r-rbc . , slope . ). the %diff-plt at , , wbc/ul were . , . and , respectively. the %diff-rbc at , , wbc/ul were . , . and . , respectively. the average total testing time was similar on both instruments; min for the facsvia and min for the adam. of the total testing time, adam required continuous hands-on time, while facsvia demonstrated % ( of min) hands-off time. conclusion: both instruments showed comparable precision, linearity and accuracy. while the average total testing time was similar on both instruments, facsvia offered a significant workflow efficiency advantage. users saved an average hands-on time of minutes that could be used on other tasks. platelet rich plasma and quality control: is there a role for the blood bank? claudia s. cohn* and mickey koh . department of laboratory medicine and pathology, university of minnesota, st george's hospital and medical school background/case studies: autologous platelet-rich plasma (aprp) is a poorly regulated blood component often produced at the patient's bedside and used for indications such as chronic and acute orthopedic injuries, wound and incision-healing and rheumatologic diseases. prp isolation can be done by apheresis, which yields a consistent, platelet-rich fraction; however, most aprp is made using small bench-top centrifuges with cartridges that deliver uneven platelet enrichment. thus, the consistency and quality of aprp is questionable and the lower yielding prp may have decreased efficacy. study design/methods: a survey was designed to assess aprp manufacture, usage and quality control (qc) measures taken prior to its use. a survey was developed with input from content experts. the survey was sent to members of best and isbt. survey respondents were encouraged to forward the survey to colleagues, thus a true denominator is unknown. a total of completed and partially completed surveys were received. results/findings: responses came from countries, but the majority of responses came from the united states (us). of the respondents, % reported aprp use in their hospital. aprp was used predominantly for outpatients, though > % of hospitals also used aprp in the in-patient setting. in most hospitals, aprp was used by - mds; however, hospitals had > mds using aprp. the aprp was used for orthopedics, wound/incision repair, rheumatology and other indications. in the us the aprp was manufactured outside of the blood bank, while outside the us aprp was isolated by blood bank personnel. nearly all the aprp manufacturing was done with no quality control (qc) measures ( %); however, respondents assessed the final product prior to release. these qc measures included a platelet count to measure the enrichment of the platelet fraction, culturing the product and infectious serology testing. in some cases, if the aprp failed qc it could still be used, pending an md's approval. in the hospitals conducting qc on the final aprp, the testing was done by the blood bank. a subset of respondents from african nations also used allogeneic prp (allprp). in contrast to the patterns of use with aprp, allprp was used primarily for inpatients for indications including orthopedics, wound/incision repair and 'other'. the allprp was manufactured in the blood bank or the donor center with no qc other than a regular check of the centrifuge used to isolate the prp fraction. conclusion: prp is used in hospitals throughout the world for a wide variety of indications. the blood bank is involved in its manufacture in some countries, but in the us aprp is made outside of the blood bank. quality control of aprp production and the final product is not done in most hospitals. to improve the consistency and efficacy of prp, more stringent qc measures need to be in place. background/case studies: the morphology of donated red blood cells (rbc) change with storage, along with a loss of deformability, increased surface exposure of phosphatidylserine (ps), and decreased intracellular atp. these changes have been associated with increased rbc clearance within hours of transfusion. analysis of morphological alterations of stored rbc with imaging flow cytometry (ifc) has identified a subpopulation of small rbc that accumulates upon storage. this rbc subpopulation has a reduced projected surface area and undergoes a spherocytic shift which is expected to induce their retention in the spleen (roussel, dussiot et al, ) . some of the storage alterations are reversible when the rbc metabolism is reestablished. as such, treatment with a rejuvenation solution (citra labs) before transfusion is expected to restore some of the rbc properties and thus potentially increase their capacity to stay in circulation and operate effective tissue oxygenation following transfusion. study design/methods: a multi-parametric analysis of rbc alterations was performed to evaluate the effect of rejuvenation on rbcs stored in sagm (n ) under blood bank conditions at day (d ), at day (d ), after rejuvenation (r), and after rejuvenation and washing (rw). morphological alterations of stored rbcs were evaluated with ifc (imagestream x mark ii, amnis v r ). results/finding: rejuvenation increased the level of intracellular atp, confirming the metabolic effect of this process. population distribution as per rbc projected surface area measured by ifc depicted a well-demarcated subpopulation of small rbc that increased with storage from . - . % at d to . - . % at d . rejuvenation markedly reduced this storage-induced spherocytic shift ( . - . %) and partially restored rbc morphology, an effect confirmed by differential interference contrast microscopy. the restoration effect of the rejuvenation process did not correct the storage-related loss of rbc elongation but was associated with a decrease in ps exposure (table) . conclusion: our multi-parametric analysis shows that some but not all storage-related alterations are therefore corrected by metabolic rejuvenation. the impact of these effects while generally positive at the cellular scale requires further analysis by specific clinical studies assessing transfusion yield and tissue oxygenation. red cell concentrate volume and manufacturing method impact post-thaw quality in cryopreserved products processed using a closed cell processor anita howell , angela hill , tracey turner , april xu , brandie dennis and jason p. acker* , . canadian blood services, centre for innovation, canadian blood services, university of alberta background/case studies: the blood service uses both top/top with whole blood filtration (wbf) and top/bottom with red cell filtration (rcf) methods to prepare cpd/sagm lr red cell concentrates (rccs). mean volume (ml) is higher in wbf units ( ) than in rcf units ( ), with similar hematocrits. a closed system cell processor is currently being implemented for cryopreservation of rccs. post-deglycerolization re-suspension in as- additive solution is performed on-instrument to a defined total end volume, as dictated by the centrifuge bowl size. the impact of the resulting variation in hematocrit on post-thaw in vitrorbc quality was evaluated to ensure that regulatory standards can still be met for rccs at the extreme edge of the input volume range. study design/methods: small rcf ( - ml) and large wbf ( - ml) rccs were stored for d before being glycerolized and frozen at - c for ! h. large rccs whose red cell mass exceeded the capacity of the ml deglycerolization centrifuge bowl were volume reduced prior to glycerolization. rccs were thawed in a c water bath, deglycerolized and re-suspended in as- . rccs were stored d and then tested for in vitrorbc quality. results/finding: small rcf rccs had lower (p< . ) hematocrit, specific gravity, hemoglobin per unit, supernatant k and na concentration, deformability (ei max ), and higher (p< . ) recovery than did large wbf units. no significant differences in hemolysis, atp, , -dpg, p , rbc indices, rbc morphology, or residual glycerol were seen between groups. the majority of units met acceptance criteria (table ) , however of large wbf units had rbc recoveries < % due to pre-glycerolization volume reduction, and of the small rcf units had hemoglobin values < g per unit. when the recovery and hemoglobin failure rates are analyzed against the organization's rcc production volume distribution, the mean recovery is projected to be well above % and the hemoglobin failure rate would be below % of units tested; compliant with regulatory standards. conclusion: the differences between groups in the cryopreserved rcc physical characteristics were expected due to the re-suspension method and differences in the input product red cell mass. the lack of significant metabolic differences between groups indicates that the differences in postdeglycerolization hematocrits are not adversely affecting product quality. . . . . . . . . elongation index ( pa) . . . . . . . . this study is funded by zimmer biomet. (hasan ) . the objective was to determine the effect of rbc rejuvenation on rbc oxygen release capacity (orc) and estimated oxygen consumption (vo ) after simulating a single unit transfusion of either standard or rejuvenated rbc stored for days. study design/method: oxygen dissociation curves (odc) (hemox analyzer, tcs scientific) were generated from fifty-two ( ) rbc units (leukocyte-reduced), cpd/as- or cp d/as- , on day , day , and after rejuvenation and washing (pw). the odc for each sample was used to determine orc (ml o /g hb) and total releasable oxygen (tro) of the unit (ml o ). orc was determined by assessing the change in % o saturation from mm hg po (e.g., lung) to mm hg po (e.g., venous blood) multiplied by . ml o /g hb (li ). a simulated baseline pretransfusion vo of ml o /min was estimated using the day orc and assuming a g/dl transfusion trigger with a cardiac output of l/min and l blood volume. paired student's t tests were used for comparative statistical analyses. results/finding: rbc rejuvenation on day restored orc and tro to levels greater than day ( table ) . orc of the rejuvenated unit was . . times and . . times greater than rbc on day and day , respectively (p< . ). vo increased after a simulated single unit transfusion of rbc (day , day , and pw) by . %, . %, and . % over the pre transfusion vo , respectively (p< . ). conclusion: these results suggest a transfusion with rejuvenated rbcs has the potential to release . times the volume of o compared to standard, untreated rbcs stored for days. inferior oxygen delivery to tissues (vo max) has been observed during exercise in healthy human volunteers after transfusion of two autologous rbc units stored for days vs days which seem dependent on genetic variability and storage time (bennett-guerrero ). therefore, transfusion practices to correct anemia may be less effective than intended due to the variable orc of standard stored rbc units. transfusion strategies should consider whether the use of rbc with increased orc may be physiologically advantageous. disclosure: this study was funded by zimmer biomet. rejuvenation solution as an adjunct storage solution maintains physiological hemoglobin oxygen affinity during rbc unit storage andrea ansari* , jay srinivasan , gustaaf de ridder , alan d. gray , matt landrigan , keaton charles stoner , angela crabtree , jessica poisson and ian welsby . duke university school of medicine, duke health pathology, citra labs, a zimmer biomet company, zimmer biomet, duke university, department of pathology, durham veterans affairs medical center, duke university hospital, duke university medical center background/case studies: deleterious changes develop during the storage of packed red blood cells (rbcs) collectively called the "storage lesion". these include altered membrane composition and decreased deformability, increased in-bag and post-transfusion hemolysis, loss of atp, snitrosohemoglobin, vasodilatory capacity, and cell surface ps expression, and depleted , -diphosphoglycerate ( , -dpg). the loss of , -dpg increases the oxygen affinity of hemoglobin, resulting in lower p (partial pressure of oxygen at % hemoglobin saturation). decreased p may negatively impact the ability for transfused rbcs to release oxygen to peripheral tissues. an fda-approved rejuvenation solution (citra labs) can restore normal levels of atp and , -dpg, normalizing membrane function and oxygen affinity, respectively. this process requires incubation at c for an hour, an impractical step in time-sensitive situations, followed by washing of the rbcs. we tested the hypothesis that rejuvenation without the incubation step ("cold rejuvenation") could prevent or reverse changes in oxygen affinity, deformability, and susceptibility to hemolysis of rbcs. study design/method: eight units of group a , leukoreduced prbc stored in as- were obtained from our local blood center. after days of storage, units were divided into separate aliquots: control (ctl), wash (w), standard rejuvenation (sr), and cold rejuvenation (cr). the rejuvenation solution ( ml) was added to the cr group, and all groups were then stored for another days at - c. on day of storage, the sr group was incubated for hour at c with rejuvenation solution, after which the w, sr, and cr groups were separately washed on a c.a.t.s v r (fresenius kabi) using the high quality wash setting. hemoglobin p was measured by tonometry using a hemox analyzer (tcs scientific). deformability (elongation index or ei) was measured by ektacytometry (lorrca mechatronics). supernatant plasma free hemoglobin (pfhb) was measured using visiblelight spectrophotometry. cell surface ps expression (ps ) was measured by annexin v flow cytometry. all group results were compared using nonparametric wilcoxon signed-rank tests with a . . results/finding: significant differences in p were noticed between all groups (table ) . ei, ps , and pfhb did not differ between groups. conclusion: cold rejuvenation prevents the increased oxygen affinity (lower p ) seen over days of rbc storage without adverse effects on deformability or hemolysis. this offers an alternative to incubated rejuvenation to provide clinicians with ready access to rbcs with a high/normal p that may better release oxygen to the tissues. cause transient and potentially fatal cardiac arrhythmias upon transfusion, particularly in infants, and massively-transfused patients, and those with compromised renal function. reactive antibodies and other inflammatory agents in rbcs can also elicit life-threatening reactions, potentially causing high fever, transfusion-related acute lung injury (trali), anaphylaxis, and even death. in this study, a multifunctional bead-based filter was evaluated for removal of k , along with free hemoglobin (hb) and other prbc contaminants that can contribute to transfusion related adverse events. study design/method: ten leukocyte-reduced prbc ( ml) units stored in as- , obtained from a regional blood donor center at expiration ( days), were passed by gravity through sorbent-devices containing ml of multifunctional polymer bead, at a flow rate of ml/min. supernatants were analyzed for k removal as well as free hb, antibodies and cytokines ( -plex, biorad). rbcs were analyzed for viability and integrity via flow cytometry and osmotic fragility assay, respectively. results/finding: filtration of the aged prbc units through the sorbent device reduced [k ] from . . to . . meq/l; equivalent to an . % reduction. free hb was reduced by . % from . . to . . mg/ml. antibodies, specifically igg, iga, and igm decreased from . . to . . mg/ml ( . %), . . to . . mg/ml ( . %), and . . to . . mg/ml ( . %), respectively. inflammatory cytokines were significantly reduced, specifically: ip- from . . to . . pg/ml ( . %), mip- b from . . to . . pg/ml ( . %), and pdgf from . . to . pg/ml ( . %). filtration had no significant impact on cell surface markers of rbc viability (< . % decrease) or sensitivity to osmotic changes. values listed represent mean sem (p < . for all analytes tested). a paired ttest was used to assess significance. conclusion: the sorbent filter was highly effective in reducing the levels of extracellular k as well as free hb, antibodies, and cytokines from prbcs without impact on rbc viability or integrity. this study demonstrates the viability of a multifunctional sorbent filter for removal of k along with other detrimental components from stored prbcs that can readily be incorporated into transfusion practices to minimize adverse effects. background/case studies: platelets carry no rh antigens, but residual red blood cell (rbc) in platelet products can immunize d negative recipients if the donor is d positive. current recommendation is to give rh immunoglobulin (rhig) to rh negative patient if they receive rh positive platelet unit to avoid potential alloimmunization to d antigen. a recent study has shown a very low frequency ( . %) of d alloimmunization when a rh mismatch platelet is transfused. restricting d negative patients to receive only d negative platelets could create shortage and cause inventory challenges. higher yields of platelets with minimum to none residual rbcs are obtained with new generations of apheresis machines. as a consequence, the need for prophylactic rh immunoglobulin (rhig) may be unnecessary with the use of apheresis derived platelets. the accurate determination of residual rbc in a platelet unit is important for patient safety to prevent rh alloimmunization. hemocytometer is considered the gold standard for cell counting. however, the rapidity and convenience offered by automated methods resulted in widespread use of automated hematology analyzers. currently there are no standardization and/or guidelines to advise what system to use for rbc quantification in platelet products. study design/method: we designed this study to quantify the residual rbc in apheresis platelets and whole blood derived platelets comparing hemocytometer and automated methods. we measured the amount of red blood cells per microliter in apheresis and whole blood derived platelet units using hemocytometer and two different automated hematology analyzers, namely, sysmex (sysmex america, lincolnshire, il) and advia (siemens healthcare diagnostics, tarrytown ny). the whole blood derived platelet units were produced using acrodose tm system technology. we conducted non-parametric permutation test based on permutations to compare sysmex and advia between apheresis and whole blood derived groups. abstract collection) rbcs to rbcs stored for days and after treatment with an fda approved rejuvenation solution. study design/method: the addition of a rejuvenation solution to stored red blood cells (rbcs) has been shown to increase atp and , -dpg profiles to fresh levels. the objective was to compare % hemoglobin-oxygen saturation (p ) and morphology profiles of fresh(day of collection) rbcs to rbcs stored for days and after treatment with an fda approved rejuvenation solution. results/finding: in vitro rbc recovery (overall) was . . %. hemolysis (%) was similar on day before and after dry-air incubation with the rejuvenation solution ( . . % vs . . %). percent hemolysis (%) decreased after washing ( . . %) and was maintained below < % for all units during storage for hr ( . . %). average atp and , -dpg were restored above the average fresh values. the morphology score decreased $ % by day , which was restored to near fresh values following rejuvenation and washing and storage hr ( . % and . %, respectively). rbc oxygen affinity, as assessed by p , was restored above fresh values. all values were significantly different compared to day (p< . , paired t-test) ( table ) . conclusion: rbc morphology was restored to near fresh and average atp, , -dpg, and p were restored above fresh values when incubated with a rejuvenation solution using the dry-air incubation process. rbc morphology, atp and , -dpg were maintained during storage hr. rejuvenation of refrigerated rbcs may offer avenues to improve rbc quality prior to transfusion. vandi ly*, dimath alyemni, warren r korn, matthew j brune and julie katz karp. thomas jefferson university hospital background/case studies: blood donors are screened with a donor history questionnaire that includes questions regarding behavioral risk factors, but none that specifically screen for the use of marijuana. therefore, there is the theoretical possibility of transfer of active cannabis metabolites through transfusion. donor plasma collected at an urban, hospital-based blood donor center was examined for the presence of active cannabis metabolites, d tetrahydrocannabinol (thc) and -oh-d -tetrahydrocannabinol ( -oh-thc). study design/method: de-identified donor plasma segments were sequestered and stored frozen until time of testing. testing for thc and -oh-thc was performed by liquid chromatography-tandem mass spectrometry (lc-ms/ms) based on a method modified from lacroix and saussereau. in summary, this method used dabsyl chloride derivatization of thc and -oh-thc to produce samples for lc-ms/ms analysis. lc used a c column. post-column detection by ms/ms used positive ion electrospray with q :q ion pairs of m/z . : . (internal standard (is), d -thc), m/ z . : . (thc), and m/z . : . ( -oh-thc). quantitative results for thc and -oh-thc were obtained from a standard curve (ratio of analyte integrals to integrals of internal standard) ranging from - ng/ ml for both thc and -oh-thc. limits of quantitation, defined as standard deviations above background, were . ng/ml for thc and ng/ml for -oh-thc. results/finding: a total of donor plasma samples were tested for thc and -oh-thc. no samples tested positive for either thc or -oh-thc. theoretical calculations according to statistics of a poisson distribution indicated that there would be a % probability of one or more positives at a prevalence of . % positive samples, and a % probability of one or more positives at a prevalence of . % positive samples. results thus indicated a boundary of prevalence of the presence of active thc-metabolites in plasma samples to be less than % among this donor population. standard pharmacokinetics of cannabis metabolism in previous studies indicate a likely time window of less than hours for post-exposure detection of thc and/or -oh-thc in plasma. conclusion: testing of donor plasma samples for active metabolites of cannabis at one urban, hospital-based blood donor center produced no testpositives. statistically, results indicated that prevalence of positivity, if greater than zero, is at most less than %. probability of occurrence of cannabis metabolites in blood donor samples is likely to be highly variable across donor centers and is largely dependent on blood donor demographics. elisabeth maurer-spurej* , ruqayyah almizraq , daniel millar and jason p. acker . university of british columbia, university of alberta, lightintegra technology inc., canadian blood services background/case studies: the controversy around the quality and clinical impact of aged red blood cell concentrates (rcc) is ongoing. current studies are limited by the lack of quality measures suitable for routine screening of rcc. based on evidence that fragments called microparticles (mp) or extracellular vesicles are markers of cellular activation or degradation, this study investigated the utility of mp screening to characterize the effect of rcc production methods and storage. study design/method: red blood cell concentrates were prepared by whole blood filtration (wbf; top/top) or red cell filtration (rcf; top/bottom) methods, centrifuged to prepare a supernatant and tested for mp content (as measured with dynamic light scattering or a tunable resistive pulse sensing technique), hemolysis, atp and red cell deformability on days , , and of storage. one rcf rcc was tested on days , , , , and and six ml aliquots were stored in parallel and tested on days , , and . all samples were tested for mp content and compared to the other quality indicators. results/finding: mp content showed a linear increase with storage time with statistically significant differences between days , and (p< . ) and correlated with supernatant hemoglobin, and inversely with atp or rbc elasticity. both mp testing methods agreed with respect to total mp content. starting levels of the quality indicators varied between donations, preparation methods (wbf rcc contained much higher levels of mp), and storage time. mp content in the aliquots were consistent at each time point but statistically higher than in the original rcc on and after day of storage. conclusion: mp content correlates with measures of hemolysis and other rbc quality indicators and could be implemented as a routine screening tool. differences in mp content between donors, processes and age could be monitored and used to inform component production decisions. measuring mp content would allow % screening of rcc products in studies and pragmatic qc initiatives which are needed to settle the controversy about the clinical effect of rcc age. single donor spray-dried plasma: the future of plasma therapy? qiyong peter liu*, jihae sohn, ryan c. carney, sruthi sundaram and mark a popovsky. velico medical inc background/case studies: frozen plasma is integral to hemostasis management in many situations but logistically cumbersome because of frozen storage and long thawing time. spray-dried plasma (odp, on demand plasma) is potentially superior because it may be stored under refrigeration near the patient and reconstituted in minutes at the point-of-care. the objective of this study is to determine if odp can be consistently manufactured at a blood center with key proteins and coagulation function comparable to ffp. study design/methods: units of never frozen plasma collected at a blood center were processed on-site at a fixed volume into odp using velico's spray dryer. odp (n ) and paired ffp aliquots were stored for - days at - c and - c, respectively, reconstituted with a fixed volume of rehydration fluid (sterile water for injection), and extensively characterized with respect to the levels of hemostatic proteins, coagulation and complement activation markers, and clotting performance. the volumes of processed plasma and rehydration fluid were pre-determined ensuring similar total protein concentration in reconstituted odp and ffp for direct comparison. results/findings: compared to ffp, odp had ! % levels of functional clotting factors (fibrinogen, factors ii, v, vii, viii, ix, x, xi and xii), plasminogen, and protease inhibitors (antithrombin iii, protein c, protein s; plasmin, c esterase and alpha -proteniase inhibitors). the level of factor xiii in odp was slightly lower, about % of ffp by both activity and antigen assays. odp was identical to ffp in the levels of albumin, immunoglobulins (iga, igg and igm), lipoproteins, calcium, citrate, and coagulation proteins evaluated by antigen assays except for factor xiii. the levels of the markers for coagulation (thrombin-antithrombin, prothrombin fragments i ii and ddimer) and complement (c a and c a) activation in odp remained similar to ffp. odp was equivalent to ffp when assessed by aptt, pt and thrombelastography. transfusion vol. supplement s abstract spray-drying fragmented a substantial number of high molecular weight von willebrand factor (vwf) multimers into smaller ones, leading to a net increase of vwf multimers in odp. the size re-distribution reduced the vwf ristocetin cofactor activity (vwf:rco) to % in odp relative to ffp, but had no impact on vwf antigen and factor viii function (stabilized by vwf). vwf-specific studies have shown that odp retains hemostatic function in supporting platelet adhesion and aggregation (see abstracts by meledeo et al/us army institute of surgical research and bercovitz et al/blood center of wisconsin). conclusion: odp can be manufactured at a blood center with a quality comparable to that of ffp. future studies will determine if the product is bioequivalent to ffp and comparable in safety and efficacy. background/case studies: the collection time of whole blood is, according to european guidelines, limited to minutes. in addition, donations with collection times between and minutes should not be used for preparation of platelet (plt) concentrates (pc) because of the chance of too much activation of plt. it seems justified to re-evaluate the quality of plt from these donations because new generations collection systems and mixers were introduced, including a more efficient needle. the aim of this study was to investigate the in vitro quality of pc prepared from - minutes buffy coats (bc) with the aim to prevent unnecessary discarding of bc and to simplify the total blood bank process. study design/method: single-donor pc (spc, n ) were prepared from one - minutes bc and ml of autologous plasma in a ml pvc-dehp container. as a reference, spc from donations with collection times of < minutes were prepared (n ). in addition, pc were prepared from bc, of which at least bc were from - minutes donations (n ). after pooling of the bc, ml of pas-e was added and a standard pooling set with a pvc-bthc storage container was used for storage of pc. all pc were stored for days at c and sampled at regular intervals for determination of the in vitro quality. aggregation tests were performed with chronolog (adp or collagen) and multiplate (arachidonic acid) aggregometers. thromboelastography (teg), using kaolin as an activator, was applied for assessment of the overall clotting capacity. values are expressed as mean sd. a non-paired t-test or a mann-whitney u test was applied for statistical analyses of normal or non-normal distributed data respectively. results/finding: volume ( vs. ml) and platelet content ( vs. x ) were similar in both groups. at the end of storage, both groups showed comparable in vitro quality (day , ph( c): . . vs . . , other data not shown). no differences in aggregation response after stimulation with arachidonic acid, adp or collagen were measured. teg parameters in both groups were also comparable. the five-donor pc fulfilled all requirements of european guidelines, aside from occurrence of small aggregates at day and/or in / pc (possibly because sometimes ab incompatibility was accepted). on day , plt showed low cd p expression ( . . %) and phosphatidylserine exposure (annexin v binding, . . %). hypotonic shock response of platelets was comparable with historical data. conclusion: single-pc in plasma as well as five-donor pc in pas-e, prepared from - minutes whole blood donations had a normal composition and showed good in vitro quality during day storage. to substantiate that the exclusion of - minutes donations for pc preparation could be stopped, further studies will be performed. the effects of a pneumatic tube system on red blood cell units amy mata* , jessie miller , ranee marie wannarka-farlinger , sandra bryant , scott a hammel , sherry stern and camille van buskirk . mayo clinic, mayo clinic rochester background/case studies: the use of pneumatic tube systems (pts) has become commonplace in many healthcare facilities throughout the world. the purpose of these systems is to transport products and specimens, resulting in reduced turnaround time for laboratory testing and to aid in the timely delivery of patient care. a downfall of ptss is that they have the potential to play a role in increased hemolysis. while several studies have been published on the effects of ptss on blood specimens, there are very few that address the effects on blood products, specifically red blood cells (rbc). the objective of this study was twofold: to determine if the pts that is in use at our facility contributes to an increase in hemolysis of rbc units and to evaluate how the pts system affects red cell microparticle (rmp) levels. study design/method: forty-one units of as- rbcs, irradiated and non-irradiated, were selected for the study. the units varied in age, ranging from to days old. specimens were obtained from each unit both prior to and after being transported through the pts, which runs underground and spans the length of a mile and a half. specimens were spun down and the plasma supernatant was removed. all specimens were evaluated for plasma hemoglobin (hgb), potassium (k), hemolysis index (hi), and rmps. the wilcoxon signed-rank test and p value were used to compare the pre and post values. additional statistical analysis was performed to compare the values after adjusting for age and irradiation. results/finding: after sending the rbc units through the pts, hgb, hi, and rmps were statistically (p< . ) higher than before. when adjusted for irradiation, the same analytes remained statistically higher, however when adjusted for age, the p-value was only significant for hgb and hi. the k values did not significantly change. rmps significantly increased, but only if the units were irradiated (p . ). (table) conclusion: the use of a pts provides an effective means to transport blood products; however, it can contribute to biological changes within rbc units. it is uncertain at this time how those changes can affect the outcome of patients who receive these products. each pts system is different in its specifications and should be validated prior to being used to transport blood products. validation of factor viii levels of thawed fresh frozen plasma after days of storage pei lun karen lim* , erma sofia sumardi , isamar eduardo ancheta , susan lim , christina yip , lip kun tan and shir ying lee . national university hospital singapore, national university hospital, national university hospital, singapore background/case studies: plasma transfusion is indicated in patients with coagulation factor deficiencies and active bleeding, or who are about to undergo an invasive procedure. fresh frozen plasma (ffp) has to be placed in the freezer within hours of processing and stored at - c or colder in order to preserve its coagulation factors. thawed ffp has an expiration period of hours hence to reduce wastage, this study aims to investigate factor viii (fviii) activity in thawed plasma stored for days and kept at to c. fviii was chosen as it is an important coagulation factor in correcting coagulopathies. arbitrary fviii level acceptance limit was set as not less than iu/dl. study design/method: randomly selected units of ffp (n ) were measured for fviii concentration based on clotting assay (stav r -deficient a transfusion vol. supplement s viii diagnostica stago). fviii levels were measured at five time points: prefreezing, , , and hours post-thawing. ffp were thawed using helmer plasma thawer (helmer scientific) at to c for minutes. an aliquot of thawed ffp from each unit was removed and measured for fviii before refrigeration ( hours post-thaw). thawed plasma (tp) units were kept in a refrigerator at to c for days for subsequent testing. results/finding: results obtained were listed in table . units to were not tested for fviii at post thaw- hour due to operational issues. the overall fviii concentration decreased at an average of % from pre-freezing to post thaw hour. after further storage of tp post thaw- hour and - hour, residual fviii level remain to be above iu/dl except unit which had a lower initial fviii concentration. at post thaw- hour, out of units tested had residual fviii activity within the pre-set standard of iu/ dl. the average decline from -hour post-thaw to -hour, -hour and hour post-thaw was . %, . % and . % respectively. there was no observed trend of any blood group having higher or lower pre-freezing fviii and this is likely due to small sample size. conclusion: decrease of coagulation factor such as fviii in ffp is expected due to its diminishing stability. nevertheless, our data showed that majority of the tp retained at least iu/dl of fviii. typically patients with factor levels below iu/dl may start to show abnormal coagulation profile. while tp is not used for specific factor replacement therapy, it may be indicated for patients with general coagulopathies and active bleeding. further study extending to measurement of other labile factor such as fv may add value to the validation study. validation of the pathogen reduction method using amotosalen/ uva: comparing pathogen-reduced pooled prp-platelets and conventional single prp platelets for quality and bacterial inactivation efficacy lubna ahmed almenawi , ayman mohamad sabri , ali abdullah alajeafi , ashwaq hasan alhekri , saleem bin mahfouz , ali hasan alkhodari , rawya saeed shealy , marcus picard-maureau* and hussain bana almalki . king abdulaziz hospital and oncology center, cerus europe bv background/case studies: the growing number of transfusiontransmitted infectious (tti) risks, including emerging and endemic pathogens, is a constant challenge for blood centers in saudi arabia. while for a limited number of these pathogens tti risk can be reduced using blood screening assays, alternative solutions are anticipated. pathogen reduction (pr) technology was identified as a potential solution. validation of amotosalen/uva photochemical treatment in our blood center was performed by comparing the platelet component (pc) quality of the standard "control" single-donor prp-concentrate in % plasma over a day storage period and the new "test" pathogen-reduced, pooled (pools of ) prp pc in % plasma over a day storage period. the efficacy of the bacterial inactivation was also assessed in our setting. study design/method: the quality parameters of leucoreduced test pcs were assessed at day of storage and compared to leucoreduced control pc at day of storage. the test pcs were pathogen-reduced with the intercept blood system (cerus corporation, concord, u.s.a.) at day ; the process was completed by day post-collection. samples were taken daily for quality analysis from test and control pc until day and day , respectively. for bacterial spiking, additional pc were spiked with each receiving ml of mcfarland ($ . x cfu) s. aureus, s. epidermidis, e. coli, p. aeruginosa or s. viridans, respectively, to challenge pr efficacy. results/finding: the average platelet loss in the test pc post pr treatment was . % . , the total average platelet loss at day was . % . . the average platelet loss in the control units at day was . % . . the average ph of the test units at day was . . and in the same range as the control pc, ph . . . glucose concentration in test pc at day ( . . mmol/l) was lower than in the day control units ( . . mmol/l). lactate levels increased during the course of storage; lactate levels at days and were outside the range of the assay (> mmol/l). cultures inoculated with pathogen reduced, bacterially spiked units were negative after days of incubation, in contrast to those inoculated with nonpathogen reduced samples from the control units, which were positive for bacterial growth. conclusion: the quality parameters of the pathogen reduced test pc were within specifications and comparable to the conventional control pc. the high efficacy of bacterial inactivation together with comparable quality parameter values suggests the use of amotosalen/uva pathogen reduction is safe and efficient to enhance pc transfusion safety. keaton charles stoner* , jay srinivasan , jessica poisson and ian welsby . duke university, duke university school of medicine, duke university hospital, duke university medical center background/case studies: the coagulation cascade relies on a complex interaction between proteins known as clotting factors. cryoprecipitate (cryo) is a plasma-derived blood product that contains several of the proteins central to the clotting cascade and is typically used as a fibrinogen replacement in bleeding patients. however, cryo contents tend to be variable, and little quantitative evidence exists regarding the exact therapeutic effect of cryo on coagulation. my study aimed to better characterize cryo for consistency across and within sources in terms of its functional effect on in vitroclot formation. study design/method: the duke proteomics core conducted a semiquantitative liquid chromatography-mass spectrometry/mass spectrometry transfusion developed an in vitromodel for a coagulopathic patient using serial dilutions of pooled normal plasma with saline and then added the equivalent of one, two, and three cryoprecipitate doses. a tissue factor-activated test on the rotemv r delta hemostasis analyzer (extem) was performed on each condition. for each source, dose-response curves for clotting time (ct), alpha angle, and maximum clot formation (mcf) were generated using linear regression models. inter-source unit variability was determined by anova and tukey's hsd post-hoc analysis (rstudio inc.). results/finding: lc-ms/ms identified proteins in cryo; of the most abundant, only fibrinogen was relevant to coagulation. notably, the american red cross (arc) single donor source had the steepest slope for mcf ( . mm/dose), indicating a greater per dose potency than the other sources. the arc single donor source had the highest mean mcf across all dosing levels, but also the highest standard deviations and response variability. the arc single donor source was significantly more potent than the australian source. conclusion: paired with our estimates regarding the variability of clot formation responses to cryo, the quantitative dose-response curves provided in this study for ct, mcf, and alpha angle can provide physicians with more information regarding cryo dosing. future studies that evaluate the therapeutic effect of cryoprecipitate versus fresh frozen plasma or fibrinogen concentrate would be of clinical importance and give us further insight into the relative utility of and dose requirements for cryo to correct dilutional coagulopathy. viral inactivation and enrichment of factor viii, factor xiii, fibrinogen and von willebrand factor (vwf) multimers from fresh frozen plasma (ffp)using, "vips plasma, virus inactivation treatment system". background/case studies: the solvent/detergent (sd) process used for plasma can safely inactivate all lipid-enveloped viruses. the method proved effective in the processing of coagulation factor concentrates by disrupting the membranes of lipid-enveloped viruses, cells and most protozoa, while leaving the labile coagulation factors intact. this study is done to assess viral inactivation and, factor viii, factor xiii, fibrinogen and von willebrand factor (vwf) multimers enrichment capacity of, "vips plasma, virus inactivation treatment system". study design/method: "vips plasma, virus inactivation treatment system" comprise of interconnected bag system where the s/d reagents are removed by filtration and the final products subjected to bacterial ( Á lm) filtration. cryoprecipitate mini-pools ( ml) were subjected to doublestage s/d viral inactivation, followed by one oil extraction and a filtration on a s/d and phthalate [di( -ethylhexyl) phthalate (dehp)] adsorption device and a Á lm filter. the initial and the final products were compared for visual appearance, blood cell count, factor viii, factor xiii, fibrinogen and von willebrand factor (vwf) multimers. initial and final products were also checked for hiv, hbv, hcv, dengue, malaria and bacterial contaminations. results/finding: our analysis showed that the treated cryoprecipitate were very clear, with negative blood count and the protein content of factor viii, factor xiii, fibrinogen and von willebrand factor (vwf) multimers were well conserved (table ) . kit ensured bacterial sterility (table ) and most importantly, final product was free of hbv, hcv and hiv (table ) . conclusion: it's the first time, "vips plasma, virus inactivation treatment system", is used in south asia for product enrichment and viral inactivation. results showed effective product enrichment and viral inactivation in our conditions. but further investigation is needed to characterize functional activity of the enrich component. irrespective of that the process may offer one additional option to blood establishments for the production of virally inactivated plasma components especially in low income countries. background/case studies: buffy coats (bc) from donors who used pain medication like aspirin and ibuprofen up to days prior to the donation are discarded, because a known side effect of these non-steroidal anti-inflammatory drugs (nsaids) is inhibition of platelet (plt) aggregation. these nsaids inhibit the enzyme cyclooxygenase- , thereby blocking synthesis of thromboxane a from arachidonic acid. however, the quality of platelet concentrates (pc), prepared from this bc is not known. the aim of the study was to investigate the in vitro quality of pc prepared from nsaid-bc and autologous plasma during storage. study design/method: single-donor pc (spc, n ) were prepared from a nsaid-bc and ml of autologous plasma. information about the type of pain medication was extracted from the anamneses form. the spc were stored for days at c and sampled at regular intervals. aggregation tests were performed with chronolog (adp or collagen) and multiplate (arachidonic acid) aggregometers. thromboelastography (teg, kaolin) was applied for assessment of the overall clotting capacity. spc in plasma from normal controls (n ) were investigated as a reference. values are expressed as mean sd or as median & iqr. a non-paired t-test or a mann-whitney u test was applied for statistical analyses of normal or nonnormal distributed data respectively. results/finding: volume ( vs. ml) and plt content ( vs. x ) were similar in both groups. on day , both groups showed comparable ph and changes in plt content (data not shown). phosphatidylserine exposure on day was significant higher in a subset of donors who had used ibuprofen (n ). aggregation tests with arachidonic acid revealed in general a low or absent response for spc with aspirin ( , - , p< . ), diclofenac ( , - ) and naproxen ( , - , p< . ), compared to normal controls ( , . no differences were detected in aggregation with adp or collagen. with teg, slightly longer r-times (initiation phase) were measured on day in spc with aspirin, diclofenac and naproxen, compared to the normal controls (only significant for naproxen). these differences disappeared during storage. conclusion: storage properties of spc prepared from nsaid-bc were comparable with spc from normal controls. main differences were observed in aggregation and coagulation properties for donors who used aspirin, diclofenac or naproxen. plt from donors who used ibuprofen showed little or no deviations. this is most likely caused by the fast (< hour) disappearance of ibuprofen from the blood circulation and the reversible binding to plt. the use of bc from donors who used ibuprofen will be further investigated in a 'worst case' (pc in plasma) and 'best case' (pc in additive solution) scenario. the effects of ibuprofen on aggregation and coagulation properties will be further investigated in a dose-response study design adding different levels of ibuprofen to plt. background/case studies: previously it was shown that donors could be classified as having platelets (plt) with good, average or poor storage properties [bontekoe, transfusion, ] . a main difference between 'good' and 'poor' storage properties involved metabolic activity, resulting in a faster decline of ph during storage of 'poor' plt concentrates (pc). this might be caused by a different functionality of the plt mitochondria and there are indications that donors with a history of 'poor' pcs are more likely to have health issues, pointing towards metabolic syndrome and type diabetes (t d). because of the strong rise of people with t d in the dutch population, the aim of this study was to characterize plt from whole blood donors diagnosed for t d, but accepted as donor. study design/method: twelve whole blood donors with t d, not using insulin, were selected and buffy coat (bc) and plasma were, after overnight hold, used for preparation of a single-donor pc (spc). an equivalent number of spc was prepared from age and sex matched control donors, derived from the same collection sessions. spc were stored for days at c and sampled on day , or and . the diabetic marker hba c was determined in red cells and cholesterol and triglyceride levels in plasma. from both groups 'good' (ph day > . ) and 'poor' (ph day < . ) storing spc were selected and analysed in more detail. results/finding: donors were of age year and primarily men ( %). donors with t d had a higher mean bmi ( . . vs. . . kg/m ) and higher hba c than controls. the spc of both groups had the same volume ( vs ml) and plt content ( vs x ) but on day glucose concentration was higher in the diabetic group ( . . vs . . mm, p< . ). on day , the average in vitro quality was comparable in both groups (data not shown). when combining a transfusion vol. supplement s the selected 'good' and 'poor' storing plt from both groups, a large difference in lactate production was observed ( . . vs . . mmol/ day/ plt). the 'poor' plt showed a faster decline of the mitochondrial membrane potential (as measured with jc- ) during storage than 'good' plt. remarkably, a difference in triglyceride levels was detected on day ('poor': . . vs 'good': . . , p< . ). conclusion: bc from donors with t d who did not use insulin and fulfilled all donor criteria, were comparable with bc from age and sex matched controls, and seem suitable for preparation of pc. when selecting the 'good' and 'poor' storing plt from the combined groups, the results of our previous study were confirmed, with significant differences in glycolysis rate and functionality of mitochondria. metabolic syndrome and t d are still suspected as health issues involved in 'poor' storage of plt because donors were of high mean age and because of the observed differences in triglyceride levels between 'good' and 'poor' stored pcs. whole blood leukoreduction failures --following manufacturer's instructions may not be enough karen klinker*, nancy m. dunbar and zbigniew m. szczepiorkowski. background/case studies: our hospital based blood donor program uses a blood collection system which leukoreduces the unit at room temperature prior to centrifugation. the manufacturer recommends minimum wait time of minutes prior to filtration. anecdotally, the vendor states waiting an hour improves the leukoreduction. we experienced leukoreduction failures in january and february of detected by our routine qc. we initiated an investigation as to the cause of these unexpected failures. study design/method: for each of the leukoreduction failures, the following factors were analyzed: collection time, length of filtration, length of wait time prior to filtration, platelet count, staff performing the process, the lot number of the collection system bag, and whether or not units collected from the same donor failed leukoreduction in the past. hemoglobin s determinations were not sought out as no repeat donor failures were noted and our donor population would suggest a minimal number of donors would be found to be hemoglobin s positive. results/finding: a relationship was established between the length of time the product rested or waited prior to filtration and leukoreduction failure. we found that shorter wait times increased the percentage of leukoreduction failures (see table ). all units that failed had wait times less than one hour. a similar trend was noticed for the previous year. the investigation showed no relationship between length of collection time, or the length of filtration time and leukoreduction failure. staff performing the filtration was ruled out as possible cause as the failures were spread out among numerous personnel and observation of their technique displayed no sample collection issues. platelet counts on the donors involved were available and none were outside of the normal range. various lot numbers of the collection sets were involved, and no donors were repeat failures. conclusion: in our small study, we found that following manufacturer's recommendations for the resting or wait time prior to filtration was insufficient to avoid excessive leukoreduction failures. we extended our minimum wait time to minutes based on our data. we have not experienced any leukoreduction failures after this change. absolute immature platelet count in diagnostic algorithm and management of pediatric thrombotic microangiopathy hamza n gokozan* , , katharine a downes , , hollie m reeves , and robert w maitta , . case western reserve university school of medicine, university hospitals cleveland medical center background/case studies: prior studies highlighted the utility of absolute immature platelet count (a-ipc) and a-ipc ratio once therapeutic plasma exchange (tpe) is initiated to differentiate thrombotic thrombocytopenic purpura (ttp) from other thrombotic microangiopathies. this can be helpful to determine those who may benefit from prompt initiation of tpe when tests such as adamts are not readily available. we report a young pediatric patient presenting with diarrhea in the setting of laboratory results suggestive of a microangiopathic thrombocytopenia suspicious for ttp in which a-ipc measurement was clinically useful. study design/methods: previously healthy month old unvaccinated girl presented with history of diarrhea for days which was bloody at onset, accompanied by fever and dehydration. laboratory results showed: white blood cell count: x /l, platelets: x /l, bun: mg/dl, creatinine: . mg/dl, lactate dehydrogenase u/l. hospital course was complicated by tonicclonic seizure episodes that stopped with anti-convulsants and acute kidney injury requiring hemodialysis. peripheral blood smear revealed schistocytes. on third day of hospitalization, platelet count decreased to x /l, adamts sample was sent out and tpe was initiated for clinical suspicion of ttp versus hemolytic uremic syndrome, atypical versus shiga-toxin mediated. immature platelet fraction (%-ipf) and calculated a-ipc (%-ipf x platelet count) were obtained with daily pre-tpe cbc. a-ipc ratio was calculated from baseline. results/findings: platelet count began to increase prior to tpe initiation ( x /l and a-ipc of . x /l). two consecutive tpe were completed which resulted in a platelet count decrease to x /l and a-ipc of . x /l. a-ipc ratio was . below the ratio of which has been reported for ttp patients. similarly a-ipc count was not below x /l threshold reported in setting of ttp with severe adamts deficiency. at this time stool culture obtained prior to start of tpe came back positive for e. coli o :h toxin. testing of c , c , factor h, factor h autoantibody, factor i and factor b were normal. adamts activity was %. patient was treated for the infection and platelet count improved within days to x /l, with resolution of her renal failure: bun: mg/dl, creatinine: . mg/dl. no additional seizures were observed during follow-up. conclusion: measurement of a-ipc can be used to aid clinical decisions in pediatric patients suspected of ttp especially when adamts testing and those for other etiologies are still pending. tpe did not seem to have a significant effect in a-ipc but decreased platelet counts in this patient. a-ipc is rapid to obtain and can provide helpful information in the setting of potentially overlapping etiologies in the setting of other testing with longer turnaround time. background/case studies: thrombotic thrombocytopenic purpura (ttp) is a thrombotic microangiopathy characterized by low adamts activity. many patients with severe autoantibody-mediated adamts deficiency at initial disease presentation may suffer from one or more recurrent episodes over the following months or years. it is unclear if disease course and characteristics of recurrent/relapsed ttp may be different from that seen at initial presentation. since absolute immature platelet counts (a-ipc) have been shown to be useful in the diagnosis and to follow response to therapy of ttp patients, we proceeded to evaluate if a-ipc pattern was different in relapsed verse initial presentation. study design/methods: our study cohort consisted of three patients (two female and one male) with acquired ttp (adamts activity < %) who underwent daily therapeutic plasma exchange (tpe). clinical course and laboratory values were reviewed. platelet count (plt), immature platelet fraction (%-ipf) and a-ipc (%-ipf x platelet count) were analyzed during treatment course. a-ipc values at presentation and peak, a-ipc peak time (days), and plt count recovery time (days) were compared between initial onset and relapse episode for each patient. a-ipc percent change in relapse episodes compared to initial presentation was calculated. results/findings: all patients had an increased %-ipf, and decreased a-ipc and plt count at presentation in both initial and recurrent episodes. once tpe treatment was initiated, a-ipc rapidly increased and reached a peak value - days prior to plt count recovery, consistent with that previously described in ttp patients. however, compared to first onset, recurrent episodes featured lower a-ipc at presentation (results shown as percent decrease, column ), increased peak a-ipc value (results shown as percent increase, column ), delayed a-ipc peak, and delayed plt recovery (table ) . moreover, recurrent episodes required more procedures compared to initial presentation (table ) . conclusion: recurrent/relapsed ttp demonstrate lower a-ipc at presentation and a delayed and increased a-ipc peak value in response to tpe compared to initial presentation. a longer treatment course was observed in recurrent patients. future studies of more relapsed ttp patients are needed. donors undergoing frequent plateletpheresis and its effect on the hematological parameters sweta nayak*, poonam coshic and r.m pandey. all india institute of medical sciences background/case studies: frequent plateletpheresis donors are assets for the blood banks. the well-being of these donors has been a matter of concern. in our study we intend to analyze the effect of plateletpheresis on the hematological parameters of these donors assessed prior to each subsequent procedure. we also try to compare the effect cell separators used for plateletpheresis on the post donation hematological parameters. study design/method: the study was conducted during february to march on all the repeat plateletpheresis donors coming to the department of transfusion medicine for the nd time within a month of the first plateletpheresis. the values of the hematological parameters including red cell and platelet indices tested prior to each plateletpheresis were entered into the excel sheet and gap between each donations were calculated. the plateletpheresis were done either on hemonetics mcs separator (hemonetics corporation, braintree, massachusetts, usa), fresinius separator (com.tec), dn (fresinius hemocare gmbh, bad homburg v.d.h, germany) and gambro trima accel, software version . after taking consent from the donors. the target collection of each procedure was a dose of x platelets in - ml of plasma. to compare the effect of the cell separators on the hematological parameters due to the plateletpheresis, parameters at consecutive donations within days were considered. data was analyzed by stata . within change in the continuous variables were assessed by paired t-test and between two groups comparison was done by independent t-test or wilcoxon rank sum test. the comparison among the cell separators was done by kruskal-wallis test or one way anova. results/finding: of the donors, repeated the plateletpheresis within a week (group i) and underwent nd plateletpheresis within - days (group ii). no significant alteration was found in the red cell or the platelet indices within either group but a significant difference in the variation of platelet counts of the groups (p . ). though above the eligibility cutoff of . lakhs/ml, platelet counts were lower than baseline in group i donors whereas it was higher at nd plateletpheresis in group ii donors. there were donors who presented to us for the rd time for plateletpheresis with a mean gap between st and rd plateletpheresis being days. no significant difference in the parameters assessed prior to any of the plateletpheresis was found except the platelet distribution width (p . ). plateletpheresis through all the cell separators had similar effects on the hematological parameters. conclusion: there was no significant change in the hematological parameters in the plateletpheresis donors who underwent frequent plateletpheresis. post donation follow-up hematological parameters were not affected by the cell separators used for plateletpheresis. efficacy of therapeutic plasma exchange on angiotensin ii type receptor antibodies in two kidney transplant recipients chisa yamada*, silas p. norman, milagros samaniego and laura cooling. background/case studies: some kidney transplant recipients develop antibody mediated rejection (amr) without detected hla donor specific antibodies (dsas) in sera. in recent years, angiotensin ii type- receptor antibody (at rab) has been reported to cause amr, especially refractory amr, possibly by contraction of renal arteries. at our institution, therapeutic plasma exchange (tpe) followed by ivig every other day has been applied to reduce at rabs in kidney transplant recipients, and we here report efficacy of tpe treatments in two cases. study design/methods: two kidney transplant recipients who received tpe treatment followed by ivig to decreased at r ab are reviewed. results/findings: case : the patient is a currently -year-old female with focal segmental glomerulosclerosis who received her first kidney transplant from a living related donor at age , and a second deceased donor transplant due to a rejection of the transplanted kidney at age . three years post-transplant, her creatinine (cr) started to rise from . to . mg/dl and a biopsy showed banff criteria grade amr, grade a t-cell mediated rejection (tcmr) and grade interstitial fibrosis and tubular atrophy. hla dsa had been negative in serum, but high level at rab was identified at > u/ ml (high: > u/ml, intermediate: - u/ml, negative: < u/ml). she received tpe treatments every other day and started losartan. after a course of tpe, at rab decreased to u/ml and histology showed improvement of amr and tcmr, however, cr kept increasing slowly to . ml/dl. in one month, her at rab increased again to > u/ml, therefore, she received more tpe treatments with a decrease in her at rab to u/ml. although at rab level increased slightly to u/ml after months, her cr has been stable at . - . ml/dl. case : the patient is a -year-old mean /-se - . /- . % * . % /- . %* * p< . a female with malignant hypertension who received a deceased donor kidney transplant at age . her cr started to rise weeks post-transplant from . to . mg/dl without detectable hla dsa. although biopsy showed no amr or tcmr, there was focally severe arteriopathy. she was found to have high at rab level at u/ml. she received tpe procedures every other day and at rab decreased to u/ml with a decrease of cr to . mg/dl and improved arteriopathy in histology. because her at rab level slightly increased to u/ml over the next weeks, she started weekly tpe treatment. after weekly tpe, tpe treatment was stopped because her at rab level remained relatively unchanged. her cr has been stable at around . ml/dl to date. conclusion: we present kidney transplant recipients who received tpe treatments for high at rab levels. a course of tpe procedures followed by ivig every other day was effective to decrease at rab levels; however, weekly tpe had no effect on reducing at rabs. tpe treatment may be also beneficial to improve histological amr and clinical kidney function. experience in management of thyroid storm by plasmapheresis tatiana belousova*, vanya jaitly, brian castillo, hlaing tint, kimberly klein and yu bai. university of texas health sciences center at houston background/case studies: thyroid storm (ts) is an extreme manifestation of thyrotoxicosis that is a serious complication occurring primarily in patients with graves' disease. clinically they may present with a wide range of hypermetabolic symptoms which may be fatal if not managed appropriately. we report two cases where ts with severe cardiac complications was managed by plasmapheresis (plex) with excellent effect. study design/method: a year old man (patient a) with a medical history of hyperthyroidism present with ts complicated with cardiogenic shock [ejection fraction (ef) < %], renal and hepatic dysfunction as well as coagulopathy. patient was persistent tachycardic while being intubated, sedated and requiring tandem heart support. a year old man (patient b) with a medical history of hypothyroidism (on synthroid for years), end stage renal disease and non-ischemic cardiomyopathy (ef of - %) presented for evaluation of dual kidney-heart transplant. he subsequently developed ts with multiorgan failure. standard steroid medication treatment showed little response. results/finding: both patients underwent urgent plex along with standard medication administration as soon as the clinical suspicion of thyroid storm was raised. a - . plasma volume, iso-volumic procedure using fresh frozen plasma as replacement was performed in the intensive care unit where the procedure associated hemodynamic impact could be easily managed. both patients showed significant clinical improvement within hours of the procedure completion. their total t , t and free t levels trended to normal or near normal range within hours (table) . in addition, the plex effect on hormone and the associated antibody removal seemed remained and no "rebound" phenomenon was observed in both cases, making repeated plex unnecessary. both patients had total thyroidectomy - weeks after the event with great clinical outcome. conclusion: our cases demonstrate that plex is a safe, effective treatment option in managing ts patient with severe cardiac dysfunction. the procedure can not only lead rapid decrease in thyroid hormone and its associated antibody levels, but also lessen the severity of tissue injury by moderating the inflammatory process and correcting complications. extracorporeal photopheresis in s ezary syndrome treatment: hospital-based blood bank experience sandra ortega s anchez* , laura martínez molina , cristina muniesa montserrat , octavio servitje bedate , silvia cosano navarro and maria isabel gonz alez medina . banc de sang i teixits, dermatology service. background/case studies: extracorporeal photopheresis (ecp) is an immunomodulatory therapy widely used since years in cutaneous t cell lymphoma, several autoimmune diseases and organ transplant rejection, and in the last years, also used in graft versus host disease treatment. the use of ecp in cutaneous t cell lymphoma (ctcl), mycosis fungoides (mf) and s ezary syndrome (ss) in their erytrodermic form are recently categorized by the american society for a pheresis (asfa) , as first line treatment alone or in combination with other therapies, with a strong recommendation: grade ib, category . since mf and ss are incurable diseases current therapies are focus in controlling skin symptoms and minimizing immunosuppression. the objective of this observational study is to assess outcomes of patients diagnosed with ss and compare them in their first evaluation once the th procedure is been performed. study design/method: ecp is a leukapheresis-based therapy, ex vivo exposition to a photosensitizer drug ( -methoxypsoralen, -mop) and uva light, and subsequent reinfusion of the treated cells which are now induced to apoptosis. volume treated varies from . to total body volume (tbv) and the schedule for ss disease is one cycle (two daily ecp procedures) twice per month. the venous access was peripheral in all cases except in where central catheter was needed. the procedures were performed with optia or amicus devices for the aphaeresis and external uva irradiation for off-line system (in / patients) and with online system (therakos) just in . main parameters for evaluation were cutaneous response rate, number of s ezary cells, previous treatments, duration of the response and possible complications during ecp treatment. results/finding: global response rate is ' % (partial remission . % and complete remission . % with maintained response). no severe side effects related with the procedure were found. the patient outcomes analyzed are similar to results in published literature. conclusion: cases treated in our hospital confirm the efficacy of ecp in ss treatment, with a good safety profile. another great advantage of ecp is the relative lack of immune suppression. many questions remain still unanswered about ecp: which schedule is the most suitable one, how we must continue or stop when partial or complete remission is achieved; and the number of leukocytes to be treated, as techniques as mini-photopheresis are also getting good results. all these questions and more make prospective studies necessary to be performed. : u/l) requiring transfusions, mild thrombocytopenia ( x /l), acute kidney injury (bun mg/dl, creatinine . mg/dl). by the third hospitalization day hgb improved to g/dl, however with worsening thrombocytopenia ( x /l) that led to clinical concern for ttp. peripheral smear showed many red cell fragments. patient was transfused with platelets day prior to first tpe. immature platelet fraction (%-ipf) and a-ipc (%-ipf x platelet count) were obtained with daily pre-tpe cbc. a-ipc ratio was calculated from baseline. results/finding: four tpe in five days were performed (hospital days - ). platelet count and a-ipc improved to x /l and . x /l respectively just prior to first tpe. response to four tpe led to a decrease in both platelet count ( x /l) and a-ipc . x /l. these dynamics did not resemble those which had been described for ttp patients with adamts deficiency. adamts obtained prior to tpe initiation was resulted at this time and was %. no causative organism or toxin was identified after urine, blood, and stool examination and culture. based on these results, tpe was discontinued which led to an immediate increase in a-ipc ( . x /l) that preceded platelet count increase to x /l three days later when patient was discharged. other laboratory values at this time were ldh of u/l, hgb: . g/dl in the setting of recovery of renal function. conclusion: timely diagnosis of ttp is essential to start of tpe. a-ipc dynamics differ in ttp compared to other thrombotic microangiopathies. in our patient a-ipc failed to improve despite tpe and improved once procedures were discontinued and were followed by increases in platelet counts three days later. when ttp is not the causative etiology, a-ipc can help adjust therapy and lead to clinical improvement. further research is needed to characterize immature platelet dynamics in non-ttp microangiopathies. infection and its role in the clinical course of idiopathic thrombotic thrombocytopenic purpura associated with severe adamts deficiency eiman hussein* and jun teruya . department of clinical pathology, cairo university, texas children's hospital background/case studies: ttp is a life threatening disease, defined by microangiopathic hemolytic anemia, thrombocytopenia and severely deficient adamts . since the introduction of therapeutic plasma exchange (tpe) as a treatment modality for ttp, its prognosis has improved dramatically. nonetheless, some patients may develop relapse or refractoriness, with potentially fatal outcomes. despite the notable progress that has been made with studies that emphasized the pivotal role of adamts , the epidemiology of ttp remains uncertain. previous studies have suggested that many factors appear to influence its pathogenesis. some studies point toward infection as a possible trigger which may contribute to the development and can ultimately influence its clinical course. one of the theories to explain this association is the possible cross reactivity between antibodies targeting infectious pathogens and those directed against adamts . the aim of this study was to prospectively examine the potential association between infection and the clinical outcome in a cohort of patients with idiopathic ttp. study design/method: patients with idiopathic ttp who underwent tpe from january through march were studied. sessions were performed daily until platelets and reticulocytes had been normal, then sessions were gradually tapered. we only included patients with adamts activity of less than %. data on infections that occurred at or within a week prior to the development of ttp were analyzed. results/finding: thirty-two patients were categorized as idiopathic ttp with severe adamts deficiency. eight patients ( %) were associated with suspected bacterial infection. four of the patients ( %) showed acute relapse coincident with bacterial infections. central line associated staphylococcus aureus infections occurred in three patients and acinetobacter urinary tract infection was reported in one patient. one patient had symptoms of respiratory infection before the development of ttp, on his initial as well as his relapsing episode. refractoriness to treatment was demonstrated in patients. it was associated with dental abscess in one patient. the other two were associated with mycoplasma pneumonia. tpe sessions were continued in all refractory patients until their death. conclusion: in patients with idiopathic ttp refractory to conventional treatment, a serious consideration should be given to non-idiopathic causes, particularly the presence of a remote source of infection, which can be an additional triggering factor for their initial and / or recurrent episodes. sandra satoe kayano*, marcos paulo colella, rafaela guerra maciel, ingrid priscila ribeiro paes ferraz and rafael colella. a c camargo cancer center background/case studies: therapeutic leukapheresis (tl) has become an ordinary procedure in low body weight children with cancer, and its use over the time has been replacing exchange transfusion. leukodepletion preceding chemotherapy helps preventing leukostasis and hiperviscosity, and aims to reduce metabolic and renal complications associated with cell lysis. the objective of this study is to evaluate the efficacy and safety of leukapheresis procedure in pediatric patients with less than kilograms using a single apheresis procedure. study design/method: in october and june , two children with possible leukemia were submitted to tl procedure. they were and months old, and weighted , and , kilograms. central venous catheters were placed, and apheresis were performed using a continuous flow apheresis system. the device was primed with ml of abo, rh and kell compatible, leukocyte-reduced, irradiated, % hematocrit packed rbcs, and the anticoagulant used was acd-a plus heparin ( ml of acd-a and , units of heparin), at a blood to anticoagulant ratio of : . a complete blood count was determined before and after apheresis. the room was heated to avoid hypothermia, and ionized calcium was measured every minutes to prevent hypocalcemia. during the collection, changes in blood pressure, oxygen saturation and heart rate were observed. net fluid balance was calculated as the sum of the volume of anticoagulant, cation and nondiverted apheresis prime solutions minus the product volume. when the procedure was completed, the blood that filled the apheresis tubing was discarded. the patients were in the intensive care unit (icu) under the supervision of a pediatric physician and icu nurse who were aware of potential adverse events, and the procedure were performed by two hematology physicians and the nurse practitioner. results/finding: the white blood cell (wbc) in blood was counted immediately before apheresis in both subjects, and were . and . / mm . the formula "collection pump flow , x inlet flow x preapheresis wbc count" was used with the goal of removing up to x leukocytes/ml. a single leukapheresis procedure was performed with total blood volume processed per patient. immediately after the -hour procedures, wbc count were . and . wbc/mm , and -hour post tl, wbc count were respectively . and . /mm . net fluid balance was zero in both procedures, and the patients required no transfusion. conclusion: tl was safe and efficient. experience with leukodepletion in infants is limited, and a procedure in children weighing kg or less needs forethought and a multidisciplinary effort, hence operators need to customize procedures for safe collection. however, despite the potential complications that may occur (placement of adequate vascular access, management of low extracorporeal blood volume, anticoagulant-related toxicity with metabolic and hematologic issues), remains an excellent source for leukoreduction in hematologic malignant diseases. background/case studies: nationwide apheresis registry can give us information on the current status and trend regarding apheresis procedures. data can be compared with other regions to find and understand differences in perspectives, indications, technology, and clinical practice. the korean society for apheresis (ksfa) has launched an online web based registry system for apheresis procedures since . we report the data from the year . study design/method: the registry is consisted of two sub-registries. one addresses the overall aspects of apheresis procedures performed at each institute, and the other is focused on therapeutic plasmapheresis procedures. data is registered by voluntarily participating hospitals in korea. results/finding: a total of , apheresis procedures were performed at hospitals. therapeutic plasmapheresis was the most frequent procedure ( . %) followed by autologous peripheral blood stem cell (pbsc) collection ( . %), allogeneic pbsc collection ( . %), donor leukapheresis ( . %), and therapeutic leukapheresis ( . %). cobe spectra ( . %) and amicus ( . %) were the most widely distributed instruments. centrifugation was the dominant technique ( . %) for therapeutic plasmapheresis. detailed information was given for , therapeutic plasmapheresis procedures performed on patients (some items were not completely filled out). spectra optia ( . %) and cobe spectra ( . %) were the most frequently used instruments for therapeutic plasmapheresis. fresh frozen plasma (ffp) was used most frequently ( . %) as the replacement fluid followed by % albumin ( . %), % albumin ( . %), and % albumin ffp ( . %). most of the procedures were performed for plasma volume ( . %). acd ( . %) and heparin ( . %) were used for anticoagulation. central venous catheter ( . %) was the dominant type of vascular access. major clinical indications were desensitization for abo incompatible renal transplantation ( . %), antibody mediated rejection in renal transplantation ( . %), thrombotic microangiopathy ( . %), desensitization for abo compatible renal transplantation ( . %), neuromyelitis optica spectrum disorders ( . %), and hyperviscosity in monoclonal gammopathies ( . %). adverse reactions were observed in . % of the procedures. allergic reaction ( . %), hypocalcemic symptom ( . %), and hypotension ( . %) were frequently reported. therapeutic effect was achieved in . % of the patients. our apheresis registry has been well run for years. recent data reflects the increase of abo incompatible transplantation in korea. revision and update of the registry planned this year will help us achieve better understanding on the apheresis status of our region. plasma exchange may not always be necessary in patients with severe hypertriglyceridemia and acute pancreatitis. jan c hofmann* and dobri d kiprov. california pacific medical center background/case studies: hypertriglyceridemic pancreatitis (hp) is characterized by severe hypertriglyceridemia (shtg: triglyceride > - mg/dl), acute pancreatitis (ap), and absence of other causes. hp is a potentially fatal complication of acute pancreatitis with an incidence of $ deaths/ , cases/year. complications of shtg include: abdominal pain (nausea/vomiting), acute pancreatitis, hepatosplenomegaly, eruptive xanthomas, lipemia retinalis, memory loss, dementia, and peripheral neuropathy. we report on the effective use of plasma exchange (pe) to treat patients (pts) with hp refractory to conventional medical therapy (lipid-free diet plus pharmaceutical interventions). study design/method: we reviewed the medical records of pts who were diagnosed with hp from january, through january, , and referred for immunotherapy evaluation. / ( %) pts received conventional therapy (ct) and pe (pe group), and / ( %) pts received ct alone (ct group). mean age was years (range - ), and % were female. baseline mean triglyceride level (normal < mg/dl) for pe group was , mg/dl ( , - , ) versus , mg/dl ( , - , ) for ct group. baseline mean lipase level (normal < u/l) for pe group was , u/l ( - , ) versus u/l ( - , ) for ct group. results/finding: all pts were treated with dietary restriction (lipid-free diet, or nothing by mouth) and aggressive lipid lowering protocols involving - medications. / ( %) of pe group and / ( %) of ct group received insulin therapy to manage symptoms (sxs) of hyperglycemia and/or diabetic ketoacidosis. / ( %) of pe group and / ( %) of ct group received heparin therapy to stimulate lipoprotein lipase release. the pe group underwent an average of . pe treatments (txs) (median of , range - daily txs) using % albumin; / ( %) required ffp to treat dilutional coagulopathy. in most cases, we did not perform pe txs when baseline triglyceride levels were < - mg/dl and lipase < - u/l ( . - . x upper limit of normal). mean triglyceride levels after pe txs were , mg/dl ( - , ) for pe group (mean decrease %); mean triglyceride levels after additional hours of ongoing ct were , mg/dl ( - , ) for ct group (mean decrease %). while the pe group achieved a greater mean decrease in triglyceride levels after pe txs (compared to the ct group after hours of ct), both groups experienced marked improvement in clinical sxs of pancreatitis and hyperglycemia (p> . ). limitations of the retrospective cohort study include lack of long-term follow-up. conclusion: this small study adds to the literature which demonstrates that plasma exchange is very effective in rapidly lowering triglyceride levels in pts with acute pancreatitis and hypertriglyceridemia. it suggests that there may be a threshold (or range) of triglyceride and lipase levels below which conventional therapy may be nearly as effective in achieving clinical resolution of symptoms. randomized controlled trials would further elucidate the appropriate use of adjunctive plasma exchange in the setting of hypertriglyceridemic pancreatitis. role of plasma replacement in therapeutic plasma exchange for hypertriglyceridemia: a single patient study geoffrey wool* and angela treml. university of chicago background/case studies: our apheresis service performs chronic therapeutic plasma exchanges (tpe) for a -year-old man with a chronic history of hypertriglyceridemia > mg/dl, diabetes mellitus type ii, and chronic abdominal pain. his abdominal pain is severe and persistent, but there is not overt evidence of chronic pancreatitis on imaging or fecal elastase testing. targeted sequencing has not revealed a pathogenic mutation to explain the patient's hypertriglyceridemia. hypertriglyceridemic pancreatitis is a category iii indication for tpe by asfa guidelines, in a patient unresponsive to optimal medical management. asfa guidelines for this disorder state that "some have used plasma as it contains lipoprotein lipase and could enhance triglyceride (tg) removal. no direct comparisons of replacement fluids have been reported". there are three apheresis physicians on our service and use of partial plasma replacement has been variable. we undertook a retrospective study of the efficacy of partial plasma replacement in this patient. study design/method: we have performed tpe on this patient. we performed a chart review to capture replacement fluid use and pre-and post-tg levels, if drawn. tpe was performed using spectra optia (terumo, lakewood, co) exchanging approximately one plasma volume, using entirely % albumin for exchange fluid ( % albumin procedures) or partial plasma replacement ( - units of thawed plasma). twenty-six tpe had pre-and post-procedure tg values available. we determined the percent tg reduction achieved by the tpe. we also determined the daily rate of tg increase until the next tpe appointment (to assess any long-term effects of plasma preventing tg rebound). significance was assessed by student's t-test (one-tailed, heteroscedastic). results/finding: twelve tpe were performed with partial plasma replacement, while were performed with % albumin replacement. table shows that partial plasma replacement was associated with significantly greater % tg reduction. the rate of subsequent daily tg increase was also lower with partial plasma replacement, but this did not meet significance. one mild allergic reaction has occurred during partial plasma replacement which responded quickly to additional iv diphenhydramine. conclusion: we have performed an ad hoc cross-over study on the efficacy of partial plasma replacement in tpe for hypertriglyceridemia. in this patient without lipoprotein lipase mutations, plasma was significantly associated with improved % tg reduction, but not with prevention of post-tpe tg rebound. safety and efficacy of local albumin replacement for therapeutic plasma exchange phandee watanaboonyongcharoen* , , metha apiwattanakul , sompis santipong , jutaluk jaipian , jettawan siriaksorn and ponlapat rojnuckarin . chulalongkorn university, king chulalongkorn memorial hospital, prasat neurological institute background/case studies: therapeutic plasma exchange (tpe) with albumin replacement has been used to treat a variety of diseases. however, there had been rising cost and supply shortage of imported albumin in our country. to solve the problem, our national blood centre had established a plasma fractionation plant to manufacture plasma derivatives including albumin. the objective of the study was to evaluate the safety and efficacy of local albumin as a replacement for tpe. study design/method: all tpes using local albumin as a replacement from two tertiary care hospitals performed from june through february were included. complete blood count and serum calcium were tested before tpe. serum albumin was tested before and after tpe. local albumin is available as a % solution. before using, it was diluted to a % albumin concentration with normal saline. all the patients were hospitalized and received oral calcium before tpe to prevent hypocalcemia. the adverse effects were recorded. results/finding: the total of tpes in patients were included as shown in the table. neurologic disorders were the most common indication for tpe, followed by autoimmune diseases. the median total plasma volume was , (range , - , ) ml. although the corrected calcium level was low (< mg/dl) in . % ( / ) before the procedure, no clinical manifestation of hypocalcemia was detected. adverse effects were observed during the tpe procedure in patients. the first patient had events of mild symptomatic hypotension. he previously took angiotensin converting enzyme inhibitor. the second patient complained nausea after finishing tpe. all reactions were mild. the incidence of adverse effects was . % ( / ). in , the incidence of tpe adverse effects was . % ( / ) when commercial albumin was used. the difference was not statistically different (p . ). median serum albumin levels pre-tpe and post-tpe were . ( . - . ) and . ( . - . ) g/dl. the increase in serum albumin after tpe was statistically significant (p< . ). eighty-two percent of pre-tpe serum albumin levels were lower than . g/dl explaining the rises of albumin after the procedures. we demonstrated that local albumin was safe and effective in maintaining albumin levels in patients undergoing tpes. safety, efficancy and cost-effectiveness of mononuclear cell collections for autologous immunotherapies: experience from a private outpatient collection facility within the eu markus dettke*. akh vienna university hospital, cyto-care.eu background/case studies: within the eu the collection of mononuclear cells (mnc) as starting source for the manufacturing of autologous cell therapies are mainly performed in hospitals or hospital-associated apheresis centers. we report about the challenges to perform the leukapheresis procedure (la) at a private held medical practice, with specific emphases on safety, cell collection efficiency, and cost-effectiveness. study design/method: we reviewed the records of altogether outpatients who underwent a total of la procedure at cyto-care, a private held medical practice/ certified cell collection facility located in vienna, austria. all patients participated in various industry-sponsored clinical p i-iii trials; the study sponsors were responsible for the manufacturing of the active cell product. disease entities were mainly prostatic cancer ( %) and ovarian cancer ( %). based on differences in the study protocols la was performed either one-time ( %), two-times ( %) or three-times ( %), with an interval of at least weeks between repeated collections. results/finding: all patients successfully completed the apheresis course. because of poor venous access, out of patients ( %) required a shortterm femoral catheter insertion. there were no serious side effects in patients who required a femoral catheter, or in patients with repeated la procedures. side effects of the la procedure mainly consisted on mild hypocalcaemia-related symptoms in % of patients. a follow-up survey one week after completion of the la revealed no infectious complications, and no patient required hospitalization. median cell yield collected per single apheresis was . x wbc consisting of . x mnc. mnc cell yields remained stable even in repeated la collections. all cell products were successful transformed into an active cellular product. analysis of the cost structure showed that the total cost of care was % lower in the setting of a private collection center compared to hospital-based apheresis centers. conclusion: leukapheresis performed in a private medical practice/ certified cell collection facility is safe and effective, with low rates of complications and high levels of patient satisfaction. this service model is costeffective and can help to reduce the cost of manufactured goods in the production of innovative cellular products. although typically associated with monoclonal gammopathies (e.g. waldenstrom's macroglobulinemia and multiple myeloma), hvs has rarely been reported in patients with disorders of immune system such as rheumatoid disease, sjogren's syndrome, hiv and igg -related diseases. therapeutic plasma exchange (tpe) is indicated in hvs due to monoclonal gammopathy (asfa category indication). however, there are limited data for the utility of tpe in hvs due to polyclonal gammopathy. study design/methods: a year old female patient with a medical history significant for seropositive erosive rheumatoid arthritis, hypertension, diabetes mellitus, cutaneous lupus and diffuse parenchymal lung disease, presented to our institution with complaints of progressive fatigue, muscle weakness, poor appetite, headache and epistaxis for a few months. fundoscopic examination showed dilated and tortuous vasculature as well as bilateral retinal hemorrhages (mixed flame-shaped and dot-blot patterns). pertinent laboratory findings included a positive anti-nuclear antibody screen with anti-histone antibodies and anti-ro antibodies. serum rheumatoid factor was markedly elevated to , iu/mls (ref. range < ) and anti-cyclic citrulline peptide antibody was elevated to , units (ref. range < ) . serum protein electrophoresis and immunofixation demonstrated a polyclonal hypergammaglobulinemia; protein precipitates were noted at the point of application, suggestive of circulating immune complexes. serum igg, igm and iga were , and mg/dl respectively. a cryoglobulin screen was negative. serum free kappa to lambda ratio was . . peripheral blood flow cytometry did not identify any monoclonal bcell population. plasma viscosity was noted to be . centipoise (cp) at admission (ref. range . - . ). pet-ct imaging was negative. the patient was treated with high dose steroids; a single tpe procedure was performed using the following parameters: volume treated - total plasma volume; replacement fluid - % albumin and normal saline in a : ratio; replacement fluid volume: % of the total volume processed. the procedure was tolerated without complication. results/findings: immediately post-tpe her plasma viscosity level dropped to . cp. serum igg, igm and iga levels decreased to , and mg/dl respectively. her rf had decreased to , iu/ml. the patient reported subjective improvement in strength. she subsequently received two infusions of rituximab separated by two weeks. her plasma viscosity has remained less than cp since tpe. conclusion: polycolonal gammomathy (e.g. secondary to ra) is a rare cause of hvs. tpe can provide transient relief of symptoms in unusual cases of hvs and may facilitate therapy to prevent recurrent hvs episodes. therapeutic plasma exchange in neuromyelitis optica spectrum disorders -experience from tertiary care centre in north india ratti ram sharma*, rekha hans, satya prakash, naveen sankhyan and neelam marwaha. postgraduate institute of medical education and research background/case studies: neuromyelitis optica spectrum disorder (nmosd) is an idiopathic inflammatory demyelinating disorder of central nervous system preferentially involving optic nerve and upper segments of the spinal cord leading to optic neuritis and myelitis. tpe is indicated in acute phase or as a maintenance therapy to treat or prevent relapses in chronic phase. study design/method: to assess the efficacy of plasma exchange in patients of nmosd not responding to high dose intravenous steroids. we did a retrospective review of tpe records for patients with nmosd over a period of three years (jan -dec ). tpe was done using, cobe spectra (terumo bct, lakewood co. usa), replacing one to one and half patient plasma volume with % human serum albumin or fresh frozen plasma on alternate days. the improvement in clinical signs and symptoms was recorded after each tpe procedure and at the end of the therapy. adverse reactions if any were also recorded results/finding: eleven patients of nmosd between to years age (m: f; : ) underwent tpe procedures with an average of . per patient. all the patients were on high dose immunosuppressant therapy without much clinical improvement. three ( %) patients had only visual symptoms, ( %) had both visual as well as muscular symptoms whereas ( %) patients had muscular symptoms only. three ( %) out of the seven tested, were positive for aqp -igg. all the patients showed significant improvement in their visual symptoms post exchange, from no vision/light perception to finger counting in two patients, recovery of colour vision and diplopia in six patients. post exchange recovery in the muscle power was observed in patients with grade- , in patient, and by grade- , in seven. adverse events were observed in % ( / ) of the procedures with allergic reactions to replacement fluid as most common event (n- ) followed by hypotension (n- ). follow up was available in % ( / ) of patients and are doing well on immunosuppressive therapy. one patient died due to respiratory failure after months and another had relapse for which he underwent second tpe cycle and continue to do well. conclusion: tpe is a safe and effective adjunct therapy to high dose immunosuppression in nmosd. trima accel software upgrade from . to . for platelet collections rachel m beck*, kimberly j duffy, sandra bryant, audrey e traun, mary m benike, james r stubbs and justin d kreuter. mayo clinic background/case studies: terumobct released trima accel software version . as an enhancement to allow for the collection of platelets (plt) with platelet additive solution (pas) and provide additional improvements to increase overall reliability. additionally, the manufacturer identified a slower centrifuge speed at low draw flow rates. this software was expected to function similarly to version . . the objective of this retrospective study is to identify any variances with the software upgrade influenced the plt products collection process or products collected. study design/methods: prior to / / , plt collections were performed on nine trima accel machines operating with version . . upgrading and validating all nine machines to version . occurred from / / to / / . the trimas were programmed with the same plt configurations both before and after software update. platelet collection data from version . ( / / to / / ) was compared to version . ( / / to / / ). incomplete collections, runs identified as having possible leukocyte contamination, duration of collection, and plt split rate were evaluated for each time period. generalized estimating equations (gee) were used to assess differences between plt collections with version . and . , adjusting for multiple visits per donor, with significance defined as p-value < . . results/findings: following the upgrade to version . , staff observed a number of changes including an increased centrifuge recovery time on a donor with a low flow and a notable increase in possible leukocyte contamination products. version . of the trima accel showed a statistically significant increase in possible leukocyte contamination from % to % of collections as compared with version . . both the duration of collections and the plt split rate remained constant even with centrifuge speed adjustments in version . . conclusion: due to fda limitations not allowing for the implementation of trima accel pas plts with the currently available pathogen reduction system, the institution decided to implement only the pathogen reduction system at this time. subsequently, the version . software is no longer required. with the noted slight increase in possible leukocyte contamination as well as the lack of enhancements for plt collection, the upgrade to version . currently does not provide added value over version . for plt collection. pulmonary and neurologic symptoms due to leukostasis. therapeutic leukocytaphersis (tl) is used as an adjuvant therapeutic modality in these patients with symptoms suggestive of leukostasis. tl procedures are performed using cell separators where anticoagulated blood is subjected to centrifugal force resulting in separate layers of cells and plasma depending on their density. there are two programs in the cell separator, a mononuclear (mnc)program which has greater centrifuge speed and efficiency for the collection of mncs and a polymorphonuclear (pmn)cell program with lower centrifuge speed for the collection of pmns. hydroxyethyl starch(hes) is preferred for the collections of granulocytes for transfusion from healthy donors. use of hes facilitates the sedimentation of the granulocyte layer and increases the efficiency of collection. though use of hes in tl was not associated with adverse events with its use as a volume expander (pagano) its use in tl varies and no reports are available on the efficiency of leukodepletion using hes for tl. study design/method: we received a request for leukoreduction in yearold lady with chronic myelogenous leukemia (cml) who had a good response to imatinib. she is weeks pregnant with an increased wbc count due to the discontinuation of imatinib. we performed tl with the cobe spectra using a replacement fluid of ml % albumin. wbc counts were monitored pre and post tl in the patient and in the collected product. we modified the collection based on these results using the mnc program with acd-a or the pmn program with acd-a . as leukodepetion was not adequate with these programs we elected to use hes after discussion with the patient and her physician. tl was performed using ml of hes with citrate and the pmn program. wbc pre procedure, immediate post procedure and the product was obtained and the efficiency of leukodepletion with the different programs was calculated. results/finding: the efficiency of % wbc depletion was calculated by product wbc to patient wbc based on blood volume and also pre to post wbc the patient tolerated the procedures well and there were no adverse reactions in the patient and in fetal monitoring during the procedures conclusion: therapeutic leukocytapheresis in cml patients is safe and more effective in reducing the wbc count with the use of ml of hydroxyethyl starch with anticoagulant. post procedure patient wbc counts sometimes may not provide the data on the efficiency of leucodepletion. background/case studies: early recognition of hypertriglyceridemia (htg) in the setting of acute pancreatitis (ap) is critical to initiate effective therapy. the role of plasmapheresis as an early/adjuvant approach in acute htg-induced pancreatitis is controversial. currently, there are no consensus guidelines in optimal therapy and is asfa category iii. reported here is a case where the tg level as well as clinical symptoms improved after one therapeutic plasma exchange (tpe). study design/method: a years old male with history of hypertension, htg, and diabetes mellitus (dm) presented to our emergency department with excruciating abdominal pain. the patient was diagnosed with htg at years old. he was treated initially with diet and lifestyle modification. however, his clinical course has been compromised after developing pancreatitis with acute episodes requiring prolong hospital admission of approximately months each which were successfully treated medically. however, the recurrent episodes resulted in chronic pancreatitis which was complicated with pancreatic pseudocyst and pancreatic insufficiency. since the first episode of pancreatitis, he was then medically managed with fenofibrate, lovaza, lisinopril, levemir and novolog. during evaluation on current admission, he was found to have a tg level of mg/dl, lipase u/l, glucose mg/dl, bicarbonate mmol/l, anion gap . ct findings were consistent with ap without evidence of necrosis and stable pancreatic pseudocyst. medical therapy was started with omega fatty acid, fibrate, statin, hydration as well as pain control. statin therapy was suspended on day of hospitalization, because he was noted to have elevated liver function tests (lft) and tpe was requested and started on day after admission. results/finding: the patient tg decreased by % ( mg/dl) with medical therapy, followed by additional % ( mg/dl) after one volume of tpe. his symptoms significantly improved and was discharged with medical treatment on day after admission. compared to previous episodes, his hospital stay was significantly decreased. tg levels remained below mg/dl at days follow up after discharge. conclusion: early tpe may be of value in treating patients with elevated tg associated with recurrent pancreatitis. plasmapheresis might be an effective early adjuvant therapy to mitigate length of hospital stay, improve cost-effectiveness and patient safety. background/case studies: from to , a national blood donor center in southeast asia conducted a program to monitor the ferritin levels of platelet blood donors. the aim of this study was to explore the trend of changes in ferritin. study design/method: in this study, we collected , cases whose ferritin levels have been monitored more than twice with an interval of detection in - days. the collected plasma samples were tested for ferritin by chemiluminescence using a commercial assay. inclusion criteria included apheresis platelet blood donors with over two results of ferritin, and first time ferritin test result was over lg/l. and the upper limit was set to be lg/ l in male and lg/l in female as described in manufactures insert. the impact on ferritin from gender, age, and the blood donation frequency were examined with anova test. the blood donations frequency was categorized into five groups: times, to times, to times, to times and more than times. the high frequency (more than times group) blood donors were analyzed ferritin changes in longitudinal data. results/finding: there were , donors included in the study, of which , were male ( . %) and were female ( . %). the mean ferritin was . lg/l in male ( % ci: . - . lg/l) and . lg/l in female ( % ci: . - . lg/l). the result of anova indicates that the group with the highest frequency (more than times) has the significant lowest ferritin level (p< . ). the average change of ferritin if donation over times would up to . and . lg/l in younger and elder y/o male and and lg/l in female. and then for high frequency (half a year more than times the group of blood donors) for longitudinal analysis and found that the long-term sustained high frequency of blood donation caused a significant decline in ferritin. the average change about ferritin in high frequencies donors (over times in $ days) was reduced from . lg/l in the first period to . lg/l in the third period ( period $ days). along with the more and more period, the decline of ferritin decreased. conclusion: this analysis revealed that frequent apheresis platelet donation would decrease ferritin of donors. but the high frequency of platelet blood donors who continue to donate after a year, the decline of ferritin slowed down. a rare case of blood donation precipitating acute delirium joseph griggs* , mary townsend and lizabeth rosenbaum . university of new mexico hospital, blood systems, inc., blood systems inc. background/case studies: we report a case of whole blood (wb) donation that precipitated a transient agitated delirium. a year-old first time male donor presented to the local blood center, completed the donor health questionnaire, mini-physical exam, and hemoglobin check, and was deemed eligible for blood donation. approximately minutes after an uncomplicated wb donation, the donor had an observed, brief loss of consciousness in the post-donation area. no fall or injury was seen. shortly after regaining consciousness, the donor became agitated, confused, and was not oriented to month or year; was unable to remember the names of friends and family members; was unable to read an analog clock; and had difficulty with word finding. the donor was transported to the local university hospital where he was noted to be combatively delirious and had altered mental status; he had to be forcibly restrained. he ultimately was sedated and intubated, and transferred to the intensive care unit. study design/method: an extensive laboratory investigation was performed including standard hematologic and chemistry panels; serologic and pcr-based studies for multiple organisms including west nile, herpes, hiv, varicella zoster, and syphilis; aerobic and anaerobic blood cultures; and a urine drug screen for multiple drugs of abuse. radiographic imaging was performed including a chest x-ray, and a ct and mri of head and spine. in addition, an eeg was performed. the inpatient neurology and psychiatry services were consulted for this patient. results/finding: after the sedation was discontinued, the patient was successfully extubated and rapidly improved. he completely returned to baseline within hours of onset of the event. laboratory investigation revealed no signs of infectious organisms or evidence of drugs of abuse. radiographic imaging and eeg studies showed no abnormalities. in addition, infectious disease marker testing performed by the blood center laboratory was negative. investigation revealed that the donor was experiencing high levels of stress at school, had an aversion to the sight of blood, and was coerced into donating by his girlfriend and peers. a week following hospital discharge, the blood center medical director contacted the donor by phone; the donor had resumed his normal routine and was attending his graduate level classes. conclusion: to our knowledge, this is the first report of blood donation precipitating a transient acute delirium. at the time of donation, the health status of all potential blood donors is assessed to help ensure the safety of the donor and the recipient. the health questionnaire, physical exam, vital signs, hemoglobin level, and infectious disease testing help to identify overt signs of medical illness that may disqualify a donor. however, routine donor screening does not explicitly evaluate mental health issues, both diagnosed and undiagnosed. although exceedingly rare, this case highlights the limitations of donor screening to identify donors who may be at risk for mental health adverse reactions when donating blood. a targeted approach to increasing the african american blood donor pool arnethea sutton* , william korzun , teresa nadder , susan roseff and elizabeth ripley . virginia commonwealth university, virginia commonwealth university medical center background/case studies: a continuous need for blood products for those who require frequent transfusions, such as individuals with sickle cell disease who could benefit from products collected from african american donors, warrants the need for targeted interventions to increase blood donations from underrepresented populations. one population in particular, african americans, only account for % of blood donors in the united states. literature indicates numerous reasons why this population is underrepresented amongst donors, including fear, lack of knowledge about the blood donation, and specific to this population, lack of trust in the medical community. study design/method: african americans in richmond and norfolk, virginia were recruited through churches and local universities. the study's aims were to develop, implement, and assess a targeted educational approach incorporating the theory of planned behavior and various teaching methods, to develop and implement a survey to evaluate participants' feelings, attitudes, and intent to donate, and to motivate african americans non-donors to attempt to donate blood. participants attended a -hour educational session where they were educated on the importance of red blood cell donations from african americans. participants completed three surveys -one before the session, one directly after the session and one, two months after the session. a two-proportion z-test was used to compare the known proportion of african americans who present to donate in the study areas to those who presented to donate in this study, while regression analysis was used to estimate the relationships among survey variables. results/finding: a total of subjects were included in the data analysis. sixteen percent of the study participants presented to donate as a result of attending the educational session. this resulted in a statistically significantly higher proportion of african americans presenting to donate than the current proportion in the areas of the state where this study was conducted. results from the first two surveys indicated that subjective norm and attitude were significant predictors of one's intent to donate blood, while perceived behavioral control was not a factor. the educational session increased survey scores related to intent to donate in comparison to scores obtained prior to the session. conclusion: this study shows that a targeted educational program can change attitudes toward blood donations in african americans resulting in an increase in new blood donors. additional studies are needed to see if this behavior will continue and whether african americans can influence their community to increase awareness and motivation for life-long blood donation. were from female basic trainees conclusion: the significant increase in hemoglobin deferrals at basic training site a from to could be a result of a change in the blood drive timing of the training schedule of that location. in , basic trainees at site a were scheduled at day of . in january , the blood drive date changed to day of . the extra three days in the basic training atmosphere, and its associated diet changes and increased physical activity may have had an effect on the hemoglobin levels in that population. at basic training site b, the significant increase from to of hemoglobin deferrals can be attributed to a larger male population presenting at this site for basic training. additionally, the percentage of female recruits donating at the blood drives decreased in . these observations support the hypothesis that the increase in hemoglobin deferrals in resulted from the implementation of the male hemoglobin standard change from . to . g/dl at basic training site b. when planning for blood drives at basic training site b, screening of an additional % of recruits must be considered when performing these blood drives, in order to meet the same collection goals set prior the implementation of the change in the male hemoglobin standard. blood donation in the donor with spinal cord injury joan-ramon grífols* , eva alonso , oscar bascuñana , monica romero , teresa vich , elena castaño , laura carbonell , eva palomas , saray almerge , francesc carpio and xavier curia . banc de sang i teixits, institut guttmann background/case studies: donation of blood components (bc) in donors with spinal cord injuries (sci) is poorly studied. paralysis is a state, not a disease, after a reasonable time since its acquisition these people should not be differentiated from the rest of the non-paralytic population in terms of bc donation. the literature reviews of blood donation suitability criteria among these people are scarce and the vegetative lability that they may present depending on the type of their sci it's obvious. in daily practice these potential donors are often rejected for donation with no specific criteria related to their sci. the objectives of this study are to establish the selection criteria for bc donation in people with sci based on medical criteria. to evaluate the rate of adverse donation blood reactions of these donors against a donor control group without sci. study design/method: our organization regularly organizes a donation campaign at a rehabilitation center for patients with sci. in this campaign some donors with sci as donors without (professionals of the center, relatives, etc.) donate blood. from january to december we analyzed the number of donors who came to give blood, the number and reasons for exclusion of those who could not make the donation, whether or not they had sci and number and typology of adverse reactions to the donation detected in both groups. donors with sci higher than t due to the high risk of autonomic dysreflexia were excluded for donation. donors with sci below t and less than one year of evolution were set as temporary exclusion criteria. the presence of neurogenic bladder was not considered a reason for exclusion. results/finding: in the analyzed period, donors came to give blood, of these, ( %) were excluded for donation for various reasons. two of the donors excluded suffered sci higher than t excluding them due their high risk of dysreflexia. another one donor excluded suffered sci lower than t but his hemoglobin levels were lower than our selection criteria. of the donors selected for donation ( . %) had sci lower than t and t . adverse reactions to donation ( . %) were recorded in our haemovigilance program, none of them in donors with sci. conclusion: according to our experience donors with sci lower than t have not had any type of adverse reaction to the blood donation. there should be selection / exclusion criteria based on the donor's paralytic conditions. the vagal syndrome that could appear as a complication to the donation in these sci donors should be approached differently to the usual protocols that we use. blood donor center's experience with changing from manual to automated blood pressures kimberly j duffy*, sandra bryant, audrey e traun, kristine i borth, mary m benike, james r stubbs and justin d kreuter. mayo clinic background/case studies: blood pressure (bp) is important for determining the health and suitability of blood donors. the manual method of reading bp can result in variability due to minor variances in the way staff perform the manual procedure. automated bp devices are able to reduce the variability in bp determination. in december of , automated bp devices were validated and replaced the manual bp method in our blood donor center. the objective of this retrospective study is to determine if the change from a manual to an automated bp process has impacted the average systolic and diastolic pressures and, additionally, if a differences in the deferral and reaction rate can be observed. study design/methods: data for the manual bp process was accumulated for an month period from january to november . the same information was assembled for the automated bp process for the month period of january to november . the automated bp process implemented in mid-december ; so the december data for both and has been excluded from the study. bp, bp deferrals, reactions, donor weights and demographics were evaluated for each time period. a donor may be included multiple times in each year and could be in both sets of data. generalized estimating equations were used to assess differences between automated and manual bp with significance defined as p < . . results/findings: significantly more people were deferred using automated bp compared to manual bp readings (p . ). both systolic and diastolic bp measured significantly higher by automated bp method than by manual method. although donors in the automated bp group experienced fewer reactions than those in the manual bp group, the reduction was not large enough to reach statistical significance. even after adjusting for gender, weight and age at donation, bp deferrals, systolic and diastolic bps all remained significantly higher (all p < . ) with the automated bp while and reactions remained non-significantly lower (p . ). conclusion: automated bp devices have improved convenience for both staff and donors. with a statistically significant increase in deferrals and marginal decrease in reactions, the use of automated bp devices may play a minor role in the safety of blood donors. for the purpose of this study, only the hemoglobin values that were below . g/dl will be compared as a surrogate for deferral. to adjust for multiple visits per donor, generalized estimating equations were used to assess significance between lancet a and lancet b, using the appropriate distribution for the data type, defining statistical significance as p-value < . . results/findings: the average hgb was slightly lower with lancet b but there was a larger change with the number of donors under . . statistically more visits with hgb less than . g/dl used lancet b than lancet a. additionally, fewer first time donors were seen during the lancet b time than during the lancet a time. after adjusting for the effects of both gender and first-time donation by using logistic regression, the risk of hgb under . was . % higher with lancet b than with lancet a. conclusion: donor's hgb was slightly lower with lancet b than lancet a, but not clinically different. slightly more lancet bs were used per visit than lancet as. in addition, more hgb deferrals were obtained using lancet b than lancet a. even after adjusting for the effects of gender and repeat donors, we saw more potential deferrals with lancet b than lancet a. the slight difference in the gauge of the lancet may have some association to free-flowing amount of blood and may affect hgb levels. prior to implementing materials at a lower cost, an evaluation of downstream consequences would be recommended. blood donors' acceptance and response towards implementation of automatic appointment booking yi lin ang*, ching lian toh and william choon hong sim. health science authority background/case studies: with surges in demand for blood due to an aging population and more hospitals being built, it is becoming increasingly important to be able to ensure that donors return on a regular basis to improve blood supply and blood stock management. disliking the obligation imposed by appointments, singaporean donors generally prefer "walk-ins" as opposed to appointment bookings. blood services group (bsg) singapore, has made a move to change donors' mindset by introducing automatic appointment scheduling. this paper aims to study donors' level of acceptance towards this initiative. study design/method: to determine the donors' acceptance rate, data was collected from january to march . after completing their donation, donors were automatically given the next earliest eligible date for their next donation. those who do not wish to accept the recommended appointment can either decline this arrangement or log into the blood bank's donor appointment booking system (donor-care) to make changes to the appointment offered. a reminder will be sent to their phone via sms and/or email to their account three days before the appointment date. data was collected from donor-care and was used to measure the number of appointments made and declined over the three months period. donors who declined appointment scheduling were verbally interviewed for their reasons. results/finding: a total of donors who has donated blood in the blood bank's main branch were used as the baseline for this study. % of donors (n ) accepted automatic appointment booking, whereas some donors (n ) were not comfortable with it. % of those who declined still preferred walk-ins (n ) based on their own time schedule, the rest decided that variable situations (n ), donation frequency (n ) and choice of preferred donation locations (n ) were reasons for declining automatic appointment booking. prior implementation of appointment booking at other blood bank branches showed that donors who booked appointment through donor-care was %. a comparison was made and found that this study shown a significant increase of acceptance rate by %. conclusion: generally, the results were positive and the automatic appointment booking system enabled bsg to predict donor attendance, ensure better manpower management to reduce donor turnaround time and thus hopefully improve donor retention. bsg is still monitoring this automatic appointment system and future study are still required to determine the effectiveness of automatic appointment booking, donor return and retention rate. currently bsg has collection centers, each managing its own appointment system. the eventual aim is to be able to have a centralized appointment booking system whereby donors can book appointments and still be able to donate at any collection site. ) , . . poisson distribution, normal distribution, logistic distribution, lognormal distribution a transfusion (p> . ) in donor and reference populations except in younger ( - yrs) male donors (p< . ; donor . %, reference . %). mean donor sbp, dbp, and pulse were . mmhg, . . mmhg, and . . bpm, respectively. screening blood pressure levels consistent with hypertension ( . % male; . % female) in the - year donor group, significantly (p< . ) higher than the reference population ( . % male; . % female). no differences were observed in the - year groups. conclusion: normal source donor demographic and physiologic characteristics often paralleled those of the reference usa populations. however there were differences including lower cholesterol levels and a higher rate of high blood pressure in younger donors and higher weights in - year old females. developing blood donor educational materials gay wehrli* , susan rossmann , louis m. katz and dan a waxman . university of virginia health system, gulf coast regional blood center -sugar land, americas blood centers, indiana blood center background/case studies: donors must have sufficient information to make a decision, time to consider options before making a decision and an opportunity to make a choice of whether to proceed with or decline donating. donor education (de) materials must address mandates set forth by regulatory agencies. these materials must be accessible and understandable by the general population. the goal of this non-experimental, qualitative design study was to evaluate knowledge acquired through standardized de materials. this study was irb approved as an exempt protocol. study design/method: we developed a de document written at an th grade comprehension level. a convenience sample of volunteers was identified for this two-part study. a focus group (fg) incorporated a pre-and post-quiz for knowledge acquisition from reading the four-page de document. the quiz was followed by a group discussion for feedback. the preand post-quiz contained the same multiple choice questions with single best answers including the option to answer, "i don't know." the de document was revised based upon the fg feedback and quiz results. the revised, . page, de document was then tested using the same pre-and post-quiz during individual interviews (ii). results/finding: demographics and quiz results are summarized in table . results from the fg and ii revealed a lack of knowledge in four areas: a donor might be asked not to donate at any time during the donation process, the need for photo identification to donate, iron helps increase a low red blood cell level, and not to donate for the sole purpose to obtain hiv testing. post-quizzes from the ii group revealed an improvement in knowledge acquisition for all four areas. feedback from both groups reiterated that the document was too long. conclusion: developing de materials requires a complicated balance of providing critical information, concisely and at an appropriate comprehension level ( th grade). testing de materials is an essential step in the development process to ensure the intended knowledge is acquired by the end user population. the next steps for this group will be to pilot the further revised, two-page de document at donation sites. effect analysis of the 'rh(-) blood supply program' establishment hyesung han*, deokja oh, buja hur and chulyong kim. korean red cross blood services background/case studies: the rh(-) blood supply program was developed in for the purpose of prompt and stable blood supply. based on the computerized system, the program operates the emergency contact/ communication. this program has major functions such as the request of the emergency blood, the recruitment and management of the rh(-) blood donors for the emergency blood donation, real-time blood supply status monitoring program and statistics program. the aim of the research is to validate the effect of rh(-) blood supply program operations and the responsiveness of the emergency blood supply under the rh(-) blood supply program. study design/method: researchers investigated the database from to after the rh(-) blood supply program was developed. investigators analyzed and compared the recruitment and blood donation of the rh(-) blood donors for the emergency blood donation and securing the blood supply upon request. results/finding: the data shows that the number of voluntary blood donors who pledge to give blood for the emergency blood donation has increased from . % to . % in and , respectively. also, the actual participation rate of rh(-) blood donations among the group who pledge to give blood for the emergency blood donation has increased from . % in to % in . moreover, the data has indicated that the blood supply has fully met the demand for the emergency blood request. conclusion: the result showed that the rh(-) blood supply program was effective for the recruitment/management of the rh(-) blood donors for the emergency blood donation. this system contributes to recruiting and managing rh(-) blood donors who pledge to donate blood and securing rh(-) blood in emergency situation . the institution that needs to meet the demand of rare blood type could possibly use the rh(-) blood supply program which leads to securing special type blood. hanwei chen*. wuhan blood center background/case studies: in china, volunteer blood donors can donate platelets by apheresis (ap) up to times per year. however, the awareness and knowledge of ap donation is much lower than whole blood donation among the chinese population. there are approximately . million doses of ap transfused within . billion people each year in china; it is one challenge to recruit new ap donors and retention them as frequency ap donors in china. study design/method: one stratified recruitment and retention strategy established and applicate at wuhan blood center since . firstly, "one-to-one" telephoning model for whole blood donors instead to donate platelet; secondly, group message for permanent ap donors and had not donated with an interval of more than days in low inventory. thirdly, specific recruiter telephone for those ap donors who had donated aps for more than times and had not donated for more than days or less than times with an interval of more than days from the last donation; the last one is preparing one letter of thanks for those ap donors who gave more than times annually which advise them to voluntarily come to the blood center for ap donation when they were available. results/finding: over the past decade, the overall donation time of ap donors increased by . times from to and the doses of ap increased by . times from to within years. the aps collected fulfilled the clinical needs. according to the donation frequency, ap donors were divided into groups: those who donated ap once, those who donated - times, - times, - times, and those who donated more than times, respectively. it was found that the number of permanent ap donors who donated ap more than times was only ( . %), but they denoted a total of doses of ap ( . %) from to . conclusion: aps increased at a rapid and steady pace in wuhan blood center from to , which not only met the clinical needs but also were supplied to other region outside wuhan. and in addition, the permanent ap donors who gained more attention donated the greatest percentage of platelets. in conclusion, stratified recruitment is one effective approaches to meet clinical needs for platelets and worth to popularize to other region. years were evaluated at sites on consecutive donations for finger stick (fs) hemoglobin (hb) per site policy. venous (ven) and capillary (cap) zpp and ven ferritin (fer) were performed per manufacturers' direction. donors were assessed for subclinical iron deficiency using ranges (fer < ng/ml and zpp levels > umol/mol heme) at hb levels. participants completed an online survey between donations to collect data on symptoms of anemia. univariate linear regression analysis was used to determine relationship between tests. results/finding: subclinical iron deficiency was present among first-time and repeat blood donors at all hb levels with both genders and all age groups. (table) there was a highly significant correlation between fs zpp and ven zpp . % (r . ) at first and . % (r . ) at second donations. at first donation when compared to fs hb, only . % (r . ) of variation could be explained by variation in fs zpp, . % (r . ) by ven zpp and . % (r . ) by ven fer. at second donation, when compared to fs hb, only % (r . ) of variation could be explained by variation in fs zpp, . % (r . ) by ven zpp and . % (r . ) by ven fer. for each donation, variation among tests (fs hb, ven fer, ven zpp and fs zpp) was significant (p< . ) suggesting strong evidence against correlation. % ( ) responded to the survey of which % ( ) reported not feeling well after donation. it should be noted that noted that % ( ) female study participants reported feeling unwell after the first donation and had ferritin levels below ng/ml but the zpp levels were less than umol/mol heme. of the % ( ) male participants that reported not feeling well none had ferritin levels below ng/ml nor ven or fs zpp levels above umol/mol heme. conclusion: subclinical iron deficiency was present at all hemoglobin levels. there was insufficient correlation with fs hb and ven fer to support use of fs or ven zpp analysis as measurement of iron stores for blood donors. symptoms reported by study participants were not consistent with laboratory results. the minimum male hb was raised from . to . gm/dl. fda imposed specific vs ranges for acceptable pulse (p) and blood pressure (bp), removing center-by-center discretion. a survey of members of america's blood centers (abc) was performed to assess the impact on donor deferrals resulting from these changes. study design/method: online survey software (surveygizmo, boulder, co) was used to solicit collections and deferral information from blood centers over two intervals, july-dec. and july-dec. (i.e., before and after the implementation deadline for the final rule respectively). information on deferral at presentations for whole blood (wb) donations and apheresis platelet (ap) donations was requested for hb thresholds and vs. the information was stratified by gender (male m, female f), and abo type. statistical analysis included t-tests for numerical and chi-square for categorical data (minitab . , chicago il). p <. was considered significant. results/findings: data were provided by of centers invited, representing , , and , , wb donations and , and , ap donations in aggregate during the two intervals respectively. gender and abo distributions appeared representative of the us donor base. among m wb donors the rate of deferral rose from . % to . % in the two intervals among aggregated donation attempts (p<. ), and for m ap from . to . % (p<. ). the mean "by center" deferral rates (table) were similar to that and significant (p<. ). mean by center hb deferral rates among f donations during the two intervals were . and . % (p . ) for wb, . and . % (p . ) for ap, respectively, absent any change in their acceptable hb thresholds. data on vs deferrals were much sparser. for p deferrals, only centers could provide specific high vs. low vs. irregular pulse deferrals; provided only a summary (i.e total pulse deferrals), and could provide none. for bp, provided detail (high vs. low), summary and none. p deferrals increased in the successive intervals among f wb donors from a center mean of . to . % (p . ) and for m wb donors from . to . % (p . ). where details were available, high and irregular pulses were responsible for most of the changes for both genders. bp deferrals were not significantly increased among wb donors, regardless of gender. the data sets and deferral rates re: vs in ap donors were quite small, possibly reflecting culling during their prior donation experience. conclusion: substantial additional donor deferrals attended the increased hb thresholds for m in the final rule, for both wb and ap. changes were more modest among female donors, consistent with the absence of changes in allowable hb levels. modest but significant changes attended more stringent requirements for vs, though data limitations restrict this aspect of the analysis. background/case studies: diabetes mellitus is reaching potentially epidemic proportions in india. given the disease is now highly visible across all sections of society within india, there is now the demand for screening of diabetes and urgent research and intervention -at regional and national levels -to try to mitigate the potentially catastrophic increase in diabetes that is predicted for the upcoming years. due to its ease of use, several studies have found that hba c testing can identify patients in the community who might otherwise go undiagnosed. we took an initiative to find out the incidence of diabetes by random blood sugar (rbs) measurement among indian blood donors and measure the hba c levels among those with rbs > mg/dl study design/methods: a prospective study was done at department of transfusion medicine and department of biochemistry from st march to st march . total of , blood donors were tested for rbs. those with rbs > mg/dl were further tested for hba c by gold standard hplc method using variant ii biorad. blood donors with > mg/dl rbs and hba c > . % were advised to consult a physician for further evaluation. results/findings: of the , donors tested, ( . %) donors showed a rbs of > mg/dl. forty two ( . %) were males and ( . %) females with a mean age of . years ( - years). of these, ( . %) were known case of type-ii diabetes mellitus (dm) on oral medications and were excluded. of the remaining , ( . %) of them had a family history of dm. of these donors, donors did not give a consent for testing for hba c. among the donors tested for hba c levels, ( . %) had hba c > . %. all the donors were counselled and referred to a physician for further management. the overall incidence of donors having dm in the population is . % ( of donors). conclusion: screening for blood glucose level by targeting the blood donors can go a long way in curbing the diabetes burden on the society. incidence of low ferritin levels in regular male blood donors with acceptable hemoglobin levels in singapore ramir alcantara* , hwee huang tan and ai leen ang . health sciences authority blood services group, health sciences authority, blood services group background/case studies: iron deficiency is a known complication of regular blood donation. in order to protect the donor's health and prevent iron deficiency, aabb increased the minimum acceptable hemoglobin level for male whole blood and apheresis donors from . to . g/dl last may . the current minimum acceptable hemoglobin for male donors in singapore is . g/dl. the aim of the study is to determine the incidence of low ferritin levels in regular whole blood and apheresis male blood donors with acceptable borderline hemoglobin levels ( . - . ) and in donors with hemoglobin g/dl and above. study design/method: during a month period, serum ferritin testing was performed on regular male whole blood and regular male apheresis donors who made at least donations in the last two years with an acceptable hemoglobin level. the donors were divided into groups according to donation type and hemoglobin range; group a (whole blood with hemoglobin . - . ) group b (whole blood with hemoglobin ! , group c (apheresis with hemoglobin . - . ) and group d (apheresis with hemoglobin ! ). the serum ferritin levels of the four donor groups were compared and analyzed. a ferritin level below ug/l is considered low and levels below < ug/l are considered having absent iron stores. results/findings: . % of donors in the study have ferritin levels below ug/l. there were more donors with low ferritin in group a compared to group b, % and % respectively (p< . ). in apheresis donors, low ferritin rates were higher in group c donors compared with group d, % and % respectively (p . ). ferritin results for the groups can be seen in table . conclusion: more than half of the donors in the study have low ferritin and of the donors with low ferritin, more than half or . % have absent iron stores. donors with low ferritin were immediately informed of their result, given iron supplements and advised to come back for donation after months or more. since donor health and safety is of paramount importance, measures to limit and prevent iron deficiency in blood donors must be implemented. due to the high incidence of low ferritin levels in whole blood and apheresis donors with hemoglobin . - . g/dl, it is recommended that the minimum hemoglobin level cut off for male blood donors in singapore be increased to . g/dl. other measures to be implemented includes better donor education on the risk of iron deficiency and the need for iron supplementation using our website and social media. background/case studies: safe blood is a crucial and irreplaceable component in the medical management of many diseases. the voluntary nonremunerated blood donation is the ideal sources of quality blood, which forms less than % of the demand of the blood in pakistan. motivation among the youth, particularly students, is essential to make voluntary blood movement more successful. to assess the knowledge, attitude and practice regarding the voluntary blood donation among the young student population of karachi so that an effective approach can be made regarding motivation enrolment of voluntary non remunerated blood donors in future in pakistan study design/method: a cross sectional prospective study was conducted among students from different universities and colleges of karachi. a well-structured and pre-tested questionnaire, in english, was used to access the knowledge, attitudes and practices about voluntary blood donation. a scoring mechanism was used to understand overall knowledge level. obtained data was analyzed. results/finding: the sample population consisted of % male and % female students in the age group of - years. only % of the students have heard about voluntary blood donation and % of the students have given blood once in their lifetime and among them % are blood donors at the moment. % of the participants believed that there is a specific reason why they don't donate blood and % believed that there is a risk involved for the donors, when donating blood. % students wanted to promote voluntary blood donation. fear and lack of awareness on blood donation are the reasons for not donating blood. students gather information about voluntary blood donation from several sources mostly schools, colleges, family and friends. ( ); miscellaneous effects were reported in courses. side effects led to interruption of supplementation in instances. ferritin levels (mgt sd) at entry into the program and at the last visit were . and . . mg/l in participants, vs . . and . . mg/l in controls. the positive impact of iron supplementation on ferritin levels was observed only in those who took ! % of the tablets. ferritin levels< mg/l were found in , % of participants and . % of controls. deferral for low hemoglobin was below % in both groups. conclusion: an iron supplementation program in a drbcd program is feasible.however, when taking into account acceptance to participate and compliance with supplementation, only % of donors obtain full benefit from such a program. using an iron preparation which is better tolerated may increase compliance. background/case studies: hereditary hemochromatosis (hh) patients are permitted to donate blood for the allogeneic blood supply as long as they are eligible for donation under cfr . and the collection is a physician-ordered therapeutic phlebotomy. blood collections establishments do not need an exception or alternative under § . to make a collection under this provision if the requirements set forth in § . (a)( ) are met. the objective is to describe current hh donors and long-term contributions of to our hospital-based donor center and hospital blood supply. study design/method: in , an irb protocol was approved for the enrollment and therapeutic phlebotomy of hh patients/subjects. this required filing an fda variance to permit hh donor blood for use in our allogeneic supply without disease labeling. the frequency of therapeutic bleeds are guided by routine clinical assessment, mcv/hemoglobin, serum ferritin, and transferrin % saturation monitoring. serum ferritin levels of - ng/ ml are targeted for maintenance phlebotomy. operationally, a custom, computerized database application is employed to ease phlebotomy management. results/finding: since inception, the cumulative number of hh subjects enrolled in the hemochromatosis protocol reached , of whom ( %) are c y homozygotes. without active recruitment, accrual rate is about per quarter, with % of subjects qualifying as allogeneic donors. the mean current age is . years, % male, % caucasian. the majority of hh donors ( of an active cohort of ) are in the maintenance phase of therapy with an average of . donations/year and a % deferral rate. over the last years, hh donors contributed approximately - % of the hospital's allogeneic blood supply, averaging whole blood units for transfusion per year. moreover, hh donor's whole blood (wb) donations provided - % of blood for in vitro research at our institution with an average of wb research donations/year. there have been no hh donor-derived transfusion-transmitted infections over years. since / / , with an increase in male hgb deferral threshold to g/dl, there has been only hh male deferral from blood donation. conclusion: a simple, safe system for donor evaluation, phlebotomy management, and transfusion of blood drawn from hh subjects was established. blood donated by hh donors remains an important resource at our hospital. hh donors benefit from careful medical follow-up of their iron status. this mutually beneficial relationship is feasible and sustainable. testing for accuracy of non-invasive blood hemoglobin methodology in a blood donor setting michele walker*, sharon garcia and mythili ram. gulf coast regional blood center background/case studies: the objective of the study was to assess the accuracy of hemoglobin (hb) levels measured on the orsense nbm- non-invasive occlusion spectroscopy device by comparing them to hb levels measured on venous samples with a laboratory hematology analyzer. in addition, the study examined operator ease of use and donor satisfaction with a finger stick-free method. study design/method: study procedures and protocol, including acceptance criteria, were defined in conjunction with the device manufacturer to determine the standard deviation (sd) of the difference between the nbm- non-invasive sample results and the sysmex hematology analyzer venous sample results. staff were provided training on the use of the nbm- non-invasive occlusion spectroscopy device. over a span of days, eligible blood donors, both male and female, were first screened by the nbm- non-invasive occlusion spectroscopy device followed by performance testing utilizing a capillary blood screening method. a venous sample was collected from each of the blood donors for the performance of hb measurement on the sysmex hematology analyzer within - hours of collecting the venous samples. results/finding: the sd of the difference between the nbm- non-invasive sample results and the sysmex hematology analyzer venous sample results was not to exceed . g/dl. the hb measurements obtained from the nbm- and the sysmex hematology analyzer were analyzed using the statistical software minitab and the sd of the difference was reported to be . g/dl. the precision of the nbm- yielded a co-efficient of variation of . g/dl and a standard deviation of . g/dl. conclusion: the operators found the nbm- easy to install, maintain, and operate with minimal training. the nbm- non-invasive occlusion spectroscopy technology showed accurate performance compared with the venous sample results. it was comparable to the capillary finger stick method and deemed suitable for screening donors. donors were satisfied with the process and appreciated the safe, painless methodology. ronel swanevelder , ravi reddy , dhuly chowdhury , don brambilla and edward l. murphy* . sanbs, rti international, ucsf/bsri background/case studies: to maintain an adequate blood supply, south african blood centers need to collect more blood from their majority black african population. success in recruiting first-time black blood donors has been tempered by lower suboptimal return rates. study design/method: we performed a prospective cohort study of firsttime, black blood donors donating during a four-month period in and followed them for one year. within days post donation, a questionnaire including questions on blood donation motivators and deterrents was administered by telephone. questions used -point likert scales to assess agreement with statements relating to domains of altruism, collectivism, selfesteem and marketing derived from local focus groups (muthivhi et al. ) . linking questionnaires to a blood donation database allowed logistic regression analysis to predict return for a second donation within one year. results/finding: we included , first-time black donors with median age and female predominance ( %). within one year, , donors ( %) attempted at least one additional donation. when likert scales were analyzed as an ordinal variable ( strongly agree to strongly disagree), donor return was associated with the following motivators "blood donation is an easy way to make a difference" (odds ratio for each likert increment (or) . , % ci . - . ), "i donated in response to adverts/campaigns on the radio, tv or newspapers" (or . , % ci . - . ). responses to altruism-associated statements were not associated with return. among deterrents, donors were less likely to donate if they agreed with the statement "i am afraid of the sight of blood" (or . , % ci . - . ) and "i wasn't treated well by the <blood center> staff" (or . , % ci . - . ). surprisingly, donors were more likely to return if they agreed with the statement "i was afraid of finding out about my hiv status" (or . , % ci . - . ). a secondary analysis treating the likert scales as -level categorical variables revealed generally similar results, with the additional finding that donors who disagreed with the statements "if i give blood then blood will be available when i need it" and "i don't know where the nearest blood collection point is" were more likely to return. conclusion: this novel design allowed us to study the link between donation motivators and deterrents and actual rather than intended return for donation. it is interesting that self-esteem and marketing predicted return better than altruism. fear and poor customer experience are recognized deterrents which could be addressed. we plan to use these data to construct black donor recruitment interventions which may be tested using randomized trial designs. willingness to donate blood during the summer christopher d bernard , ramya ghantasala , obhijit d hazarika , nicole leonard , cori a polonski , zachary b wunrow , michelle heleba , jan k carney and mark k fung* . university of vermont larner college of medicine, american red cross blood services background/case studies: each year donation rates fall in the summer months straining blood banks' capacities to meet local demands. in hopes of identifying factors to increase summer donations, our study investigated donor reported barriers which influence summer donations habits. study design/method: an anonymous question survey investigating various donation factors was administered across multiple blood donor centers in a state-wide region. questions addressed donor demographics, frequency of blood donation, preference in appointment making modalities including smartphone app use, summer travel habits, willingness to donate during vacation, and factors that deter donors from donating on vacation. results/finding: a total of surveys were received. survey respondents across multiple demographic groups cited similar barriers to summer donation, namely "too busy" ( . %) and "traveling is a time for me to relax." ( . %). of the respondents who travel in the summer, very few reported donating while traveling ( . %). summer donation rates between summertime travelers ( . %) and non-travelers ( . %) were essentially equivalent. the most preferred methods of scheduling appointments were via the regional blood donor center website ( . %) and phone ( . %). willingness to use a regional blood donation smartphone app was highest among respondents ages of to ( - %) and lowest among ages and older ( - %). of respondents with no prior knowledge of summer seasonal shortages ( %), / rds indicated newfound motivation to donate. background/case studies: viral infections (adenovirus, ebv, cmv, bk, hhv , and rsv etc.) have been implicated as major contributors to posttransplant morbidity and mortality in hematopoietic stem cell transplantation (hsct) from unrelated donors. investigators have shown that in-vitro expanded virus specific cytotoxic t lymphocytes (ctls) generated from donors with specificity for one or more viruses are safe and effectively treat viral infections in the hsct setting in recent clinical trials. present clinical trials have shown that ctls can be rapidly produced by a single stimulation of donor peripheral blood mononuclear cells (pbmcs) with a peptide-mixture spanning the target antigens in the presence of potent prosurvival cytokines interleukin- (il- ) and il . others have used banked third party epstein barr virus (ebv)-specific ctls generated from third party ebv-seropositive blood donors with encouraging results. study design/methods: eligible and consented blood donors were tested for cmv antibodies by serology. cmv-seropositive whole blood (wb) units underwent buffy coats processing from non-leucocyte reduced wb units collected in fenwal triple blood-packs tm that underwent hard spins at rpm for minutes with separation after each spin on a compomatev r g . plasma and buffy coat was separated from red cells after the first spin. the second spin lead to the separation of the buffy coat from plasma. the buffy coats were submitted to the gmp stem cell lab for processing of cytomegalovirus-specific ctls. hla typing at high resolution for hla-a/-b/-drb loci was obtained for all donors. results/findings: forty five eligible healthy blood volunteers ( m [ %]: [ %] f); median age years (range - ) donated a unit ( ml) blood from which buffy coats (average volume ml) were processed. the buffy coat process was previously validated on wb units. the mononuclear cells (lymphocytes and monocytes) recovered from the buffy coats are listed in figures and . all of the buffy coats received by the gmp stem cell lab were adequate in cell numbers to be processed. the processing of buffy coats from whole blood is a viable option for the concentration of pbmcs specifically for production of viral specific ctls as third party off the shelf products as well as use in other research projects that require pbmcs from healthy adults. background/case studies: the goal of this presentation is to describe the journey and challenges towards tjc, patient blood management (pbm) certification. transfusion-related health risks and increasing economic pressures have driven hospitals to recognize evidence-based blood management as an important cost-saving strategy. providence holy cross medical center (phcmc), as the providence california region alpha site, has embarked on this journey. our goals are pbm certification and reduction of the number of unnecessary transfusions by % within months of the program launch while improving patient outcomes. this paper will discuss our journey toward certification and the various hurdles being overcome. study design/method: tjc, aabb, and the society for the advancement of blood management have served as our primary resources for identifying current evidence-based transfusion practices and management methods. we needed to identify our organizational gaps in data gathering and analysis. then we could determine baseline performance and set improvement targets. from our internal assessment, we learned we had to start from scratch as we had no easily accessible data metrics and gaps in education to our staff. we took the following steps to develop our pbm program: formed an interdisciplinary pbm team consisting of physicians, nurses, blood bank staff, and data analysts constructed a report on rbc transfusions to help identify outliers and opportunities background/case studies: the maximum surgical blood ordering schedule (msbos) is a list of surgical procedures performed at a hospital along with a recommendation for pre-transfusion testing and rbc allocation before each surgery. the extent to which hospitals have an msbos and its design was explored in this survey. study design/methods: the survey was designed, piloted and refined by members of the best collaborative and invited colleagues. it was then encoded in online survey software and the link distributed to best members and colleagues who were encouraged to respond and to further distribute it. the survey was open for days. results/findings: there were completed responses, of which ( %) indicated that their hospital had an msbos and ( %) did not. the majority of hospitals without an msbos were academic centers ( / , %) from oceania ( / , %) or europe ( / , %), had between - beds ( / , %); the majority of these hospitals transfused between , - , rbcs ( / , %) per year. / ( %) are going to implement an msbos in . of those with an msbos, the majority / ( %) were from north america. the majority were academic hospitals ( / , %) with - beds ( / , %) that transfused ! , rbc units per year ( / , %) offering a wide range of surgical services. on average there were procedures listed in the msbos'. the msbos recommended no pre-transfusion testing for a mean of % of the procedures listed, a pre-operative type and screen for %, crossmatching rbc units for %, and for % of procedures a different recommendation was made. most ( / , %) of the msbos' were created by a combination of obtaining consensus between the surgical services and blood bank and use of procedure-specific transfusion data; only / ( %) of msbos' were created solely by using procedure-specific data, and most ( / , %) do not use patient-specific data in making a testing recommendation. most msbos' are updated less frequently than annually ( / , %), and the hospital transfusion committee is often ( / , %) involved in updating it. the msbos' are generally available electronically in both the operating rooms and in the blood banks. it was the opinion of the majority of respondents ( %) that the msbos was used regularly by only a limited number of surgeons and anesthesiologists, % of respondents felt that it was regularly used by all surgeons and anesthesiologists; % felt that it was not used at all at their hospital, % did not respond. conclusion: an msbos was available in only about half of the respondent's hospitals and in only the minority of cases was it felt to be regularly used. however, % of the hospitals currently without one indicated that it would be implemented in suggesting that these hospitals perceive the value of having one in place. implementing and following an msbos can be an important step in peri-operative patient blood management and in streamlining the operations of the blood bank vis-a-vis pre-operative testing. blood management -one hospital system experience leana serrano rahman*, mallika gupta, susan solometo, ronald walsh and joan uehlinger. montefiore medical center background/case studies: our system, a pioneer aco, is a -bed tertiary-care referral center dedicated to serving patients from across the new york city area and beyond. the comprising four hospitals see , hospital admissions and nearly , emergency department visits annually. we have active programs in high risk ob, stem cell transplant, solid organ transplant (heart, liver, and kidney), ct surgery, ecmo, oncology and critical care. transfusion medicine plays a key role in the support of these services. blood product spending in was approximately $ . m. in nov. , an interdisciplinary committee was created in an effort to improve patient care (by reducing blood product exposure) and reduce blood product expenditures. the vice president-sponsored multidisciplinary committee was composed of representatives of: surgery, anesthesia, blood bank, pediatrics, perfusion, cardiothoracic surgery, critical care, medicine, and emergency department. study design/method: first important step: "know your numbers"-although the committee had multiple sources of data, there was no "one report" that could display all of the pertinent information. baseline numbers were imperative to the committee's ability to effect change. a home grown one time only report revealed which services and clinicians were the highest volume users. the initial plan was to target their use with education. an initial goal was set to reduce expenditure by $ . m. the journey continued with regular bimonthly meetingsto brainstorm strategies and monitor utilization. utilization was analyzed using a home grown crystal report "transfused patients by location". this report was further compared to utilization patterns ( and ), by "dollars spent" and "total units per patient" by the project manager using excel. key initiatives developed by the committee . development of evidence based transfusion triggers. . education on evidence based transfusion triggers across multiple campuses, specialties and resident programs . clinical information system (cis) "soft stops" when ordering blood products outside guidelines. rbc order set defaulting to " " unit instead of " " units. . updated guidelines posted to easy to find internal intranet spots results/finding: despite higher patient volumes and a more complicated patient mix in , we were still able to reduced blood product expenditures by $ , when compared to . conclusion: in spite of limited resources, the committee was able to effect change by capitalizing on current stakeholders fully supported by leadership and project management. cord blood pathway to reduce iatrogenic blood loss in neonatal intensive care patients tracy shachner* , anna w rains and christopher t clark . university of tennessee graduate school of medicine, univeristy of tennessee medical center, univeristy of tennessee graduate school of medicine background/case studies: anemia due to iatrogenic blood loss in preterm and low birth weight infants is a major contributory factor leading to red blood cell transfusion in this patient population. methods to reduce phlebotomy for laboratory testing can reduce iatrogenic anemia. at a universitybased teaching hospital, a pathway to collect cord blood samples on all newborn deliveries was established. the cord blood sample is used for initial blood bank laboratory testing on newborn patients transferred to the neonatal intensive care unit (nicu), preventing need for additional blood draw. the blood tubes are saved for week post-delivery, with cost of $ . per delivery tray for sterile tubes. with an initial negative antibody screen on cord blood sample, no additional phlebotomy is required for blood product selection or compatibility testing in this population until four months of age. study design/method: labor and delivery data from our facility in was analyzed, and the gestational age and birth weight of all infants transferred to the nicu was collected. from this data, we were able to calculate the total blood volume of these infants using medcalc system. by using the blood volume values, and assigning a value of . ml as the minimum amount of blood that would be drawn to perform an antibody screen, we calculated the percent of an infant's blood that would have to be drawn if the cord blood pathway was not established. transfusion results/finding: in , there was a total of , infants delivered at our facility. out of all the deliveries, ( %) infants were transferred to the nicu. of those infants, % received at least one red blood cell transfusion and % received at least one platelet transfusion. of the infants transferred to the nicu, ( %) had a percentage of blood volume that would have had to be drawn for blood bank testing greater than or equal to % (which we considered to be significant), had the cord blood pathway not been in effect. the percentage of blood volume preserved in these infants ranged from . % all the way up to . %. in those infants, the birth weight ranged from - grams, and the gestational age ranged from weeks to weeks and days. conclusion: the established cord blood pathway has proven to be a relatively cost-effective method to prevent iatrogenic blood loss secondary to blood bank testing in a population of nicu infants who are most susceptible to iatrogenic anemia. the infants that were most likely to benefit from this policy are premature infants who are low birth weight (less than grams). development of a standardized response team for massive hemorrhage events outside of an operating room setting james burner* , shannon davis , suzan new , vaishali patel and oren guttman . university of texas southwestern medical center, ut southwestern medical center background/case studies: managing a massive transfusion protocol (mtp) in an operating room (or) is a relatively frequent occurrence with team members well trained in their specific roles. however, in the event of mtp activation outside of an or, sufficient and/or appropriately trained individuals may not be present. this can lead to a scene of confusion and chaos with potential for patient harm. study design/method: a failure mode effects analysis was performed to develop a standardized process for managing mtp outside of an or setting. with participation from anesthesia, surgery, transfusion medicine, patient safety and quality and nursing, every step of the hospital's mtp was analyzed for potential errors. the results were used to create a "code hemorrhage" team trained to respond to any massively hemorrhaging non-or patient. results/finding: code hemorrhage represents a multi-system team critical event requiring coordination of different sub-teams (primary resuscitation, surgical/interventional, transfusion services, blood preparation, equipment management, medication management, and lab requisition/monitoring). our code hemorrhage protocol utilizes critical care trained nurses from the hospital's rapid response team who play two key new coordination roles: hemorrhage coordinator and electronic medical record (emr) coordinator. their combined roles serve to reduce the cognitive load of the various teams, prevent duplication of resources/efforts during mtp and enable enhanced closed loop task performance. the hemorrhage coordinator establishes reliable : communication between the primary resuscitation team and transfusion services, and aids in multi-team on-site coordination. the emr coordinator enters all orders into the emr, sends/communicates laboratory results and ensures blood products are available to the resuscitation team. the primary resuscitation team includes a team leader (medical decision making and cardiac life-support management); a proceduralist (establishing venous and/or arterial access), event documenter (real-time documentation of actions, medications, events, etc.), medication manager (registered nurse who prepares and administers medications) and equipment technologist (managing rapid blood product infusion devices). additional secondary roles will also be assigned, such as blood product checker(s) (verifies blood product prior to transfusion) and blood bank runner (courier sent to retrieves blood product shipments). conclusion: the code hemorrhage protocol is designed to ensure timely, efficient delivery of blood products to massively bleeding patients outside of an or setting. future work will assess its overall effectiveness by comparing blood product utilization/wastage and patient outcomes before and after implementation. background/case studies: preoperative anemia affects up to % of surgical patients and increases the risk of red blood cell (rbc) transfusion. both preoperative anemia and perioperative rbc transfusion are associated with increased risk of adverse outcomes following surgery. preoperative treatment of anemia includes oral and intravenous (i.v.) iron and erythroid stimulating agents (esa) such as erythropoietin (epo); however, the optimal treatment strategy for preoperative anemia remains to be established. our objectives were to evaluate the efficacy and safety of esa and iron therapy based on their effects on the prevalence of rbc transfusions and adverse thrombotic events. study design/method: we searched the cochrane central register of controlled trials, medline and embase from inception to july ; reference lists of published guidelines, reviews and associated papers, as well as conference proceedings. no language restrictions were applied. we included randomized controlled trials in which adult patients undergoing surgery received either an esa and/or iron before surgery, versus iron or no intervention. three authors independently reviewed the studies and extracted data from included trials. risk of bias was assessed for all included studies. where applicable, we pooled risk ratios of dichotomous outcomes and mean differences of continuous outcomes across trials using randomeffects models. our primary outcome was the number of patients transfused with red blood cells. secondary outcomes included risk of mortality and other thrombovascular events (stroke, myocardial infarction, deep vein thrombosis, and pulmonary embolism). results/finding: a total of randomized controlled trials ( , conclusion: amongst patients undergoing surgery, the administration of an esa in addition to oral or i.v. iron was associated with a reduction in patients requiring rbc transfusion. intravenous iron was less effective at reducing rbc transfusion. neither treatment was associated with any clear increase in risk of adverse thrombotic events. additional large prospective randomized controlled trials are required to determine the optimal management strategy for patients undergoing surgery with iron restricted anemia. evidence based blood therapeutics scott neeley* and stephanie rogers . dignity health st joseph's medical center, dignity health background/case studies: over million units of packed red blood cells (prbc) are transfused annually in the united states and there is no clinical basis for as many as half of these transfusions. no randomized prospective trial has ever demonstrated a clinical benefit for transfusion in mild to moderately anemic patients and yet there is a large body of evidence which has shown that due to a variety of reasons including an immunomodulatory effect and the storage lesion, blood transfusions can cause considerable harm, including higher risk of hospital acquired bacterial infections, transfusion related acute lung injury/acute pulmonary edema, acute myocardial infarction, higher recurrence of rebleeding and higher cancer recurrence. study design/methods: a system wide goal was launched across hospitals to decrease the number of prbc transfusions given to clinically stable patients with hemoglobin (hgb) levels > . g/dl. the numerator consisted of all prbc units transfused to patients with a hgb of . g/dl or greater prior to transfusion and the denominator consisted of all prbc units transfused. exclusions included cardiac surgery, nursery, nicu, pregnancy, post-partum hemorrhage, massive transfusion protocol and transfusions in which or more prbc units were transfused in one episode. data was extracted directly from the electronic medical record and hospitals received patient level detail every month for all prbc units transfused to patients with a hgb of . g/dl or higher prior to transfusion. an extensive educational campaign re: evidence-based transfusion practice was launched for physicians and nurses, including the development of a blood therapeutics toolkit, development of standardized dignity health blood therapeutics guidelines, a one day blood therapeutics advanced training symposium, on-site visits to hospitals including cme presentations, online physician and nursing educational videos, communication tools including infographics and " is the new " buttons, development of a patient education resource and bi-monthly webinars with various educational topics and speakers. additionally, the ehr powerplans were revised to ensure available selections for "transfusion indication" (required field) were aligned with evidence based guidelines. facilties were encouraged to develop multi-disciplinary blood therapeutics committees to review all transfusions given to patients with pre-transfusion hgb > . g/dl on a routine basis, providing feedback to providers whose transfusions were deemed not in accordance with current evidence-based guidelines. results/findings: from fy to fytd , there was a % reduction in prbc units transfused to patients with hgb > . g/dl, starting at a baseline of % down to %. this represents an fy annualized savings of $ . m, from a baseline of units per , patients days down to an average units and approximately , fewer units transfused per month. conclusion: blood transfusions, while life saving, should be regarded as an organ transplant and as such they carry considerable risk. transfusions to stable, non-bleeding patients with hgb levels > . g/dl are not in accordance with evidence-based guidelines and should be avoided due to the associated potential harm. furthermore, this potential harm is dose dependent, so if the decision to transfuse is made, one unit of prbc should be transfused rather than two. three af studies (sdm - . ) reduced rbc units and two studies decreased the percentage of patients transfused (or . ). forty-three studies showed that intravenous tranexamic acid reduced the percentage of patients (or . ) and rbc units transfused (sdm - . ). qualitative/meta-analyses were translated into recommendations by an expert panel and approved by the lmbp workgroup for reducing rbc transfusion. recommendations are: early assessment and effective am; rt, hb alerts in cpoe/cds; reduction of blood loss and af assessing the percentage of patients and rbc units transfused across cases, physicians and service areas over discrete periods of time with feedback to physicians for continuous quality improvement. conclusion: conclusion: the lmbp a- method led to evidence-based recommendations for reducing transfusion. critical laboratory support is needed to achieve continuous quality and patient safety. background/case studies: reducing the inappropriate use of blood products via the implementation of evidence based guidelines is a main tenet of patient blood management. the use of electronic decision support tools such as best practice alerts (bpas) to enforce red blood cell (rbc) transfusion thresholds have been shown to reduce use by informing ordering providers when or when not to transfuse. the tools in use to date have not provided a dose of rbcs to transfuse, so in fact providers can continue to over-transfusion based on the number of units of rbc given. a therapeutic hemoglobin/hematocrit (hgb/hct) targeted approach to rbc indications/ orders allows for the calculation of a dose of rbcs to achieve the desired target and could further reduce the use of rbc units. our group has developed a computer algorithm to calculate rbc dose based on patient specific data drawn from the electronic medical record (emr) that has been used in select patient populations but has not been prospectively applied to hospital wide clinical practice. this study describes our initial experience with the use of this algorithm in non-surgical rbc transfusion. study design/method: the blood utilization calculator (buc) is a mathematical formula that draws patient specific information including index hgb/ hct and calculates a dose in number of units of rbcs to transfuse in order to achieve a selected target hgb/hct. hgb/hct target based indications for rbc transfusion were designed and used as the basis for rbc order set with in the ethe buc was embedded within the emrs rbc order set to provide a recommended transfusion dose in number of units when any nonsurgical rbc indication was selected. the target hgb/hct for these indications was g/dl/ % or g/dl/ %. the number of rbc units ordered and transfused were tracked prospectively for each of the orderable indications. comparison of units transfused per month before and after the buc implementation was performed using student's t-test. results/finding: historically, the three non-surgical rbc indications represented approximately % of the total rbc transfused. prior to the buc the mean number of non-surgical rbc units transfused was units/ month. after the first months of buc activation the mean number of units was units/month a reduction of units/month or % of nonsurgical blood use (p . by t-test). non-surgical rbc use now represents approximately % of the total rbc use hospital wide a % reduction. this change represents a significant cost savings in rbcs over time. conclusion: the use of target based transfusion indications and an electronic decision support algorithm to calculate a recommended transfusion dose can significantly reduce the non-surgical rbc transfusion rate providing enhanced patient blood management and potential cost savings. implementation of patient blood management at a community hospital - month report card richard gammon*. oneblood, inc. background/case studies: a collaboration between blood center between (bc) as consultant and three hospital ( beds) healthcare system (hcs) to implement a patient blood management (pbm) program was undertaken. this is a review of the first months. study design/method: during year one pbm working group was established. achievements included physician engagement programs, creation of transfusion committee and providing nursing education. auditing processes were implemented with nonconformance letters sent to physicians and nurses when compliance with informed consent, transfusion tags and thresholds and discharge instructions was not achieved. in year two, it created best practice alerts (bpa) when an order did not meet transfusion threshold criteria. bpa showed first line of associated procedure, link to the full procedure, three most recent lab results (e.g., hemoglobin & hematocrit for red blood cells (rbc)) and allowed ordering physician to cancel order after review. a blood administration video was created. it was mandatory that all physicians granted privileges complete within six months. low vital sign compliance required action that included reducing requirement from five to three during transfusion and formation of working group (wg) to address knowledge and practice gaps. in year three, as historically at this hcs very few jehovah's witness patients (jwp) presented, pbm wg was involved with implementation of a bloodless medicine program. all steps of care were addressed including identifying jwp at registration, creating a transfusion special arm bands, forming a bloodless medicine physician group, implementing nursing bpa in the electronic medical record, creating advanced directives and marketing to the public. results/finding: the following were monitored for compliance ( q vs. q ): present and completed consents ( vs. %), present and completed nursing flow sheets ( vs. %), transfusion thresholds supported ( vs. %), discharge instructions provided ( vs. %); ( q vs. q ) vital sign compliance ( % vs. %). jwp increased from to ( / - / ). cost savings were realized by decreased utilization and implementation of bpa. (table - q ) conclusion: pbm implementation at a hcs is a continuous and multiyear process. even with a robust program challenges such as vital sign compliance remain. improving patient outcomes in the golden-hour beatrice lebeuf*. medical city plano background/case studies: in emergency medicine, "the golden hour" refers to the critical one-hour time period following traumatic injury in which the patient has a higher likelihood of survival. nearly half of all trauma related deaths occur in the first hour after injury -half of those deaths are the result of major hemorrhaging. rapid administration of blood products is vital to the survival of these patients. we implemented bloodtrack emerge (haemonetics, braintree, ma) in our trauma emergency department (ed) as part of a quality improvement initiative to more efficiently provide group o rbcs and thawed/liquid plasma for incoming trauma patients to support ratio-based transfusions and ensure the proper handling and traceability of this regulated resource. study design/methods: we treat approximately - trauma patients monthly. an assessment of our current blood supply chain revealed a multistep, manual process that took about minutes to prepare and physically transport a cooler from the blood bank to the ed. coolers of blood were provided for incoming trauma patients, whether they ended up needing transfusions or not. this practice worked to ensure available blood supplies during critical moments, but resulted in inefficiencies and unnecessary inventory tie-ups, with only percent of coolers fully used. it also consumed valuable staff time as technologists typically made - trips per month from the blood bank to the ed. plus, there was no effective way to maintain traceability, control access to coolers or monitor usage. results/findings: since our november implementation, bloodtrack emerge has freed up technologists to perform important tasks, tightened traceability and inventory control procedures and contributed to the medical city plano's verification as a level trauma center. rather than preparing coolers of blood in case they may be needed in emergency situations, bloodtrack emerge provides ed staff ready access to emergency units whenever they're actually needed -and frees up an estimated - hours of tech time per month during which they can perform other tasks. audio and visual alerts notify the blood bank when emergency units are removed, allowing a quick response. plus, by stocking emergency blood supplies in the ed, the blood bank isn't unnecessarily tying up group o rbc units. today, the blood bank stocks and maintains - units of group o rhd negative, units of o rhd positive, and units of group a thawed plasma/ liquid plasma in bloodtrack emerge. conclusion: implementing bloodtrack emerge has enabled us to more effectively provide blood products for incoming trauma patients to support ratio-based transfusions, improve staff efficiencies and proactively respond to emergency situations. background/case studies: platelets are a limited resource for which the benefits of transfusion must be weighed against the risks. in , the aabb published platelet transfusion guidelines to assist providers. at our academic medical center, a computer provider order entry (cpoe) system combines institutional transfusion guidelines with a patient's most recent lab results to guide transfusion decisions. discordant information activates an "override" system, in which providers are prompted to select a prefixed indication for transfusion (e.g. count < k/ml [prophylaxis]) with the option to add a free-text comment. the order is placed and data is stored for later review. study design/method: override platelet orders placed from june -october were reviewed using the following data: prefixed indication, most recent platelet count, free-text comment, and ordering service/department. one of five "codes" was assigned to each order: i-indicated or ni-not indicated (based on institutional/aabb guidelines); nmi-need more information; p-protocol (e.g. liver transplant), and nic-non-indication comments (e.g. reserve for or). free-text comments were categorized and assigned one or more keywords in order to determine the common reasons for overrides. results/finding: over a -month period, , cpoe override platelet orders occurred. the percentages of code assignments by month are provided in table below. overall, ( %) were assigned as not indicated (ni). the top keywords assigned to free-text comments were "platelet count less than. . ." ( ), "active bleeding" ( ), "platelet count of . . ." ( ), and "downtrend" ( ), many with specified platelet count goals. certain platelet count goals and reasons for transfusion (e.g. "downtrend," "anticipate drop," or "per service,") are not included in institutional or aabb guidelines. of note, ( %) of overrides were placed by hematology-oncology providers. conclusion: a majority of override platelet orders were determined to not be indicated based on institutional and aabb guidelines. of concern were keywords such as "downtrend" and "anticipate drop," as these are not indications for transfusion and expose patients to unnecessary transfusions. it is unclear whether trainee progression throughout the year had any effects on ordering practices and associated override patterns. this review suggests the potential benefits of provider education initiatives at all levels of experience (with particular emphasis on hematology-oncology) in order to improve blood product utilization practices. background/case studies: early diagnosis of iron deficiency anemia (ida) by clinical laboratories (cl), with effective prevention and treatment in primary care may have an impact on packed red blood cell (prbc) transfusion, as well as intravenous iron therapy and, most importantly, applying lower transfusion triggers. they all help to avoid not essential transfusions, but also promote health and wellbeing by improving iron status in the population. results are described after implementing a process to prevent ida, its early detection and treatment for years - . study design/methods: performance measure after educational and organizational intervention. setting: public integrated healthcare system located in north africa bordering morocco, isolated by km sea distance to nearest continental spain airport, with a general hospital blood transfusion service and a establishment for blood donation and component production. cl involved in anemia detection and diagnosis receives four primary care centers and hospital based samples, and shares common leadership with both blood establishments. process: guidelines for first step cl diagnosis of ida and call for attention, primary oral iron prevention and treatment in first level care, and early intravenous iron complex for inpatients (sucrose) and outpatients (carboxymaltose). transfusion was avoided for stable ida patients without active bleeding or coronary heart disease, with a safety hemoglobin (hb) threshold of , g/dl. severely anemic patients were closely followed to asses hb increase and referred for etiology studies when hb> g/dl. background/case studies: bedside nurses are critical in safeguarding the delivery of appropriate patient care. more recently, nurses have also begun to play an important role in patient blood management (pbm) programs at the administrative level, although to our knowledge little has been published on the influence nurses may have on transfusion practice at the bedside. the goal of this study was to evaluate the impact nurses have on patient expectations and physician ordering practice. study design/method: a short electronic survey ( questions) was prepared to assess how often bedside nurses discussed transfusion necessity and the persons (patient or physician) with whom they discussed it with, as well as what was discussed, and what they felt were appropriate lab thresholds for transfusion. the survey was distributed to all registered nurses via email from floor leaders. responses were also solicited by hospital volunteers and lab staff with electronic tablets and included coverage of the night shift. results/finding: there were a total of complete responses ( %). the nurses had a range of experience from less than one year to forty years. ninety percent stated they discussed transfusion necessity with patients, % with physicians, and of these, % reported doing so proactively before an order was placed. ninety-six percent said they would discuss transfusion to suggest their patient required a blood product; only % responded that they would suggest product was not needed. nursing perception of acceptable transfusion thresholds had a wider distribution, with the most commonly reported values being hemoglobin of - g/dl ( %), platelet count of - , ( %), and inr of greater than . ( %). conclusion: this study demonstrates that nurses are willing to discuss transfusions with both patients and providers, although they appear to be most comfortable doing so in the setting of perceived transfusion necessity. the limited number of survey responses suggests a discomfort with their level of education in transfusion practice. this, along with the distribution of perceived thresholds and the reluctance to recommend against transfusions, presents an opportunity for education to further empower nurses in providing appropriate patient care within the guidelines of pbm programs. background/case studies: the use of red blood cell per , inhabitants may vary folds between european countries, revealing that there may be substantial room for blood optimization strategies. patient blood management (pbm) is an evidence-based, multidisciplinary approach aiming to preserve and optimise patients' own blood in order to improve clinical outcomes. the objective of our study was to assess the effect of a nationwide pbm program on public health in portugal. study design/method: the first phase of this research project involved a group of key opinion leaders (kol) in a stated preference inquiry to assess the relative value of specific pbm strategies, grouped in pbm pillars, to highlight the need for strategy prioritization in the implementation of a nationwide pbm policy. adaptive conjoint analysis techniques were used to elicit kol preferences. in the second phase a decision analysis model was used to estimate the impact of pbm implementation in the following therapeutic areas: surgery (orthopaedic, cardiac and urologic), cardiology, oncology, gastrointestinal bleeding, abnormal uterine bleeding, haemodialysis, inflammatory bowel disease and pregnancy. model inputs included effectiveness data regarding transfusion utilization, health resource consumption and mortality obtained from portuguese national health databases and literature review. the public health value of pbm implementation in portugal derives from the comparison of two scenarios: "current clinical practice" and "with pbm implementation". results/finding: kol elicited iron administration followed by restrictive transfusion of red blood cell as the most preferred pbm strategies ( . % and . %), for the remaining strategies weights varied between . % and . %. we estimate that , patients would be eligible for pbm strategies in one year time horizon, resulting in premature death avoided ( . % reduction) corresponding to a gain of approximately , life years and a reduction of , ( . %) disability adjusted life years (daly) relative to the current clinical practice. a decrease of , in-hospital days is expected mainly due to a . % reduction in hospital length of stay and a . % reduction in -day readmission rate. in this population the overall transfusion rate could decrease to . % from the current . % ( . % reduction) implying , blood transfusion avoided and , red blood cells units spared. conclusion: we anticipate that the implementation of a nationwide patient blood management program will represent a paramount improvement in clinical outcomes in terms of morbidity and mortality and may have a substantial public health impact while contributing a more efficient use health resources. results/finding: adult liver transplants were performed during the evaluation period. preoperative hemoglobin, creatinine, meld score, spontaneous bacterial peritonitis (sbp), preoperative hemodialysis, gender, and portal vein thrombosis (pvt) gave the strongest model predicting rbc usage. if the model predicted < ml of rbcs, all cases with ml transfused were captured and only . % of the time > ml were used. if - ml rbcs were predicted to be transfused, > ml were used % of the time. if predicted usage was > ml, % of the time it exceeded ml. conclusion: a model using specific preoperative factors can be used to predict intraoperative rbc usage. patients at risk for > ml of rbc transfusion can be identified with reasonable accuracy using this model at our institution. use of this model might help improve preparation and utilization of the blood bank. review of blood ordering practice for elective surgeries in a maternity hospital qi raymond fu*. kk women's and children's hospital background/case studies: pre-operative over-ordering of blood is common, resulting in waste of blood bank resources. blood units are withdrawn from the pool, leading to constraints in allocating the limited blood resources to meet the needs of other patients. the cross-match to transfusion (ct) ratio is often used in benchmarking efficient blood utilization within the hospital blood transfusion service. according to the american association of blood banks (aabb), a ct ratio of less than . is favorable, and anything above indicates over-ordering and cross-matching of blood. to achieve this, it is necessary to review pre-surgical blood ordering practice in a maternity hospital. study design/methods: data on elective surgeries requiring blood for standby was collected retrospectively over a month period (jan to mar ). details of total blood cross-matched, issued, transfused and returned were analyzed along with the ct ratio. results/findings: during the month period, there were patients undergoing obstetrics and gynecology procedures requiring blood on standby. a total of units of blood were requested. units were crossmatched, of which units were sent to the operating theatre (ot). only . % of blood issued to ot were transfused (n ) while the rest were unutilized. the observed ct ratio was . . conclusion: although only % of total blood requested was crossmatched, the ct ratio remains above the recommended guideline of ! . , with almost % of cross-matched blood unutilized. there is a need to improve and standardize the blood ordering practice to achieve costeffectiveness and reduce unnecessary workload. establishing and adhering to a maximum surgical blood order schedule (msbos) could help in conserving blood and prevent over-ordering of blood. background/case studies: total knee arthroplasty (tka) is a major orthopaedic procedure with increased perioperative blood loss. this perioperative blood loss could be more significant in patients undergoing bilateral tka in a single stage. the increased blood loss in bilateral tka often requires blood transfusion which results in high post-operative morbidities. study design/methods: in this retrospective study patients who received tranexamic acid (txa) (study group) and patients who did not receive txa during surgery (control) were evaluated for blood loss and transfusion requirement. the study group received a single bolus dose of txa gm iv before tourniquet deflation on first side knee. statistical background/case studies: blood product utilization is an increasing concern for hospital systems attempting to reduce transfusion-associated risks. one strategy to optimize utilization is to employ clinical decision support in the form of alerts to clinicians ordering blood products. we investigated whether an alert targeted to a patient's transfusion indication could alter provider ordering behavior. study design/method: this retrospective, observational study over the course of seven months included the inpatient adult medicine floors and intensive care units at a large academic hospital. each time a crossmatch for packed red blood cells (prbcs) was ordered via the hospital's electronic ordering system, an indication (e.g. "hemodynamically stable with hemoglobin < . g/dl") must be selected. if the indication selected contains a threshold hemoglobin concentration, and the patient's most recent hemoglobin on record was greater than this threshold, an interruptive alert displaying the patient's hemoglobin was activated. ordering providers were then given three options: cancel the order, select a more appropriate indication from a list, or provide an explanation via free text as to why transfusion was being requested outside of approved indications. an alert encounter was defined as all activations on a patient within a six hour period without an intervening transfusion results/finding: over seven months, there were unique alert encounters. of these, ( . %) led to a crossmatch being ordered while ( . %) led to the order being canceled. providers were more likely to cancel transfusions in response to alerts for hemodynamically stable patients with lower hemoglobin thresholds ( . g/dl) than for more complicated patients (bleeding, cardiovascular disease, or preoperative) with higher hemoglobin thresholds ( . or . g/dl background/case studies: the maximum surgical blood ordering schedule (msbos) is a list of surgical procedures performed along with a recommendation for the extent of pre-transfusion testing to be completed before the surgery begins. with improved patient data management systems it is now possible to create an msbos based on actual red blood cell (rbc) utilization data on a per-patient basis. this study investigated the transfusion patterns at academic hospitals with data-derived msbos. study design/method: the hospitals were in groups, with one shared msbos for each group. three of these hospitals were large academic centers while one was a children's hospital. at each center the msbos recommended no pre-transfusion testing if % of patients had been transfused for a specific procedure in the previous year, a pre-operative type and screen (t&s) if - % of the patients had been transfused, and a crossmatch of the median number of rbcs transfused if ! % of the patients had been transfused. data were collected at each center over a month period between january to march and included a maximum of cases per hospital during that one month to ensure equal representation between centers results/finding: between these centers there were a total of cases analyzed. some of the more frequently performed surgeries included orthopedics ( % of cases), general surgery ( %) and cardiac surgery ( %). there were t&s ordered for these cases, of which were positive for antibodies on the day of surgery. of all the t&s ordered, % were ordered in accord with the msbos recommendation, % were ordered when the msbos did not recommend one, and in . % a t&s was not ordered when the msbos recommended one. background/case studies: peripartum blood transfusion is more common in south africa than in the usa and recent studies have demonstrated that antenatal anemia is a strong risk factor for such transfusion (odds ratio . for prenatal hemoglobin (hgb) - . ). we therefore analyzed the etiology and characteristics of antenatal anemia according to hiv status at a large hospital with a hiv prevalence of % among obstetric patients. study design/method: we studied a sample of anemic (hgb< . g/dl) pregnant women who were referred to an antenatal anemia clinic at a large hospital in south africa. clinical information was abstracted and blood was sent for laboratory studies. t-tests were used to compare continuous variables between groups. results/findings: a total of women were enrolled, with median age (interquartile range - ) years, median gravida / para and median gestational age weeks. mean hgb before referral was . g/dl and most were already taking oral iron therapy. a total of women were hiv positive with mean cd lymphocytes counts of cells/ul; ( %) of hiv positive subjects were on anti-retroviral therapy (art) prior to the pregnancy and ( %) were on art during the current pregnancy. iron deficiency anemia was the overwhelmingly prevalent diagnosis, present in ( %) of women. there was concurrent chronic disease (n ), infection (n ), vitamin b deficiency (n ) and antenatal hemorrhage (n ); had other/unknown/missing causes of anemia. there were few pregnancy related complications. hiv positive women had higher levels of c-reactive protein but slightly lower levels of transferrin, soluble transferrin receptor and rbc folate than hiv negative women (table) . conclusion: iron deficiency is the overwhelming cause of antenatal anemia among south african pregnant women. compared to hiv-negative women, hiv-positive women had evidence of increased inflammation, relatively little differences in iron studies after early treatment with iron and lower red cell folate. a high proportion of hiv positive women were receiving art, consistent with national guidelines. future studies will examine longer-term responses to iron therapy to assess its potential in decreasing the incidence of peripartum blood transfusion. background/case studies: a month old boy presented to our institution after a month hospitalization in japan. he was admitted there, several weeks after his unremarkable term birth to an ab rh positive woman, with lethargy, failure to thrive, bloody mucoid stools with eosinophilia, and an elevated serum white count. he was found to be anemic and thrombocytopenic and required multiple transfusions. also, he had a diffuse, scaling, erythematous rash over his inner thighs. study design/method: initial workup was suspicous for an allergic/necrotizing enterocolitis. the patient had an elevated ldh and potassium, and concern was raised for leukemia with possible tumor lysis syndrome. a sample sent to our blood bank showed an anti-e, with a positive dat (igg and complement), and was positive for e, e, and c antigens. concern for a maternally-induced antibody was raised, as was the possibility of a red cell antigen passively transfused from blood products administered at the japanese hospital; both possibilities were excluded. further workup revealed no infection or hematologic proliferation. biopsy of his rash showed spongiotic dermatitis. his clinical course deteriorated, and he developed hepatomegaly and jaundice. a concern for wiskott-aldrich syndrome was raised, and workup showed normal immunoglobulin levels, but with elevated ige ( ku/l; rr: - . ). anti-platelet antibodies were identified. three days after admission, testing was sent for genetic alterations of foxp , while a japanese-speaking physician at our institution read a prior flow cytometry study showing a deficiency of foxp cd lymphocytes. the majority of these indications are seen in adults and for which a reported plasma wastage is $ . %. fortunately in pediatrics the incidence of these indications is low despite the heterogeneity of the patient population. during the utilization review process at our primary pediatric institution, we noted a mean wastage of . % over the last years. with recent changes in clinical practice (liver transplants and increased trauma) and recent evidence that faster plasma improves massive transfusion protocol (mtp) outcomes, our facility decided to implement the use of thawed plasma and benchmark mtp plasma wastage. study design/method: blood utilization review revealed an increase in the overall percentage of plasma wastage from to , with a peak of . % (range . %- . %). a single cause could not be readily identified prompting us to query children's hospital association (cha), as our initial external pediatric benchmarking, to determine if our wastage was comparable to other children's hospitals in addition to reviewing our "time of plasma availability" for mtps. results/finding: in , mtp was activated times. in cases the patient did not receive any blood product and in cases plasma was already available at the time of rbc allocation/issue. this left cases to evaluate. the median time to plasma availability was minutes (range minutes - minutes). the mean plasma wastage for mtp activations was % (range - %). of the cha replies, were using thawed plasma and their wastage was </ %. conclusion: increasing clinical evidence supports the prompt transfusion of plasma in the setting of mtp to decrease morbidity and mortality. our brief review suggests that implementing thawed plasma will allow a potential decrease in plasma availability of minutes. our implementation began with thawing a single unit of plasma for "level neuro trauma" alerts, and we are now keeping a single unit of ab plasma thawed at all times. plasma is now available at the same time as rbcs thus providing more effective and balanced blood product support while reducing wastage (preliminary estimates are . %) to a level consistent with external benchmarking. prospective evaluation of this practice is ongoing at our institution and includes collaborative clinical assessment with the trauma team. ultimately prospective, multi-institutional studies in pediatric institutions are necessary to define best clinical practice and effectively benchmark blood bank practice. background/case studies: bacterial and fungal infections remain a significant cause of morbidity and mortality in severely neutropenic patients with hematological disease receiving high-dose chemotherapy and hematopoietic stem cell transplantation (hsct). granulocyte transfusions (gtx) have been used for over years, although effectiveness, indications, and both patient and donor safety remain debated, particularly in children. to contribute to this discussion we demonstrated cases of gtx in pediatric patients with age from months to years old, neutropenic, with severe infection resistant to antibiotics and/or antifungal therapy, undergoing hsct and chemotherapy. study design/method: donors: data concerning a total of granulocytes donations from to were collected from health donors ( male/ female). donors had matched blood type and serology status for cytomegalovirus when the patient was negative. granulocytes were mobilized with a single dose of rhu-g-csf (filgrastim) subcutaneously, mcg/kg/dose (donors up to kg, maximum of mcg) and oral dexamethasone ( mg), h prior to apheresis. apheresis was performed using cobe spectra v r or spectra optia v r with citrate as anticoagulant. all products were irradiated with gy. results/finding s: granulocytes number increased times from basal levels and the median of cells collected were . x (range . - . ). in general donors reported no significant adverse reactions. from donations, only in was reported light paresthesia during the procedure. patients: all patients were medicated prior to transfusions with diphenhydramine and acetaminophen. the product was transfused within hours after collection, with efforts to transfuse as soon as possible. details about the transfusions are described on table . adverse reactions for patients were minor and not frequent ( in gtx) including vomiting, hypotension and headache, which resolved without serious or lasting complications, assuming that gtx is well tolerated. conclusion: our study provides further evidence that gtx transfusions can be safely performed on pediatric patients. irina kumukova* , elena kurnikova and pavel trakhtman . russian institute for paediatric haematology, oncology and immunology, instituteffor pediatric hematology, oncology and immunology background/case studies: infections cause major mortality among persons with prolonged neutropenia related to hsct and chemotherapy and among patients with granulocyte disorder. the granulocyte transfusion therapy, a logical approach in treating patients with infections, who are refractory to antibiotics. we want to demonstrate our granulocyte transfusions experience in children who have neutropenia or defects of the immune system, with severe infections study design/method: we analyzed the retrospective data for the period / - / . donors underwent granulocyte mobilization with g-csf in a dose - mg/kg s.c. - hrs prior to donation; granulocytes were collected with cobe spectra, caridianbct. the analysis was performed through microsoft excel, nonparametric mann-whitney's and spearman tests results/finding: was performed granulocytes collections in donors ( f, m), ages to . the mean increment of wbc after stimulation was , x /l ( , - , x /l; f- , x /l; m- . x /l); in the age groups - years (n ) and over years (n ), the mean increment of wbc was respectively , x /l ( , - , x /l) and , x /l ( , - x /l). the mean volume of treated blood was , a , ) . the mean number of total wbc in the product was , x /l ( . - x / l; f- , x /l, m- , x /l). only three donors ( , %) had apheresisrelated adverse effects the analysis included granulocyte transfusions in cases of infectious complications in patients. the mean number of transfusions was . ( - ). each patient received transfusions from mean donors ( - ); the granulocyte transfusions started at the mean days ( - ) after infection; the mean dose of wbc on the transfusion was . x /kg ( - . x /kg). wbc increment was able to estimate after transfusions. wbc increment was recorded after transfusions ( . %); the mean increment was , x /l ( , - , x /l). efficacy was determined by the number of patients who survived an infection episode and amounted to . %. in the cases of severe sepsis, the efficacy of transfusions was . %. the frequency of post-transfusion reactions was %, (n ): febrile nonhemolytic reactions , % (n ); pulmonary complications , % (n ); the anti-hla antibodies formation , % (n ) conclusion: no significant differences in the mobilization of granulocytes and products' volume, between men and women, or between age groups and we did not find significant relationship between baseline wbc and their increment after stimulation. the mean total wbc in the collected product and the mean volume of treated blood from men and women were significantly different. perhaps the reason for lower cell products from women is to treat smaller blood volume. the lack of increment wbc after transfusion is not equivalent to lack of its efficacy. we found a significant association between transfused dose and increment of wbc. we deliberately did not estimate our data on the treatment of patients with sepsis and other severe infections between patients receiving and not receiving granulocyte transfusions because the need for transfusions of granulocytes indicates more severe patients, when they have infections in a neutropenia or dysfunction of the immune system, and refractory to antibiotics cp hemolytic disease of the newborn due to anti-rh keegan barry-holson* and jennifer andrews . stanford university, dept of pathology, stanford university, dept of pathology & pediatrics background/case studies: anti-rh is a rare antibody against a high incidence red blood cell (rbc) antigen (ag) present in people with common rh phenotypes. this ag is missing in individuals who are rh null or have rh deletion phenotypes, including d--/d--. d--individuals strongly express d and lack c, c, e, and e ags. the clinical relevance of anti-rh has primarily been reported in pregnant women, causing mild to fatal hemolytic disease of the newborn (hdn). we present a rare case of hdn due to anti-rh alloimmunization during pregnancy in a d--mother. study design/methods: an o-positive full term infant was born to an opositive g p > mother with a negative st trimester antibody screen and no prior transfusions. she had two prior pregnancies, the first resulted in a normal term singleton, and the second resulted in a spontaneous miscarriage during the st trimester. father's blood type is unknown but presumably he has rh antigens. the infant was transferred to our institution at hours of life because he was found to have anemia (hemoglobin . g/dl), severe hyperbilirubinemia (total bilirubin (t bili) . mg/dl), reticulocytosis ( %) and a positive direct antiglobulin test (igg ). he was admitted to our neonatal intensive care unit for potential need for exchange transfusion given concern for hdn. he was treated with intravenous immunoglobulin and triple phototherapy on the day of admission, temporarily blunting his hemolysis. t bili rose to a maximum of . mg/dl on day of life and phototherapy was restarted. his t bili subsequently stabilized and he was discharged home and followed in clinic. meanwhile, his mother donated blood given there were no compatible red blood cells available in the united states via rare donor query. nine days after discharge, he was readmitted for worsening anemia (hemoglobin . g/dl) and was given steroids and washed maternal red blood cells. he was discharged and followed in clinic for several months with ultimate resolution of his anemia and hyperbilirubinemia. results/findings: at delivery, the mother's antibody screen was positive and anti-rh was identified; no other alloantibodies were detected. antibody identification was performed using polyethylene glycol, low ionic strength solution and ficin enhancement. maternal serum was pan reactive against panel cells and non-reactive against d--cells. anti-rh sera did not react against maternal rbcs. phenotyping of the mother revealed that she was d c-e-c-e-. molecular testing confirmed her d--genotype; molecular beadchip test yielded no type due to low signal for e, e, v and vs ags. genotyping for rh variant and targeted genomic rhce testing failed to detect several rhce exons. father was unavailable for further testing. conclusion: we report a rare case of hdn due to anti-rh antibody in a d --mother. we hope to obtain further laboratory studies in maternal relatives given the rarity of this phenotype in the general population. these studies have important implications for genetic counseling for mother's sisters. management of severe autoimmune hemolytic anemia: a case report of an infant treated with manual whole blood exchange with rapid clinical improvement yunchuan delores mo* , cyril jacquot , valli criss , philippe p pary , jay greenberg , naomi lc luban and edward cc wong . children's national medical center, quest diagnostics background/case studies: management of severe autoimmune hemolytic anemia (aiha) presenting with life-threatening anemia is challenging, particularly in the pediatric population. mortality rates in aiha are typically low; however, in children, the rate may be as high as - %. although corticosteroids and immunomodulatory therapies are first line modalities, several case reports describe the use of manual whole blood exchange (wbex) to successfully treat aiha in older children and adults refractory to first line treatment. to our knowledge, this is the first case report in which an infant with severe aiha has been successfully treated with manual wbex in an acute care setting. study design/methods: case report format. results/findings: a month-old previously healthy female patient presented to the emergency department with hemodynamic instability and a nadir hemoglobin (hb)/hematocrit (hct) of . g/dl/ . %. wbc counts ( x /l) were mildly elevated and platelet counts ( x /l) were within normal limits. her history was notable for upper respiratory tract infection days prior to the onset of anemia. laboratory studies on admission showed hyperbilirubinemia (total . mg/dl, direct . mg/dl), normal ldh ( u/l), and undetectable haptoglobin (< mg/dl) indicative of ongoing hemolysis. clinical symptoms included diffuse jaundice, hemoglobinuria, lethargy, and emesis. she was admitted to the pediatric intensive care unit for further management, including right internal jugular central venous catheter placement due to poor peripheral vascular access. the patient's blood group was o, rh (d)-negative with a positive antibody screen and panel demonstrating a strong panagglutinin ( - reactivity) with positive autocontrol. dat was positive for anti-igg and negative for c despite a positive cold antibody screen. the patient weighed . kg with an estimated total blood volume of ml. she initially received simple transfusions totaling ml/kg of least incompatible group o rh(d)-negative rbcs with no incremental response. manual wbex was then performed with ml of reconstituted whole blood consisting of o, rh(d)-negative rbcs and ab fresh frozen plasma (ffp) to an hct of %, utilizing the central venous catheter. no adverse events took place over the course of the hour exchange. her one hour post-exchange hb was . g/ dl and a subsequent antibody screen demonstrated reduced intensity of the panagglutinin ( ). after initiation of steroid therapy (methylprednisolone, mg/kg/day), she continued to improve clinically. one week later, the patient was discharged home with a hb of g/dl. one month later, she experienced recurrent hemolysis requiring re-hospitalization, at which time she had normal igm and iga levels with markedly elevated igg levels ( mg/dl). at a subsequent follow-up visit months after her initial presentation, her anemia had resolved and she had been completely weaned off steroids. conclusion: we demonstrate a case of severe neonatal aiha successfully treated with manual wbex. the main advantages of wbex include removal of both autologous rbcs and plasma as well as infusion of allogeneic rbcs. in this case, manual exchange transfusion avoided the need for an automated apheresis procedure requiring citrate anticoagulation. in summary, manual wbex is a potentially safe procedure that may be performed in young children with severe aiha. abstract operating room, each experienced blood-colored urine, laboratory evidence of hemolysis, and acute kidney injury. clerical and serologic investigations revealed no cause for hemolysis. mechanical hemolysis from transfusion rate, catheter gauge, or a recently introduced one-way valve was considered. study design/methods: in vitro simulated transfusions were performed via syringe. measurements included hematocrit (hct), free hemoglobin, and visual hemolysis index. washed and unwashed red blood cells (rbcs) were tested with or without a one-way valve, using a or gauge (g) intravenous (iv) catheter. each one-way valve was used to test three identical samples. constant pressure was applied manually (rapidly, . /- . ml/ second) or with a mechanical syringe pump (slowly, ml/min). a subset of the manual transfusions was timed. control samples for baseline measurements were collected by gravity drip, without passing through the one-way valve or catheter. results/findings: the one-way valve increased hemolysis markedly during rapid transfusion using both catheters as well as both washed and unwashed rbcs (see table) . with the g catheter, the mean change in hct was - . /- . % with the one-way valve and . /- . % without (p< . ). comparing the one-way valves tested, differences in hemolysis were observed (change in hct; p< . ). during rapid manual transfusion with a g catheter and unwashed rbcs, hemolysis was greater for samples that took longer to transfuse . ml when using a one-way valve (change in hct versus time: r - . , p< . ) compared to a significantly different (p . ) slight increase in hemolysis for samples that took less time to transfuse . ml when not using a one-way valve (change in hct versus time: r . , p . ). correlations between time and hemolysis were similar, but insignificant using g with washed rbcs and the g iv catheter. conclusion: mechanical hemolysis should be considered when investigating possible hemolytic transfusion reactions, especially with high rates of transfusion and use of a one-way valve. during rapid manual transfusion with the one-way valve, greater resistance was associated with increased hemolysis. background/case studies: gerbich (ge) antigens expressed on glycophorin c are present in . % of the population. ge antibodies cause delayed hemolytic transfusion reactions and hemolytic disease of the fetus and newborn (hdfn). ge antibodies also suppress erythropoiesis resulting in late-onset anemia. we report a case of hdfn due to anti-ge . study design/methods: a woman of paraguayan origin with prior terminated pregnancies presented at weeks gestation with passive anti-d and an anti-ge titer of . she was d-and ge:- ,- , by antigen typing. her obstetrician scheduled maternal blood collection near her due date for possible neonatal transfusion, but the woman went into labor at weeks. cord blood was dat positive for igg; the eluate confirmed anti-d and anti-ge . the birth hemoglobin (hgb) was . g/dl, reticulocyte (retic) was . %, bilirubin (bili) was . mg/dl; the infant was discharged. on day of life, the infant was referred to pediatric hematology for lethargy and poor feeding, with hgb . g/dl, retic . %, and bili . mg/dl. ge -blood was not available from the blood center or rare donor registry. the mother was b rh-and baby was b rh . obstetrics had to authorize maternal blood donation due to her hgb of . g/dl. maternal blood collection and rbc washing was expedited and the infant received ml of maternal rbcs within hours, at which time his hgb was . g/dl. post-transfusion hgb was . g/dl. one week later, the infant was symptomatic with hgb . g/dl, retic . %, bili . mg/dl. a nd aliquot of ml washed maternal cells was transfused. two weeks thereafter, the infant had hgb . g/dl, retic . %, anti-ge titer , and needed another transfusion. the maternal blood stored for just weeks had hemolyzed necessitating a nd maternal donation for baby's rd transfusion. at weeks, the infant's anti-ge titer was , hgb . g/dl, retic . %; no transfusion was necessary. at weeks of life, hgb was . g/dl, retic was . %, and the baby was thriving. results/findings: serologic studies at the hospital and reference blood center confirmed the antibodies and risk of anti-ge hdfn. molecular analysis revealed that the mother was homozygous ge -negative ge* .- , the father had homozygous wild type ge* , and the infant was heterozygous ge* /ge* .- . conclusion: the infant had hdfn due to antibodies to the high prevalence ge antigen. the continued need for transfusion was consistent with hemolysis and suppression of erythrocyte production caused by anti-ge . hemolysis of stored maternal blood was consistent with the absence of glycophorin c. this case demonstrates that cooperative multidisciplinary care among the blood bank, donor center, obstetrics, and hematology in a rare case of hdfn resulted in a successful neonatal outcome. background/case studies: patient blood management is a collaborative approach to optimize transfusion therapies to improve patient outcomes. in pediatrics, blood management is not 'one size fits all' given the paucity of clinical trials to guide evidence-based practice. in addition, pediatric care encompasses a very heterogeneous patient population such that applying one set of guidelines is difficult. because there are no standard, evidence-based clinical best practices regarding blood product usage in all children, unnecessary variation is occurring at our institution. we designed a robust analytics process to study baseline clinical practice and examine blood product usage, and plan to target the three pediatric sub-specialties with highest usage to establish standards in order to decrease variation/unnecessary transfusions. study design/methods: a data base encompassing all admissions and outpatient visits to a large, tertiary care academic children's and women's hospital was established, and included all relevant patient demographics, diagnostic and procedural codes, attending physician and specialty for each visit/admission, relevant hematology/coagulation laboratory results and blood product orders. we focused on rbc orders given the tripicu randomized clinical trial results ( ) supporting a hemoglobin trigger of g/dl in stable critically ill children and ffp since anecdotally we noted many children receiving this product for only minimally elevated international normalized ratio (inr) values without bleeding. results/findings: in , , rbc orders occurred and the top three patient groups were: % in congenital heart disease patients, % in hematology/oncology patients and % in neonates in the neonatal intensive care unit (nicu). average hemoglobin of every patient was . g/dl as measured in the hours prior to rbc order placement. in , ffp orders occurred and the top three patient groups were: % in neonates in the nicu, % in congenital heart disease patients and % in pediatric intensive care patients. average inr of every patient was . as measured in the hours prior to ffp order placement. conclusion: we have designed a robust data base that is continually updated for children in a large, tertiary care academic children's hospital. this serves as an important benchmark in pediatric blood utilization, and we plan to leverage usage patterns to make relevant practice changes in the care of children with a heterogeneous set of illnesses. background/case studies: bacterial contamination of plts remains an ongoing threat to transfusion recipients. recently, a psoralen-based pr technology that reduces the replication potential of pathogens in stored plts was fda approved. we describe our approach to phasing pr-plts into our inventory, including preliminary results of an ongoing qa study of neonatal and pediatric (peds) recipients of pr-plts. study design/methods: before the arrival of pr-plt, we undertook an educational campaign for hospital administrators, it staff, laboratory staff, clerical/clinical aides, nurses, and physicians. we also contacted risk management and the hospital ethics committee. phototherapy devices used at our hospital were confirmed to be compatible with the psoralen-based pr-plt product. shortly following the arrival of pr-plt, we introduced day bacterial "safety measure" testing of our conventional (c-plt) supply. a peds qa study monitored plt utilization and adverse transfusion event reporting relating to both pr-and c-plt transfusions. this study evaluated neonates ( - months of age), infants (> - months of age) and children (> months- years of age) who received at least one transfusion of pr-plts. results/findings: risk management and the ethics committee agreed that both pr-plts and bacteria tested c-plts would be the hospital standard of care. pr-plts were phased in and transfused to patients based on abo compatibility and expiration date, per routine, without regard for patient age or medical condition. after months, pr-plt represented % of our platelet inventory (average daily plt inventory: units). we encountered no complications with the pr platelet phase-in, either from a clinical, informatics or logistical perspective. due to the dual inventory, many peds patients in all age groups were transfused with both pr-and c-plts (table) . two potential transfusion reactions (trs) were reported over the study period in teenage recipients, one associated with a c-plt and the other with a pr-plt. in both cases, the symptoms were ultimately attributed to an underlying medical condition. no rashes were observed among transfused neonates ( - m) who received any pr-plts and phototherapy. background/case studies: packed red blood cell (prbc) transfusions are believed to improve oxygen delivery particularly in vulnerable patients such as neonates and children. however, evidence shows that hemoglobin (hgb) in prbcs has increased oxygen affinity and thus reduced oxygen delivery to tissues due to decreased , dpg levels. standardization of prbc transfusion practices in this population and the scientific evidence on which current practice is based is limited. additionally, due to small transfusion volumes, infants may be exposed to multiple blood donors, increasing their potential for adverse events. study design/method: medical records of pediatric patients receiving prbc transfusion over a month period were retrospectively reviewed. a total of patients were identified as receiving allogeneic prbc transfusion. patients who received autologous blood (cell salvage) were excluded. patient characteristics, length of stay, prbc transfusion volume, pre-and post-transfusion hgb, and adverse events were collected. results/finding: the average pre-transfusion hgb was . g/dl with post-transfusion hgb rising to . g/dl. the mean prbc volume transfused was . ml using a dose of ml/kg for all patients. complications noted were; volume overload, thrombosis, fever/infection, hemolysis, necrotizing enterocolitis (nec), and death (table) . conclusion: evidence based transfusion guidelines are lacking in neonates and infants. a typical dose of - ml/kg in a kg patient, for instance, would translate into full prbc units (about ml) in an average size adult. the current standard dose of - ml/kg yields very high increases in hgb and may put these patients at risk of adverse outcomes, especially thrombosis due to increased blood viscosity. additionally, many of these patients received volume reduced products which delivers a higher hgb concentration per transfusion. dosing should be based on goal hgb and patient condition rather than weight based, though the hematocrit level at which the benefits outweigh the risks remains unclear. pneumoniae has rarely been associated with warm autoimmune hemolytic anemia, with only case reports suggesting this association. however, each of these cases is confounded by other findings in addition to a mycoplasma infection. we describe a unique case in which a pediatric patient has clear evidence of severe hemolysis, a very strongly reactive warm autoantibody, and clinical and laboratory evidence of a mycoplasma infection without a detectable cold agglutinin. study design/methods: the patient is a month-old, previously healthy female infant who presented to the hospital with a -week history of fever, fatigue, decreased appetite, and pallor. she was only treated with acetaminophen. she also developed clear rhinorrhea the day before hospital admission. at the time of her admission, laboratory testing (outside hospital) revealed a hemoglobin and hematocrit of . g/dl and . %, respectively, platelets of , , and a reticulocyte count of . %. all other elements of the complete blood count were within the normal reference range for age. a complete metabolic panel revealed no abnormalities except for a total bilirubin of . mg/dl with a direct fraction of . mg/dl. a filmarray respiratory panel (biofire diagnostics; salt lake city, ut) detected mycoplasma pneumoniae, while all other pathogens ( total) were non-detectable. the patient was started on a -day course of azithromycin (zithromax). results/findings: prior to rbc transfusion, blood bank evaluation revealed that the patient was o-positive and had a stronglyreactive antibody screen. further testing demonstrated an antibody reactive with all reagent red blood cells. the dat was strongly reactive for igg but very weakly reactive for c . an eluate was reactive with all reagent red cells tested. finally, a cold agglutinin study was negative with undiluted serum. in addition to starting azithromycin, the patient was given iv methylprednisolone. during her -day hospital course, the patient received rbc transfusions on the day of admission and several rbc transfusions thereafter (see table ). despite transfusion, her hemolytic process persisted, so she was infused with a dose of iv immunoglobulin on hospital day . her hemoglobin rose to . g/dl on hospital day and increased to . g/dl on hospital day . at that time, the patient was discharged from the hospital with instructions to wean her oral steroid dose over the next weeks. she was followed closely by the hematology clinic and was found to have a stable hemoglobin (up to . g/dl on day after her hospital admission) with no recurrence of her hemolytic process. conclusion: m. pneumoniae infection is a typical cause of cad and has only rarely been associated with warm autoimmune hemolytic anemia. our case demonstrates clear evidence of severe warm autoimmune hemolysis in a previously healthy infant. with the increasing use of multiplex respiratory viral and bacterial pathogen detection systems, the once rare phenomenon of a m. pneumoniae infection associated with warm autoimmune hemolytic anemia may become a more recognized entity. ) and may serve to more reliably reflect when the neonates at risk for hyperbilirubinemia. the difficulty in eliminating the cord blood testing is the neonatologists' reliance of using abo incompatibility as part of the neonates risk assessment rather than using the point of care bilirubin testing. currently the ts requires all positive dat tests to be communicated to the nursing staff immediately. given that the dat strength positively correlates with the percentage of neonates diagnosed with hyperbilirubinemia, the ts staff may also consider notifying nursing staff only for those patients whose dat is or . platelet and leukocyte immunohematology, testing and genetics table . of pairs, pairs were complete match ( / ), pairs were partial match ( / ), pairs were complete mismatch ( / ). the matching rate of hla-dpb in our study is %. conclusion: the matching rate of hla-dpb in / hla matched unrelated hematopoietic stem cell transplantation is low and the gene frequency of hla-dpb in unrelated hematopoietic stem cell transplantation was obtained ,which will help to study on the relationship between hla-dpb and unrelated hematopoietic stem cell transplantation. this work was sponsored by national science foundation of china ( ) background/case studies: thrombotic thrombocytopenic purpura (ttp) is caused by severely reduced activity of the von willebrand factor-cleaving protease adamts . therapeutic plasma exchange (tpe) as well as immunosuppression minimize the morbidity and potential mortality of this presentation. absolute immature platelet counts (a-ipc) have been shown to help diagnose and follow ttp patients' responses to therapy. we report the case of a man with relapsing ttp, low adamts with high inhibitor, treated with mycophenolate mofetil in which a-ipc-indicated an unexpected response to therapy. study design/method: a year old male with a -year history of ttp, presented with status epilepticus complicated by acute respiratory failure admitted with suspicion for relapsing ttp. patient had been treated in prior admissions with tpe, prednisolone, rituximab, and cyclophosphamide with clinical improvement. he was on mycophenolate mofetil maintenance therapy which he last received just prior to day of admission due to consistently low platelet counts, adamts < % and inhibitor of . . on day of admission platelet count was x /l which decreased within five days to x /l leading to initiation of daily tpe along with mycophenolate mofetil discontinuation just prior to tpe start. immature platelet fraction (%-ipf) and calculated a-ipc (%-ipf x platelet count) were obtained with daily pre-tpe cbc. a-ipc ratio was calculated from baseline. abstract results/finding: a-ipc and platelet count were x /l and x /l respectively. counts improved rapidly post-tpe initiation and after one tpe his a-ipc tripled to . x /l achieving the ratio of previously shown to be diagnostic of ttp. on day his a-ipc and platelet counts had improved to . x /l and x /l respectively. absence of anti-pf antibodies ruled out heparin-induced thrombocytopenia at this time. on day he had an unexpected decrease in both a-ipc and platelet count to . x /l and x /l respectively, worsening by day to . x /l and x /l respectively despite daily tpe. patient received additional tpes that failed to improve a-ipc or platelets which on day were . x /l and x /l respectively. a-ipc had remained at this level for days suggesting that the observed decrease was irreversible. adamts activity remained < % low with a high inhibitor. patient's clinical condition continued to deteriorate and family placed patient on comfort care. conclusion: ttp patients have low a-ipc and plt counts at presentation, with the former improving first post-tpe initiation. despite appropriate therapy leading to early improvement of platelet count, patient's counts declined rapidly leading to suspicion for platelet production suppression as indicated by the sustained very low a-ipc. in the setting of ttp, or relapsing ttp use of immunosuppression should be closely followed and a-ipc may aid in establishing early if therapy is affecting platelet production. application of luminex bead technology to detect hpa- a, hpa- a, and hpa- a antibodies su-dan tao*, ying liu, yan-min he, ji he and fa-ming zhu. blood center of zhejiang province, key laboratory of blood safety research, ministry of health background/case studies: detection of antibodies against human platelet antigens (hpas) is crucial for patients' refractory to platelet transfusion therapy. in the text, luminex bead coupled with anti-gpiib/iiia and anti-gpia/iia monoclonal antibody was implied to detect hpa- a, hpa- a, and hpa- a antibodies, and the sensitivity of luminex bead technology was compared with monoclonal antibody immobilization of platelet antigens (maipa) assay. study design/method: monoclonal antibodies p and gi , specific for platelet glycoproteins gpiib/iiia and gpia/iia, were separately coupled to luminex xmap beads. four standard sera, containing anti-hpa- a, anti-hpa- a, anti-hpa- a and anti-hpa- b respectively, were bought from nibsc; three negative sera without hpa antibodies were prepared from ab type blood donors. platelets (containing hpa- aa, hpa- ab and hpa- aa) were collected and reacted with anti-hpa- a, anti-hpa- a, anti-hpa- a and anti-hpa- b standard sera respectively, then the antigen-antibody reaction complexes were lysed and the lysates were incubated with luminex beads to specifically capture antigen-antibody complexes via the epitopes on platelet glycoproteins. the beads-antigen-antibody complexes were then subjected to flow cytometric analysis on a luminex . the hpa- a serum was diluted to serial dilutions (from neat to / ) to test the sensitivities of maipa and luminex beads assay. the two methods were then used to test five blinded samples which were collected from fmait patients. results/finding: luminex bead technology showed that the mfi values of hpa- a, hpa- a, hpa- a standard sera samples reacted with the coupled beads were significantly higher than the negative controls ( . vs . ), which implied that the luminex bead technology could specifically identify negative and positive sera of anti-hpa- a, anti-hpa- a, anti-hpa- a. furthermore, because the platelet was hpa- aa, the hpa- b serum did not react with the coupled beads with mfi was comparable to negative control ( . vs . ). the sera were re-tested by maipa and the results of which were comparable to luminex bead technology, illustrating that detecting hpa antibodies by luminex beads technology was successful. the sensitivity of luminex bead assay and maipa to detect anti-hpa- a was / ( . iu/ml) and / ( . iu/ml), respectively. no cross-reactivity was observed with the samples containing hla, abo or other platelet antibodies. all results of five blinded samples tested by luminex assay showed that four sera were positive for gpiib/iiia antibodies which were consistent with maipa results. conclusion: the luminex beads coupled with gpiib/iiia and gpia/iia monoclonal antibodies could be successfully used to detect hpa- a, hpa- a and hpa- a antibodies via the epitopes on platelet glycoproteins. the sensitivity of luminex technology was higher than maipa technology. (ahus) is a thrombotic microangiopathy (tma) characterized by the triad of microangiopathic hemolytic anemia, thrombocytopenia and renal failure in the absence of infectious toxin. the literature suggests the presence of pathogenic mutations in complement proteins in % of cases of ahus. there is a lack of well-defined recommendations regarding testing for genetic ahus. complement pathway mutation analysis is an expensive test so appropriate utilization is crucial to prevent undue health care costs. we reviewed the indications for genetic testing to understand physician ordering practice and determine the frequency of pathogenic mutations in the population. study design/method: we performed a retrospective review of all cases referred for complement pathway mutation analysis to a national reference laboratory from january to december . clinical history was solicited by genetic counselors. cases were classified by the authors as primary ahus (tma and renal failure without identifiable cause), secondary tma (tma and renal failure with identifiable cause previously associated with tma) or non-tma. the test panel identified variants in complement proteins (cfh, cfi, mcp, factor b, c , c bp, thbd, dgke, cfhr , cfhr , cfhr and cfhr ) that were classified as vus (variances of uncertain significance), pathogenic or benign by the american college of medical genetics. chi square analysis/fishers' exact test was used to determine differences in proportion of patients with pathogenic mutations and primary ahus versus secondary tma. independent sample t-test was used to compare differences in continuous variables between primary ahus and secondary tma. results/finding: of patients tested, pathogenic mutations were detected in % ( / ) and vus in % ( / ). % ( / ) of patients did not fulfill criteria for tma; no pathogenic mutations were found in this group and ( %) had vus. % ( / ) of patients had primary ahus; of these, % ( / ) had pathogenic mutations and % ( / ) had vus. % ( / ) of patients had secondary tma; of these, % ( / ) had pathogenic mutations and % ( / ) had vus. in patients with pathogenic mutations, % ( / ) were children, . % ( / ) had a positive family history of ahus and % ( / ) had recurrent disease. patients with primary ahus had a significantly lower age at presentation ( vs. yrs; p-value: . ) and a higher proportion of pathogenic mutations ( % vs. % p-value: . ) compared to patients with secondary tma. gender distribution, hemoglobin nadir and serum creatinine levels were similar between the two groups. conclusion: we found a lower frequency of patients with pathogenic mutations compared to reported literature. our data suggests that patients with secondary tma should be carefully evaluated prior to ordering genetic testing and those without tma should not undergo this test. counting of platelets in platelet concentrates on hematology analyzers pentraxl and sysmex xn compared with a flow cytometric method farshid ezligini , kjersti roen eriksen , annette vetlesen , thomas larsen titze and geir hetland* , . oslo university hospital, university of oslo background/case studies: hematology analyzers are made for counting of whole blood samples but are often used for quality control of blood components such as platelet (plt) concentrates (pcs). a flow cytometric method for counting of plt in pcs has been developed as validation tool (van der meer et al, transfusion ). therefore, it is pertinent to evaluate plt counting in bcs on hematology analyzers with this validation method in a flow cytometer. study design/methods: samples from ten apheresis pcs and buffy coat-derived pcs were subjected to plt counting on hematology analyzers pentraxl (horiba abx, montpelier, france) and xn sysmex toa (kobe, japan) (both impedance score), and additionally, diluted and stained with anti-cd a fitc in truecount tubes (bd biosciences)(internal bead standard) for measuring in a gallios flow cytometer (beckman coulter, indianapolis in, usa). results were analyzed by paired samples test and shown in bland-altmann plots. results/findings: mean plt values x /l sd were , (<) , (<) and for counting by sysmex toa, pentraxl , and the gallios flow cytometer, respectively. sysmex count was the very lowest a transfusion vol. supplement s abstract ( . % less than for flow cytometry), but all plt counts were significantly different (p< . ), although least so ( . %) between pentra and flow cytometry. conclusion: as validated by the flow cytometric method, pentraxl seems suitable for routine quality control of pcs both because of the small difference and lower counts compared with flow cytometric method, which is too cumbersome in a routine setting. the much lower plt count on sysmex may reflect its optimization for plt counting in whole blood rather than in pcs. fast, precise & easy hpa typing with real-time pcr jonathan downing , arishma lata , roland russnak , zachary antovich , heather dunckley and thierry viard* . new zealand blood service, linkage biosciences background/case studies: the interaction of membrane-bound plateletspecific glycoproteins with the extracellular matrix plays a significant role in hemostasis. human platelet antigens (hpa) found within these glycoproteins can stimulate production of antibodies in recipients of transfused platelets or in fetus of mothers with incompatible hpa. thus, platelet incompatibility is associated with various forms of thrombocytopenia, posttransfusion purpura and other blood disorders. the new zealand blood service performs hpa typing on a pool of platelet donors to provide compatible transfusions where the need arises. the molecular basis of most hpas has been characterized as generally caused by a single-nucleotide polymorphism (snp). hpa typing has typically been performed using pcr-ssp, a method that utilizes time-consuming post pcr analysis steps. the aim of this study was to evaluate the use of real-time pcr-based techniques in a transfusion laboratory setting. study design/method: we evaluated a commercially available solution which consists of reactions that identify both variants of relevant snps located within hpa genes (hpa- through hpa- , and hpa ). genomic dna purified from blood samples, previously genotyped for hpa- ,- ,- ,- ,- and - by our in house pcr-ssp method were used in this study as validation samples. results/finding: results of the validation samples were % concordant with typing obtained by pcr-ssp. the real-time pcr approach overcomes the major challenges of hpa molecular typing by providing an automated solution resulting in increased laboratory productivity and decreased turn-around time. the analysis is facilitated by a software which generates the results. with less than minutes of hands-on set-up and no further operator intervention with the reagents, complete molecular genotyping results are provided in approximately minutes. further, since amplified products are never handled, the risk of laboratory contamination is significantly reduced. the real-time pcr approach with automated analysis was implemented by the new zealand blood service tissue typing laboratory in late and to date has tested dna samples from blood donors ( donors were tested in duplicate). concordance between the sample replicates was %. there were occasions where the assay had to be repeated, giving a repeat rate of . %. occasionally a reaction peak was insufficient to trigger the software automatic allele call and a manual interpretation was required. this occurred most commonly with the hpa- ( . %) and hpa- ( . %) assays. conclusion: real-time pcr with automated analysis provides an effective, robust an accurate method for molecular hpa genotyping. with its minimal hands-on time workflow, it is also very easy to implement and offers a cost effective alternative to classical methods used in a transfusion laboratory setting. genetic variation of cd antigen deficiency expression in jiangsu chinese han population qing chen* , jianyu xiao and chengyin huang . jiangsu province blood center, jiangsu province blood center background/case studies: cd has been implicated in the platelet refractoriness, neonatal alloimmune thrombocytopenia, and posttransfusion purpura, especially in the non-caucasian. cd deficiency varies widely among different ethnic populations, with the frequency of - % in asians and . % of african americans, respectively. however, there is little information on the molecular basis of individuals with cd deficiency in jiangsu chinese han population. study design/method: to investigate platelet cd expression levels and to determine the molecular basis of cd deficiency on the platelet surface of the han population in jiangsu region. cd expression levels on platelets were detected by flow cytometry among blood donors in jiangsu region. donors without cd antigen expression on their platelet surface were further to be determined the expression of cd antigen on their peripheral blood monocyte cells. the coding exons of cd gene and adjacent introns were amplified and sequenced in cd deficient individuals. results/finding: among these blood donors, cd -deficient and cd -expression individuals were . % ( / ) and . % ( / ), respectively. the frequencies of type i and type ii cd deficiency among the study population were . % ( / ) and . % ( / ), respectively. among individual with platelet cd expression, according to mean fluorescence intensity (mfi) value, , and individuals showed low, moderate and high expression levels of cd , respectively, and their mfis were . . , . . and . . (p< . ), respectively. the type i cd deficiency individual were heterozygous for - a>g and - c>g, respectively. among type ii cd deficiency individuals, two harbored a t insertion at position in exon which caused frameshift at codon ; one has a t>c exchange at position in exon which resulted in a tryptophan to arginine substitution at codon ; one has a a insertion before the th bp of the start codon atg in the promoter region; one were heterozygous for t>c and t>g, respectively. conclusion: platelet cd surface expression levels were diversified in the jiangsu chinese han population. the frequency of the type ii cd deficiency was higher than that in type i. the study findings indicated that the frequency of cd deficiency in the chinese population is slightly lower than that in other asian countries. background/case studies: cd -deficient phenotype can be immunized by pregnancy or transfusion, and involved in neonatal alloimmune thrombocytopenia, platelet transfusion refractoriness and other disorders. the frequency of platelet cd -deficient individuals widely varies among ethnic groups, with % to % in japanese, % in sub-saharan africans, . % in african americans, and . % in caucasians. although some studies of cd deficiency are focused on the asian populations, relatively little information has been reported in the chinese population. here we investigated the cd expression on platelets in large samples of the eastern chinese donors. study design/methods: peripheral blood samples were collected from unrelated platelet-apheresis donors in the eastern china. the expression of cd antigen on platelets was determined by flow cytometry using fluorescein conjugated monoclonal antibodies (fitc-anti-cd and peanti-cd ). the isotype control (fitc-mouse igg) was also analyzed to calculate a reference range of cd -nagtive phenotype. for those donors with cd -negative platelets, cd antigen expression on monocytes was analyzed further to distinguish between cd type i and type ii deficiency. flow cytometric parameters were statistically analyzed by mann-whitney test. the work was supported by national natural science foundation of china ( ) and zhejiang high-level innovative health talents. results/findings: the mfi (mean fluorescence intensity) of platelet cd in all samples showed a continuous distribution profile, and no obvious fluorescence-gap could be utilized to distinguish negative from positive phenotype. on account of this limitation, we classified the cd phenotypes using the (mean sd) of the background mfi observed in isotype controls. forty-three samples were detected as cd deficiency on platelet, in which one sample was cd negative both on platelet and monocyte. the frequency of cd type i and type ii deficiency in the eastern chinese donors was . % and . %, respectively. the average mfi of cd deficiency samples was significantly lower than cd positive samples ( . . vs . . , p< . ). conclusion: the frequency of platelet cd deficiency in the eastern chinese donors was close to japanese and african americans. it means that the possibility of cd antibody occurred by pregnancy and transfusion in this population is existed. it is useful to find and register cd -deficient donors by large-samples screening for potential immune thrombocytopenia patients with cd antibody. background/case studies: cd (gpiv, chromosome q . ) is an kda glycoprotein expressed on multiple cell types including platelets (plts), monocytes (mono), & erythroblasts. although rare among whites, cd deficiency (cd -n) is observed in - % of africans (t g) & is classified as either type i (cd -n plt, cd -n mono) or type ii (cd -n plt, cd mono). an acquired type ii cd -n phenotype can also be observed in the setting of myelodysplastic syndrome (mds). type cd -n individuals can develop anti-cd alloantibodies with plt refractoriness & neonatal alloimmune thrombocytopenia. we report a case of profound plt refractoriness caused by anti-cd in a patient with newly diagnosed mds. study design/method: hla antibody testing was performed with a commercial bead-based fluorescent assay. cd phenotyping (plt, mono) of patient & family members was performed by flow cytometry (fc). cd staining of bone marrow was performed by immunohistochemistry. plt crossmatching (plt-xm) was performed by the american red cross. pltspecific alloantibody testing & cd dna sequencing were performed at a commercial reference laboratory. results/finding: the patient was an year-old, group o african-american male who presented with blurry vision & lightheadedness. complete blood count findings were significant for hemoglobin . g/dl & plt count k/ml. bone marrow biopsy & cytogenetic analysis revealed multilineage dysplasia, - % blasts & a complex karyotype with del( )(q q ) consistent with mds. plt refractory work-up was initiated after repeated plt transfusion failures with corrected count increments (ccis) < . hla antibody testing was negative (class i panel reactive antibody (pra) %). the patient was plt-xm-incompatible with most donors ( / ). a trial of group o, plt-xm-compatible plts was unsuccessful (cci ). subsequent testing for plt-specific alloantibodies identified anti-cd . fc-phenotyping showed no cd on patient's mono or plt, consistent with type i cd -n. preliminary dna results show that the patient is heterozygous for t g. because cd -n apheresis plt were unavailable from blood suppliers, the patient's children & grandson were screened as possible donors: all showed normal cd expression on plts. trial of eltrombopag & romiplostim was attempted with no improvement in plt count. repeat hla antibody testing (day ) demonstrated new class i alloantibodies (pra %) in response to transfusion ( apheresis plts, rbcs). given his plt refractoriness & poor prognosis, the patient opted for hospice. conclusion: we describe a patient with cd -n & severe plt refractoriness in the setting of new mds, and q-chromosomal abnormalities. the absence of cd on plt & mono support congenital type cd -n although a contribution by the patient's underlying mds cannot be excluded. rapid platelet donor classification: hla & hpa profiles by "leansequencing" without dna purification dipika patel , kristopher fernandez* , eric senaldi , pascal george , michael seul and ghazala hashmi . biomolecular analytics, central jersey blood center background/case studies: prophylactic platelet transfusion is the standard of care for managing thrombocytopenia. in the emerging paradigm of personalized medicine, the selection of cellular products in accordance with patient immunomolecular signatures has the potential to reduce the rate of antibody-mediated platelet clearance and thus to improve treatment efficacy. while the benefits of customizing transfusion therapy have long been recognized (gmur http://bit.ly/ q heq), the routine, real-time selection of platelets by immunogenetic profile has remained impractical by current methods of dna analysis. to address this issue, we evaluated a process of platelet donor classification using buccal swab samples from apheresis platelet donors for determining the combined hla class i and hpa signature without dna purification using a novel "leansequencing" process. study design/method: under a study protocol and informed consent, we evaluated a process for collecting and classifying buccal swab samples from $ adult donors who had made ! donations in the previous months. samples (labeled with study barcodes) were shipped weekly to biomolecular analytics ("bmx") for preparation of "crude extracts" for leansequencing: this novel process combines a proprietary sample pooling strategy with a protocol that eliminates many traditional sample "clean-up" steps. briefly, after preparation of crude extracts, samples were amplified, pooled and analyzed (in separate runs) for , , , , , , , , , and for hla class (a,b,c) , the latter using a proprietary design that limits analysis to informative alleles in the hla sequence; this "information-theoretic" design permits direct allele and haplotype reconstruction using bmx-proprietary software. a subset of crude extracts was purified and analyzed side by side with positive and negative controls. results/finding: crude extracts from buccal swabs produced viable profiles for hpa as well as hla class i with significant savings in time-to-result. as an illustration, the table reports allele frequencies for platelet-antigens ("hpa") that are consistent with a predominantly caucasian or hispanic platelet donor population (http://bit.ly/ pdplf ) in hw equilibrium. similarly, hla-class i haplotype frequencies were determined. conclusion: leansequencing lends itself to the rapid determination of hla-class i and hpa signatures of platelets; the process with its streamlined lean protocol achieves additional time (and cost) savings by accommodating crude extracts produced from buccal swab samples collected and handled in accordance with the process validated in this project. the process could be readily implemented to another site using the elements and process developed. the "pool & plex" process and the early donor recruitment enables economies of scale for matched donor procurement. the serological characteristics and heritage background of a novel hla allele, hla-a * : chuan-fu zhu*, yong-hong song, xiang-min nie and wen-ben qiao. blood center of shandong province background/case studies: there are , hla alleles documented according to the imgt / hla sequence database in janury , and more than % of them were identified in the last years. besides sequences many of the novel hla alleles have not been analyzed their serological reactivities. hla-a * : allele was fist detected in our laboratory during our hla typing for china bone marrow donor program(cmdp). for further study, the serological characteristics and heritage investigation were performed. study design/methods: the routine hla tying for the potential donors from cmdp were performed by bi-allelic sequence-based typing method,using a commercial kit (rose europe gmbh, frankfurt, germany). in the case of no full matched hla typing results, group specific hlassure-se sbt typing kit (texas biogene inc., taipei, taiwan) was employed to identify the nucleotide sequences of the novel allele. fresh blood samples were collected of the proband and his family members with the consent, in order to nanalysis the serological reactivities and the possible haplotype associations to the novel allele. the hla serological specificity was indicated by one lambda(asn d)hla kit. results/findings: no full matched result was obtained at hla-a locus in hla typing for a donor,which suggested the possible existence of a novel allele. the latter nanalysis indicated that the proband have a nove nucleotide sequences at hla-a locus, the new sequences was most close to those of hla-a * : : : , but nucleotide substitution in exon , by nt c-a (codon acc-aac), which resulted in one aminoacid substitution ,thr-asn. the novel hla-a allele was officially named as hla-a background/case studies: anti-d is a frequent cause of hemolytic disease of the fetus and newborn (hdfn). as a rule, immunization occurs in d negative pregnant women, but occasionally anti-d is also observed in carriers of d variants. currently, maternal plasma analysis for determination of the fetal rhd status became an exciting new tool for the management of d-negative pregnant women, but one of the challenges in non invasive fetal rhdgenotyping is the presence of d variants in the pregnant women. we present a case of a year-old pregnant woman typed as ab , who delivered a baby affected by severe hdfn. the newborn was typed as b and presented a positive direct antiglobulin test (dat) with an anti-d identified in the eluate. the baby was treated by exchange transfusion and the mother's sample was investigated. study design/method: serologic testing was done by hemagglutination in gel cards. genomic dna was extracted from whole blood by spin column and all rhd exons were sequenced by sanger sequence method. results/finding: the mother's rbcs reacted with the four monoclonal anti-d used (igm clones p x and rum and the blends clones th ms and d d ) and were typed as c-c e-e . an anti-d was identified in her serum. molecular analysis showed the c>t and a>c in exon , the snp t>c changes in exon and the t>g nucleotide change in exon . the set of snps found is similar to the molecular background of dol , except for a>c change. conclusion: this novel set of snps found in this mother is related to a novel rhd allele leading to a partial d antigen involved in the production of an anti-d that can cause severe hdfn. this finding shows the need to elucidate the clinical significance of different rhd genotypes in various ethnic backgrounds. the and erytra v r (the routine reference platform) was performed. a total of immuno hematological tests ( abo/d grouping (including newborn samples), extended erythrocytic phenotype, antibody screening, antibody identification, dat) and crossmatches were performed on patient's whole blood samples. the erytra eflexis v r performance was evaluated according to a protocol that was designed to simulate the routine workload using the system in its two different configurations. concordance between systems was assessed and discrepancies were analyzed. the following performance metrics were assessed: time to first result (ttfr), turn-around time (tat) for the total workload from first result to last result (throughput, results/h), and manual "handson" time required as well as walk-away time, considering the two different configurations of the system. for the ease of use evaluation, different usability features were ranked and the number of steps and timing of the following activities were tracked: sample sort and loading, routine testing, post-run procedures, consumables used, and space requirements. a threshold for in vitrodetection of anti-d gamma globulin was also determined. v r analyzer and the reference method were obtained in . % of the abo/d tests (n ), , % of the antibody screening tests (n ), , % of the antibody identification tests (n ) and % of the dat tests (n ). there were discrepancies ( abo/d for the same patient, for antibody screening and antibody identification: in both cases, the erytra eflexis v r could conclude whereas erytra could not due to a poor reaction. use of the stat mode (incubator is reserved for urgent tests) proved its usefulness when testing several samples (time saving was more than min). detection threshold of the d antibody was assessed at . ng/ml ( . ui/ml) whereas the french recommendations are ng/ml. the possibility of interchanging the trays (reagents/sample) makes also possible to optimize the analyzer operation. the impressions of the technical staff were positive regarding esthetic and functional design, intuitive and easy use, as well as flexibility. v r results demonstrated velocity, sensitivity, as well as the ability to easily perform the routine workload of a medical analysis laboratory. erytra eflexis v r meets both the requirements for french regulatory in immunohematology and for iso accreditation. background/case studies: kell system antibodies inhibit erythropoiesis causing severe anemia in hemolytic disease of the fetus and newborn (hdfn). we report a case of hdfn secondary to anti-kpb that resulted in multiple intrauterine transfusions of kp (b-) donor cells and hemolytic anemia upon birth. case: a year old g p presented during her fifth pregnancy with anti-kpb with an initial titer measured of . by history, the anti-kpb developed during her third pregnancy which ended in a spontaneous abortion before antibody titers could be initiated. the patient's antibody titers peaked at during the fourth pregnancy which resulted in a healthy male without anemia or jaundice. . in the latest pregnancy, ultrasound was initiated with elevated middle cerebral artery doppler exams ( . moms) peaking at weeks. this resulted in three intrauterine transfusions. due to potential labor and the finding of reversed diastolic flow on middle cerebral artery doppler studies, a finding that has been associated with impending intrauterine fetal demise, caesarean delivery was performed at weeks gestation. the baby boy required phototherapy for hyperbilirubinemia. the indirect bilirubin at birth was . mg/dl with . g/dl hemoglobin. the baby typed as o positive, kp (b ) with a micro positive dat. the antibody workup revealed an anti-kpb. continued hemolysis required one more transfusion at weeks of age. the positive dat and passively acquired anti-kpb were no longer detected by weeks of age. his hemoglobin recovered to . g/dl with an indirect bilirubin of . mg/dl at weeks of age. all clinical signs of hemolytic anemia were resolved. study design/method: serologic testing included peg iat by tube methods. acid elution was performed using immucor gamma elu-kit ii. molecular testing was performed using immucor bio-array hea platform. results/finding: antibody identification on the mother was performed as well as alloadsorption studies to rule out other underlying alloantibodies. a new weakly reacting anti-s was detected on the day of the delivery. the baby typed as s positive however the anti-s was not detected in an eluate prepared from the baby's red cells. all of the intrauterine transfusion units were s negative. conclusion: to our knowledge only five case reports have been described for anti-kpb which resulted in moderate to severe hdfn. pregnant mothers with anti-kpb detected should be monitored closely. background/case studies: in some clinical cases, the c d-specific dat may be too insensitive to detect low, but significant levels of c d, or it may be inconclusive due to spontaneous red cell (rbc) aggregation. further, the dat is not well suited to quantify the number of immunoprotein molecules on rbcs, since a " " reaction corresponds to about molecules/ cell. a number of flow cytometric methods for the detection of rbc-bound c d have been published. however, these are mainly designed to quantify the fraction of rbcs with c d-sensitization. the aim of this study is to present a flow cytometric method for the quantification of the level of rbcbound c d. study design/method: ten microliters (ul) of : (after documenting experimentally that this amount ensured maximum binding of anti-c d) mouse monoclonal anti-human anti-c d (abcam, clone c ) were added to ul of a . % rbc suspension. after incubation for minutes at c, samples were washed x , and ul of : diluted anti-mouse-f(ab) -pe (ro , dako) were added. after incubation at c, samples were washed and resuspended before being acquired on a flow cytometer (becton dickinson facscanto ii). to enable calibration of fluorescence signals in antibody binding capacity (abc), a calibration standard (dako qifikit) stained with ro was run in parallel with all experiments. background fluorescence (in abc) was subtracted to yield net abc values corresponding to specific staining with anti-c d. the assay, in parallel with our routine dat (dc-screening i, id-card, gel card, biorad) was applied to a series of a rbcs stained with levels ( fold dilution, : - : ) of o serum with high titer anti-a. to estimate the normal range of rbc-bound c d, edta-stabilized samples from healthy donors were tested. finally, the assay was applied to a sample from a patient with clinical aiha with an inconclusive dat due to unspecific dat polyreactivity. results/finding: the correlation of the net level of rbc-bound c d (values ranging from to , abc) with level of -serum dilution (used to sensitize a rbcs) proved to be highly linear (logarithmic vs. logarithmic plot; r . , p < . ). compared with dc-screening , the sensitivity of the flow cytometric assay was superior. it detected c d sensitization at least dilution steps further. the median normal level of rbc-bound c d was abc (range - abc, n ). the assay enabled demonstration of specific c d-sensitization in the patient; the level of rbc-bound c d in the sample was significantly elevated ( , abc). conclusion: the presented flow cytometric assay is capable of quantifying the level of rbc-bound with a high degree of linearity and analytical sensitivity. further, it is capable of quantifying the level of rbc-bound c d in dat polyreactive samples. background/case studies: abo blood group system of red blood cells (rbcs) consists of a and b oligosaccharide antigens and anti-a and anti-b antibodies against these antigens, which are present in the sera of individuals who do not express the antigen(s)(landsteiner's law). because of the expression of those antigens on some epithelial and endothelial cells in the body, the abo matching is critical not only in blood transfusion, but also in cell/tissue/organ transplantation. in spite of the fact that both antigens and antibodies are involved, these genetic traits are specified by a single genetic locus of abo. forssman (fors) system is another rbc blood group system which consists in a glycosylation polymorphism specified by the gbgt gene. in humans, the abo and gbgt genetic loci are located on chromosome q , and the functional alleles encode a and b glycosyltransferases (at and bt) and forssman glycolipid synthase (fs), which catalyze the last biosynthetic steps of a and b, and forssman (fors ) oligosaccharide antigens. the molecular genetic bases for allelism of those two systems in humans have been well-elucidated. the abo and gbgt genes are also present in some other species in addition to humans. however, the presence/absence and functionality/non-functionality are species-dependent. molecular mechanisms/forces that created this species divergence, including human polymorphism, were unknown. study design/methods: utilizing genomic information available from gen-bank and ensembl databases, the gene maps of the chromosomal region surrounding the abo and gbgt genes have been constructed of vertebrate species. results/findings: extensive similarities were observed in the kinds, numbers, and orders of genes, as well as their chromosomal locations. however, numerous differences were also identified. these include chromosomal rearrangements, as well as the insertions and amplifications of specific genes. interestingly, the abo and gbgt genes were found located at the boundaries of chromosomal fragments that seem to have undergone frequent inversions/translocations during species evolution. conclusion: genetic alterations, such as deletions and duplications, are known to be prevalent at the ends of rearranged chromosomal fragments. therefore, the species-dependent divergence and polymorphism within species of those clinically important glycosyltransferase genes may have been resulted, at least partially, from unstable chromosomal structures neighboring those genes. alloimmunization despite phenotype matching in a patient with sickle cell disease and a complex rhce genotype jessica kneib* and emily coberly. university of missouri health care background/case studies: red blood cell transfusion plays an important role in the treatment of patients with sickle cell disease. sickle cell patients have a significantly increased risk of alloimmunization compared to the general population, and the standard of care is to provide phenotypically matched units for at least c, e, and k antigens to reduce this risk. unfortunately, the genotype and true alloimmunization risk may not always be accurately represented by the red blood cell phenotype, particularly in patients with complex partial rhce variants. study design/method: a year old female with a history of sickle cell disease, stroke, and iron overload presented for routine exchange transfusion. transfusion vol. supplement s the patient's blood type was o positive and her red cells had been previously phenotyped as c-, c , e-, e and k -. an antibody screen was positive, and antibodies against c and e antigens were identified in the plasma. the patient had only received phenotypically matched units negative for c and e antigens for all previous transfusions at our institution, based on her known red blood cell phenotype. blood samples were sent to a reference laboratory for molecular testing to look for partial rhce variants that might explain the antibody development. results/finding: molecular testing was performed to reveal the presence of two different partial rhce alleles, resulting in a predicted phenotype of d , c-, e-, partial c , partial e . the probable rhce genotype, rhce*ce-jal/rhce*ce g, results in partial expression of both c and e antigens. in addition to the known risk of alloimmunization against the absent c and e antigens, this result indicates that the patient is also capable of forming alloantibodies against the absent portions of both c and e antigens. based on these results, the patient's anti-e was determined to be an alloantibody and not an autoantibody. conclusion: although phenotypically matched units are standard of care for patients with sickle cell disease, the red blood cell phenotype may not accurately represent the alloimmunization risk in patients with complex partial rhce genotypes. in this case, molecular testing confirmed that the patient is at risk of developing alloantibodies against c, c, e, and e antigens. as the patient had already made alloantibodies against c and e antigens, it was determined that she would require units that were molecularly matched to her rhce variants for all future transfusions. this case demonstrates that phenotype matching for sickle cell disease patients may not be adequate to prevent alloimmunization in individuals with partial rhce variants. altered splicing in the rhd*weak d type allele associated with the skipping of exon in a pregnant woman and her newborn carolina bonet bub* , maria giselda aravechia , thiago costa , marilia sirianni , eduardo bastos , leandro santos , lilian castilho and jos e kutner . hospital israelita albert einstein, hemocentro unicamp background/case studies: rhd*weak d type is a variant commonly found in caucasians associated with a weak d phenotype. as previously reported (vege et al, transfusion ) the c. g>c change (p.g a), which characterizes the rhd*weak d type allele is a splicing variant that induces skipping of the whole rhd exon . we report an altered splicing in the rhd*weak d type allele associated with the skipping of exon in a pregnant women and her newborn with weak d expression. study design/method: the d antigen expression was evaluated with commercially available monoclonal anti-d reagents: blended igm/igg (clones th- /ms- ), igm (clones ms and p x ) and igg (ms ) in tube and on gel cards. c, c, e and e phenotyping were performed in gel. rhd genotyping was performed with the rhd beadchip platform from immucor. direct automated sequencing of the rhd exons and flanking intron regions was performed by the sanger dideoxy method. results/finding: both pregnant women and newborn samples were phenotyped as d w c-c e e . the samples showed weak hemagglutination reactions ( / ) with all anti-d clones used. rhd beadchip results showed the ls* signal indicating a possible deletion of exon in both dna samples. sequencing showed the c. g>c change and the intronic c. - t>a and c. - t>c substitutions, which are associated to the rhd*weak d type allele. conclusion: our results showed that c. g>c associated with c. - t>a and c. - t>c variations had probably a functional impact on splicing inducing exclusion of exon in both dna from mother and newborn. this finding is important to develop assays and interpret genotyping results, as current guidelines do not recommend anti-d igg prophylaxis for women with weak d type . background/case studies: sickle cell disease (scd) patients require red blood cell (rbc) transfusions to minimize disease-specific symptomatology. previous studies have shown that more than % of children with scd receive at least one rbc transfusion in their lifetime. both simple transfusions and erythrocytapheresis are associated with increased risk of rbc alloimmunization. published literature is lacking on the frequency of alloimmunization and geographical associations in pediatric populations, which is made difficult to compare due to lack of standardized categorization of what represents a pediatric patient population across studies. therefore, we looked at the alloimmunization rates of pediatric patients with scd in the unites states (us) and other countries. study design/method: a literature search was performed for studies published on alloimmunization rates of scd pediatric patients including hbss, sickle beta-thalassemia and hbsc. we evaluated the overall alloimmunization rates as number of alloantibodies per transfused patient and alloantibodies per transfused units across world literature and compared them using chi-square analysis. results/finding: fourteen studies reporting data to derive alloimmunization rates of pediatric scd patients were found. these included eleven us studies with , patients and studies from other regions (brazil, egypt and france) with patients. majority of patients included in the studies had hbss disease. patients received either episodic, chronic simple transfusions or erythrocytapheresis. age range for the us studies was to years and for the other countries to years. available data from us studies included a total of alloantibodies, the most frequent of which were antibodies to c, e, kell, m, s and kidd antigens ( . %, . %, . %, . %, . % and . % respectively). alloimmunization rates were calculated as antibodies per patient in some studies and antibodies per transfused units in other studies. we evaluated rates using both approaches as per available data. us had an alloimmunization rate of . % ( . to . , % ci) vs. . % for non-us studies ( . - . , % ci) (p . ) and a transfusion vol. supplement s more alloantibodies per transfused patient ( . vs. . , p . ). similarly, the number of alloantibodies per transfused units in the us, evaluated from five studies, was higher compared to a large french patient cohort ( Á vs. . , p Á ). average number of rbc units transfused per patient in the us was also higher compared to data from france ( vs. , p . ). conclusion: despite limited studies available to compare alloimmunization rates in pediatric scd patients in the us and other countries, the overall rates are higher in the us. though no definitive reasons could be concluded from the available data, limiting the number of rbc exposures, i.e. units transfused in non-critical conditions could lead to lower alloimmunization rates. results/findings: a post-transfusion sample was referred to the irl for a trxn investigation. there were no clerical errors; however, hemolysis was present in the post-transfusion plasma/serum. abo/rh and crossmatches using lo-ion tm were repeated on the pre-and post-transfusion samples with no discrepancies. the post-transfusion dat was positive with a negative eluate. the hospital requested another unit before the investigation was complete. antibody identification on the post transfusion sample with lo-ion tm was negative. suspecting a weak antibody, additional investigation using peg tm on both samples revealed an anti-c. no additional clinically significant alloantibodies detected in the pre-or post-transfusion samples using peg tm . conclusion: the patient experienced an acute hemolytic transfusion reaction due to anamnestic interaction of anti-c in the patient's plasma/serum against c antigen on the transfused cells. anti-c was not detected by our routine antibody identification techniques. the mma confirmed anti-e, -m and -c were clinically significant. laura bailey* , melissa grohotolsky , lisa deblass , bala carver and kip kuttner . health network laboratories, miller keystone blood center background/case studies: the en a antigen is a high prevalence antigen in the mns blood group system. the antigens of the mns system are carried on glycophorin a (gpa) and glycophorin b (gpb). anti-en a is a rare immune igm/igg antibody made by individuals who lack all or part of the gpa protein. anti-en a has been implicated in fatal htr and hdfn. the en(a-)phenotype can result from either a rare deletion of the gpa protein or the even rarer m k phenotype. because individuals with the m k phenotype lack both the gpa and gpb protein their red blood cells type as m-, n-, s-, s-, u-, en(a-), wr(b-) and have reduced sialic acid. study design/method: year old white mennonite female g ,p presented to her midwife for prenatal care with the intent of home delivery. she had a positive antibody screen by solid phase at the hospital transfusion service. an antibody identification panel was done in gel. testing for antibodies against selected cells (u-and u var ) in tube with peg enhancement and phenotyping was done. based on mns phenotype, anti-en a was suspected. the specimen was referred to an immunohematology reference laboratory (irl). the testing included phenotyping with unlicensed antisera, ficin treated panels by tube technique, allogeneic adsorptions for antibody exclusion and identification and antibody titration. following identification of anti-en a by the irl the midwife was advised to refer the patient to a maternal fetal medicine specialist at an academic center close to the patients' home. the midwife was also advised to consider autologous blood donation and /or testing of siblings. results/finding: testing by the hospital blood bank demonstrated positive reactivity in the antibody screen. the gel antibody panel ahg phase resulted in panagglutination and a negative autocontrol, suggesting a high prevalence antibody. the phenotype was performed and determined to be m-, n-, s-, s-, u -. outdated u variant reagent cells reacted in peg igg phase ruling out anti-u. anti-en a was suspected and the sample was referred to the irl. allogeneic adsorptions were performed to rule out antibodies to common red cell antigens. lack of reactivity on a ficin panel eliminated the presence of anti-u,-wr b . phenotyping with unlicensed anti-u was negative and unlicensed glycine soja demonstrated reactivity, suggesting that the patient is en(a-). the patient's phenotype is consistent with the m k phenotype. based on the lack of reactivity on the ficin panel, the antibody was identified as anti-en a fs. since anti-en a is extremely rare, this specificity could not be confirmed due to the lack of en(a-) cells and appropriate antisera. the baseline antibody titer was at igg phase without enhancement. conclusion: this case study describes the workup of a rare antibody in a prenatal patient at a tertiary care hospital. studies performed after the patient was transferred closer to home confirmed the anti-en a (fs) and genotyping was performed. three titers were performed for the remainder of the pregnancy and held at . although anti-en a has been implicated in hdfn, a healthy infant was delivered without complications. this patient should be monitored closely through future pregnancies. autologous donation and/or sibling testing should be considered in order to provide compatible blood for intrauterine transfusion or transfusions at or after delivery. background/case studies: a year old caucasian male diagnosed with hemolytic anemia and no previous transfusions was referred to the immunohematology reference laboratory (irl) for antibody identification and rbc genotyping. initial serologic testing by the referring facility and the irl demonstrated anti-d, anti-c and/or anti-g specificity with a positive auto control and igg dat. anti-g has an anti-d, -c specificity and is most frequently found in rr individuals exposed to r'r cells. the g antigen is present on rbcs expressing either rhd and/or c and very rarely on d-c-g (r g r) cells. both rhce*c and rhd genes encode ser which determines g expression. rare rhd variant antigens lacking ser are g-. study design/methods: serologic evaluation included tube testing using gamma lo-ion tm (immucor, inc., norcross, ga) enhancement, elution studies (gamma elu-kitv r ii (immucor, inc.)), edta glycine acid treatment (gamma ega tm kit (immucor, inc.)), allogeneic adsorptions with papain treated intact rbcs, reagent and patient-derived rbcs and antisera. molecular testing was performed with bioarray precise type ivd hea assay (immucor, inc.). results/findings: molecular testing revealed an rhce*ce genotype (with a c-e c e-predicted phenotype) and an otherwise unremarkable rbc typing report. serologically, the antibody(ies) demonstrated an anti-d, -c, -g specificity in the serum and eluate using r o r, r r , r'r, r g r and rr cells. this patient is predicted to be r r (dce/dce) therefore, anti-c is possible but an allogeneic anti-d or -g is exceptionally unlikely. allogenic adsorptions using papain treated r o r and r'r cells excluded anti-c and anti-d, leaving anti-g as the only explanation of the initial findings. reactivity with the patient's ega treated (dat negative) cells against the "neat" serum, eluate and anti-g antisera confirmed auto anti-g. conclusion: warm autoantibodies are common findings and often have an rh specificity; however, these antibodies usually demonstrate a broad but weaker specificity in the eluate or in the serum when enhancements are used. this anti-g had no reactivity with g-cells. the differentiation of anti-g from anti-d and anti-c is generally academic as transfusion recommendations are the same: provide rhd-, c-units. it is relevant and clinically important to determine the presence or absence of anti-d in rhd negative women of childbearing age who present with an anti-g specificity. if anti-d is a transfusion vol. supplement s excluded these women should receive rhig as part of their prenatal care. in this case differentiating anti-d, -c from an auto anti-g was necessary to provide transfusion recommendations. providing rhd-and c-units to give serologically compatible rbcs could result in formation of an allogeneic anti-e. automated eluates: comparison of solid-phase red cell adherence and gel automated eluate testing jayanna slayten* , christa voliva , kathy fletcher , heather vaught and tracie ingle . indiana university health, indiana university health (iu health) background/case studies: acid eluates (elu kit ii. immucor. norcross, ga) are to be tested via tube iat method in parallel with the recovered last wash per the manufacturer's package insert. finck et al (immunohematology ; : - ) demonstrated acid eluates may be tested in other platforms such as manual gel microcolumn assay (id-mts.igg card. ortho clinical diagnostics. raritan, nj) and automated solid-phase red cell adherence systems (echo. immucor. norcross, ga). our study looked to compare the use of the automated gel microcolumn analyzer (vision, ortho clinical diagnostics. raritan, nj) to the solid-phase red cell adherence analyzer (echo, immucor. norcross, ga) for the testing of acid eluates in a regional midwestern transfusion service. study design/methods: twenty patient samples, less than days from collection and drawn in edta, were used to prepare acid eluates (elu-kit ii. immucor. norcross, ga) while retaining the last wash to be tested in parallel. two samples were > dat positive, were weakly dat positive and were dat negative. the prepared eluates were observed for color (bluegreen/bg, blue-brown/bb, blue-purple/bp), and the ph was documented for the prepared eluate (whatman . - . ph. whatman international. maidstone, england). the prepared eluates and last washes were tested on the vision and echo against an antibody screen. if the antibody screen was positive, the sample was tested against an antibody panel to determine specificity/pan-reactivity. prior to the eluates being tested on the automated platforms, they were spun for minutes twice to remove any rbc debris which could cause false positive reactions. results/findings: the eluates prepared ranged in color: bb, bg and bp. the ph of all eluates ranged from . - . with the highest percentage of eluates at a ph of . ( %). sixteen of the eluates tested yielded the same results in both automation platforms (concordance of %). four eluates with different results are summarized in table . conclusion: the study demonstrated that both analyzers may be used for eluate investigations. both methods yielded apparent false positive results on samples which were initially dat negative. the echo was more sensitive, yielding false positive results ( ) when the vision was negative, while the vision was false positive with one eluate with echo negative. there was no apparent association in the non-correlating eluate results in relation to color of eluate, age of sample when eluate was prepared, or ph of the eluate. a larger study may be able to better elucidate the apparent false positive results noted in this study between echo and vision eluate study. background/case studies: blood agglutination observed by landsteiner in led to the discovery of human blood groups. in the abo system > alleles have been described. the glycosyltransferase encoded by most results in weakened expression of a or b or the null (group o) phenotype. as testing methods and reagents improve, donors may appear to change their abo type. here we describe a frequent group o blood donor ( units over years) who is actually a w . study design/methods: donations were tested with the pk instrument (beckman coulter inc.). routine forward and reverse abo testing was used to investigate the discrepancy. molecular studies were performed by dna sequencing of abo introns , and and exons and . specific primers located in the flanking intron regions of the blood group gene were used to amplify relevant exons by pcr. the template used is genomic dna extracted from whole blood collected in edta. pcr-amplified exons are subjected to bidirectional dna sequence analysis using standard sanger dideoxy chemistry. seqscape software (abi) was used to analyze sequence data by comparing the obtained sequence to a reference sequence from ncbi. results/findings: serologic results are shown in table . tests with anti-a, -a , -b anti-a,b were negative as were the a cells in reverse testing. the results of dna sequencing of abo introns/exons are shown in table . the significant changes were found in exons and . in exon there was a nucleotide (nt) deletion of g which resulted in a shortened transcript due to a stop codon, and another nt substitution lead to the amino acid change gly ala. mutations in exon included a nt substitution causing a pro -leu change and a nt deletion c resulting in shortened transcript. conclusion: serologic testing of the donor plasma with a cells was nonreactive revealing the abo discrepancy. molecular testing confirmed the donor genotype is heterozygote a/o [abo*o. . /abo*aw. ] which predicts a w phenotype. normally, donor rbcs are tested with anti-a and -b and the reverse type confirmed by testing the with a and b cells. this abo discrepancy was caused by the presence of anti-a in the plasma causing the forward and reverse type to be interpreted as group o. according to fda guidelines, the donor is technically group a, and as such all donations need to be labeled as group a. the donor was contacted and instructed to cease donating blood for transfusion. if donations continue, the unit labeled group a would likely test as group o at the transfusion facility resulting in an fda reportable error. there are numerous reports in the literature of the relative insusceptibility of a cells to destruction by anti-a, however, there is one hemolytic transfusion reaction to a x blood transfused to a patient with a potent anti-a titer > : . (schmidt, nacarrow et al. ) . a review of transfusion recipients of the donor reported here did not reveal any untoward reaction after transfusion. a transfusion vol. supplement s extraction of gdna from edta-anticoagulated whole blood from pilot tubes derived from the unit. dna extraction from whole blood is performed on up to blood tubes using the biorobot universal system (qiagen). there is no information on the maximum acceptable age of the blood for this purpose, either from the vendor or in peer-reviewed literature. we set out to assess if blood up to days post collection yielded suitable gdna for downstream rbc genotyping. study design/method: edta blood tubes collected from random blood donors were used to extract dna from microliters of whole blood on day , and days post collection. blood samples were stored at - c before and after extraction. tubes were brought to room temperature and rocked before loading on the biorobot. extraction was performed using the mdx blood minikit (qiagen). resulting dna samples were assessed for gdna yield and absorbance a /a using a nanodrop (thermo scientific). the extracted gdna was tested using precisetype hea molecular beadchip ("hea", immucor) and failure rates on both the biorobot and the hea were assessed. results/finding: all three extractions were successful with no invalids (result ) on the biorobot universal report. no evidence of visible clots or splatter during extraction was noted by the technologist. out of the samples, samples were chosen at random and concentrations were measured using nanodrop for each of the extracted plates. dna concentrations ranged from . to . ng/ul. all readings with the exception of ( . ng/ ul) had concentrations > ng/ul. interestingly, the one that was < ng/ ul on day , yielded > ng/ul on day and post collection. over the next months, sets of samples were extracted and tested by hea. eighty-three ( . %) failed extraction and ( . %) failed hea. none of the samples that failed extraction were or days post collection; none of those that failed hea were days post collection; . % were > < days post collection. conclusion: based on these results it can be concluded that edta blood tubes up to days post collection can be used as a source of gdna for rbc genotyping without negatively effecting the concentration of the resulting dna samples and the validity of the resulting genotyping. case study: investigation of persistent negative antibody screens on patients receiving daratumumab raeann thomas , carlos villa , rachel davis-rauser* , helen carpenter and vrunda patel . university of pennsylvania, hospital of the university of pennsylvania background/case studies: daratumumab is an anti-cd monoclonal antibody therapy that received fda approval for treatment of multiple myeloma in . communications suggest all patients receiving therapy would have a positive antibody screen because cd is a common antigen expressed on red blood cells. currently, patients have been treated with daratumumab at a large academic medical center. a wide variation of reactivity was observed, including patients who were found to have consistently negative antibody screens. while there are several potential causes, neutralization of anti-cd antibodies could easily be tested by applying established techniques used for neutralizing antibody reactivity. study design/method: samples received were drawn as a standard of care. indirect antiglobulin testing was performed using solid phase red cell adherence and gel. neutralization was performed by adding equal volumes of negative daratumumab treated patients' plasma with positive daratumumab treated patients' plasma. a dilution control was made by adding saline to each positive patient's plasma. samples were incubated for hour at room temperature and antibody screens were repeated. serial two-fold dilutions were also tested to determine if the neutralization could be titered. testing was repeated using various positive patient samples to determine if negative/positive combinations resulted in different reactivity. results/finding: all control samples remained positive. positive/negative samples were negative in solid phase testing across all patient combinations at : dilutions. variable reactivity was observed in gel. serial dilutions showed that neutralization for negative patients was observed up to a : dilution. conclusion: results suggest that patients' plasma may have a substance that neutralizes the antibodies. there is a possible correlation with patients who have persistent negative antibody screens and patient response to daratumumab. additional studies are necessary to uncover how this correlates to patient outcomes. further studies using a standardized daratumumabspiked sample will be conducted. background/case studies: the mns blood group is a red cell antigen system located on glycophorin a (gypa) and glycophorin b (gypb). individuals lacking gypa or both gypa and gypb on their red blood cells may develop a rare antibody against the en (a) antigen. the en (a) antigen is a highprevalence antigen, located on gypa. we present a case with a rare red cell phenotype and alloimmunization to the en (a) antigen. a y/o g p at approximately weeks gestation was discovered to have an anti-en (a) antibody in her plasma on a prenatal type and screen. this was worrisome for both mother and fetus, as the en (a) antibody is of igg isotype and has been implicated in both acute and delayed hemolytic transfusion reactions and hemolytic disease of the fetus and newborn (hdfn) [ , ] . further testing with red cell antisera revealed that the patient lacked m, n, s, s, and u antigens. a multiplex, allele-specific, pcr platform we commonly use to detect the presence or absence of red cell gene sequences failed to amplify genes specific for the m, n, s, s, and u antigens. these findings were consistent with a null phenotype for both gypa and gypb antigens, i.e. m (k) m (k) phenotype. the patient's husband and father of her unborn baby demonstrated a m n-s s phenotype by the same serological and molecular means. given the exceedingly rare incidence of en (a-) individuals (positive frequency > . ), clinical encounters with alloantibodies to this antigen are limited in our experience and in the literature [ , ] . however, the existing data gives credence to its association with transfusion reactions and hemolytic disease of the fetus and newborn (hdfn). the consensus in this case was to work her up as a high-risk pregnancy with frequent intensive monitoring which involved frequent monitoring of antibody titers. if transfusions were required for the mother or fetus, our options were to either search for rare units lacking the en(a) antigen via rare blood donor registries or directed donations from family members who match the patient's phenotype. at term, the patient underwent induction of labor and successfully delivered a health baby boy by vaginal route. the delivery was without event. no transfusions were necessary antepartum or postpartum. study design/methods: n/a results/findings: n/a conclusion: anti-en(a) is a rare antibody and there is limited data about its potential clinical sequelae, which is concerning in a pregnant woman. providing this patient with rare en(a) negative red cells via national or international blood donor registries would have been an arduous task if needed. this patient had many compatible family members available and willing to donate blood. the m(k) null allele (s) within this family is likely due to a genetic recombination among the gypa and gype genes rather than a mutation in both the gypa and gypb genes [ ] . this results in the absence of glycophorins a and b and the constitutive antigens of the mns blood group system. our patient was exposed to the en(a) present on glycophorin a on her unborn baby's red cells (inherited from father) in utero with subsequent alloimmunization. in conclusion, this case report demonstrates a clinical approach in identifying a rare anti-en(a) antibody in a prenatal sample. the clinical finding of a rare antibody in which there is limited data requires leveraging every resource available in order to predict its behavior and provide safe blood products to patients who may require it. background/case studies: transfusions are essential for patients with scd and thalassemia to maintain growth and development during childhood and to sustain good quality of life during adulthood; however, the development of red blood cell (rbc) alloantibodies and autoantibodies complicates transfusion therapy in such patients. routine phenotyping of blood recipients and the use of phenotype-matched blood units for transfusion has been useful to lower the occurrence of red cell alloantibodies in chronically transfused patients with thalassemia and scd. nevertheless, extensive phenotyping is expensive, laborious and cannot be performed in certain situations. the molecular understanding of blood groups has enabled the design of assays a transfusion vol. supplement s that are being used to better guide matched red blood cell transfusions and to maintain an inventory of units dna typed. based on this, our aim was to evaluate the clinical outcomes of molecular matching performed at different levels during years for patients with scd and thalassemia. study design/method: blood group genotypes were determined in dna samples from chronically transfused patients with scd, in patients with thalassemia and in dna samples from blood donors. laboratory developed tests (ldts), hea beadchip tm , rhd beadchip tm , rhce bead-chip tm , and sequencing were used to determine the genotypes among patients and donors. molecular matching was performed in levels: ( ) rh and k matching; ( ) extended matching and ( ) extended matching including rh variants. we considered the total of red blood cell units requested for each patient and a number of donations per year for the compatible donors. results/finding: according to the patients needs we performed molecular matching for % of our thalassemic and scd patients at level , % for scd patients and % for patients with thalassemia at level and % for patients with scd and % for patients with thalassemia at level . the patients were transfused with a median of . rbc units. after three years of molecular matching, we observed that this transfusion strategy avoided new alloantibodies development and hemolytic transfusion reactions in all studied patients. conclusion: molecular matching has shown clinical benefits to the patients with scd and thalassemia, contributing significantly to reduce the rates of alloimmunization to - % with c e k matching and < % with extended matching. improvements in the clinical outcomes of the patients have also been observed as shown by an increase in their hb levels and reduction in the % of hbs in scd patients, better in vivo rbc survival and diminished frequency of transfusions. allahna lilly elahie* and sandra fazari. hamilton regional laboratory medicine program background/case studies: the ideal manual backup method for an automated antibody detection system is an important choice. currently, our backup method is saline tube ( drops plasma, minutes incubation). the change to either a low ionic strength solution (liss) or polyethylene glycol (peg) method would reduce incubation time to minutes and specimen volume to drops, both important laboratory considerations. objectives of this study were to compare the relative sensitivity, specificity, positive predictive value (ppv) and negative predictive value (npv) of peg and liss, and to determine the most appropriate manual backup method for the existing automated solid phase system. study design/method: a total of specimens were compared utilizing: automated solid phase red cell adherence assay (sprca) with manual tube peg and liss, some samples were not sufficient quantity to test in liss. identification panels were used to determine: clinically significant antibodies, warm autoantibodies, and nonspecific reactions. calculations were based upon comparison to sprca. results/findings: a total of clinically significant antibodies were detected using sprca technique, as well as warm autoantibodies and nonspecific reactions. peg demonstrated the highest sensitivity and lowest specificity while liss was least sensitive and most specific for clinically significant antibodies. for warm autoantibodies, liss was more sensitive than peg with both being % specific. both reduced the detection of nonspecific reactions. while peg had more nonspecific reactions ( versus ), it identified more clinically significant antibodies ( ) than liss ( ). (table) conclusion: ultimately, the decision to choose a manual backup method must be based upon the highest sensitivity for clinical significant antibodies so as to minimize failure to detect one. peg was selected as the backup manual method even though peg has a higher sensitivity to nonspecific reactions. this study clearly demonstrates the interplay and tradeoffs between methods, which are important to understand and consider when making method choice decisions. comparison of thiol reagents in denaturing cd on rbcs patricia a arndt* , anthony salazar and regina m. leger . american red cross blood services, long beach memorial medical center background/case studies: monoclonal anti-cd , e.g., daratumumab (dara), which is used to treat patients with multiple myeloma, causes positive indirect antiglobulin tests (iats) due to expression of cd on red blood cells (rbcs). this serologic reactivity cannot be removed by adsorption so other methods have been developed to detect/identify underlying alloantibodies. one popular method is to denature the cd antigen by treatment of rbcs with thiol reagents, e.g., dithiothreitol (dtt) or aminoethylisothiouronium bromide (aet). chapuy et al described ( ) and validated ( ) ), and % aet (ph . ) as per the aabb technical manual, th ed. these treated and untreated rbcs were stored in alsevers at c and tested on days , , , and by two methods: ) polyethylene glycol (peg) iat using plasma from two myeloma patients who had received dara (plasmas from total dara patients were tested with reactivity - ), and ) flow cytometry using phycoerythrin (pe)labeled anti-cd . rbcs were also tested on days and or with a serum containing anti-k by peg iat. results/findings: the . m dtt in ph . pbs had a final ph of . and the ph of the commercial . m dtt was . . results are in table ; flow cytometry results from days , and (data not shown) were similar to those from days and . rbcs treated with . m dtt (both sources) or aet were nonreactive with anti-k and plasma from all dara patients and gave very low results (% positive events) with pe anti-cd by flow cytometry for up to days after treatment. rbcs treated with . m dtt reacted similarly to untreated rbcs with anti-k and dara plasmas, and showed only some weakening ( - %) of reactivity with pe anti-cd . background/case studies: clinically significant hemolytic disease of the fetus and the newborn (hdn) is often caused by feto-maternal rhd incompatibility. with the discovery of cell-free fetal dna (ccfdna) in maternal plasma, it became possible to determine the rhd genotype of the fetus using non-invasive techniques. however, the reliability of the non-invasive prenatal rhd test (nip rhd) is dependent on sufficient amounts of cffdna in the maternal plasma sample. recent studies show that the fraction of ccfdna in maternal plasma varies significantly between pregnant women and is inversely related to maternal body mass index (bmi). thus, high maternal bmi, may impair the validity of nip rhd. the aim of this study was to examine the effect of maternal bmi on the correctness of nip rhd and the correlation of maternal bmi with fraction of ccfdna to total free dna in the sample. study design/method: measurements of body height and weight of pregnant rhd negative women in gestational week were obtained from patient records and used for the calculation of maternal bmi. data on bmi were combined with the results from nip rhd (real-time pcr targeting rhd exon and ) and sample fraction of ccfdna (measured as threshold cycle [ct] value of rhd) to total free dna (measured as ct of ccr ) in gestational week . the correctness of nip rhd was determined by correlation with postnatal serological rhd determination. results/finding: a total of pregnant women were included. nip rhd was positive in / ( %), negative in / ( %) and inconclusive in / ( . %). compared to the postnatal rhd type, / ( . %) of nip rhd results were false positive (fp) and / ( . %) were false negative. in / ( %) of inconclusive nip rhd, the postnatal rhd type was positive. mean bmi (n ) at gestational week was . ( -and -percentiles: . - . ). there was no difference in mean bmi between individuals who tested inconclusive or false negative by nip rhd compared to the remainder (p , ). the fraction of ccfdna was calculated for randomly selected nip rhd true positive cases. median ccfdna ratio was . (the distribution had a highly positive skew, -and -percentiles: . - . ). there was no statistical correlation between bmi and fraction of ccfdna to total free dna (r , ; p . ). conclusion: neither the correctness of nip rhd test result nor the fraction of cffdna to total free dna appear to be correlated to maternal bmi with regard to maternal plasma samples drawn in the th gestational week. delayed hemolytic transfusion reaction due to anti-lan antibody: a case report. adla dh angelina*, suneeti sapatnekar and suzanne bakdash. cleveland clinic background/case studies: lan is a high-prevalence antigen and the sole member of the lan blood group system. anti-lan is a very rare igg antibody, with conflicting information regarding its clinical significance and potential for hemolysis. we report a case of delayed hemolysis in a patient with anti-lan antibody. study design/method: the patient's medical record and available literature were reviewed. results/finding: an year old man, o-positive, with a history of heart disease and bladder cancer was admitted for radical cystectomy. the antibody screen and panel were panreactive by multiple test methods (gel, liss, peg) with negative autocontrols and dat and a saline antibody titer of , suggestive of an antibody to a high-frequency antigen. anti-lan antibody was identified by a reference laboratory. only in , donors are lan-, but two frozen rbc units were locally available and transfused postoperatively. the patient's siblings were tested; one o-positive, lan-sibling was identified. nine months later, the patient was admitted for surgical management of metastases. at this time, the antibody screen was weakly reactive with cell and new antibodies were ruled out. blood conservation measures were instituted, including limited blood draws and cell salvage for surgery. due to bleeding during and after surgery, lan-rbc units were transfused over days, including rare donor units and units from the sibling donor. another surgical procedure was then performed; by post-operative day , the patient had symptomatic anemia with hemoglobin (hb) . g/dl and serially increasing troponin. no lan-rbc units were available. four rbc units untested for lan were transfused without adverse event; the units were presumed lan but crossmatch compatible and phenotypically matched for the patient's other antigens. a post-transfusion hb of . g/dl was maintained for days. the antibody screen was negative on day post-transfusion, but strongly panreactive on day , with a positive dat (igg , c ) and anti-lan antibody identified in the plasma and eluate. there was also evidence of extra-vascular hemolysis, including a progressive decrease in hb from . g/dl on day to . g/dl by day with no bleeding identified, and increase in total bilirubin and ldh (peak . mg/dl and u/l on day ) with normal haptoglobin. the patient was febrile with leukocytosis, but had negative cultures and no other evidence of infection. a lan-rbc unit was transfused on day with good response (hb . g/dl). the patient remained stable and was discharged to a skilled nursing facility days later. conclusion: transfusion of lan rbcs caused a resurgence of anti-lan antibody and a delayed hemolytic transfusion reaction days after transfusion. the rarity of lan-units may require a patient with anti-lan to be transfused with lan units, but close monitoring for delayed hemolysis is necessary even if the antibody is not demonstrable at the time of transfusion. delayed serologic transfusion reaction caused by auto-anti-f. karen yunker* , andrea gerner , lynne stewart , carol sostok , mollie bell and gregory r halverson . st. elizabeth healthcare, hoxworth blood center background/case studies: anti-f was first described in by rosenfield and coworkers in the serum of a hemophiliac who had been multiply transfused. the f antigen is comprised of the c and e antigens alligned in cis on the same chromosome, and is the th antigen assigned to the rh blood group system (isbt rh ). it is capable of causing significant transfusion reactions and mild hdfn. we report in this case a year old caucasian male, admitted for evaluation of suspected t-cell lymphoma, who appears to have had a delayed serologic transfusion reaction (dstr) due to auto anti-f. abstract study design/method: antibody screen and compatibility testing was performed by automated solid phase (echo and neo, lmmucor, inc). red cell phenotyping was done by standard tube testing with commercial reagents following the manufacturers instructions. molecular genotyping was performed using the bloodchip assay (grifols, san marcos tx). elution studies were performed using the elu-kit ii (lmmucor, inc.) results/finding: the initial antibody screen (as) was negative and the patient was transfused unit o-rbcs. two weeks later the patient received an additional o-rbc. within days the hgb had decreased from . to . g/dl, the as and direct antiglobulin test (dat) were now positive, and ounits were incompatible. anti-f was identified in the patient's plasma and eluate. three additional units were requested for transfusion. due to the rarity of o-f-rbcs, the patient was transfused r r (dce/dce) rbcs with no reported complications. the patient was discharged to follow up in clinic. molecular genotyping showed the patient was rhd deleted (rho* del) and had normal rhce (rhce*ce/rhce*ce) genes which predict a d-c-e-c e f phenotype. the rh phenotype and as was repeated on a sample collected days later. the c typing was micro positive, mixed field only after minute incubation. the other rh antigens were not mixed filed, and the as was non reactive. however, the dat was weakly positive with anti-lgg. no elution study was performed. conclusion: the expected post hour hgb increment from the receipt of a standard unit of blood should be near g/dl (or % hct.) throughout this patients hospitalization, the post-transfusion increments did not fully achieve this expectation. the first transfuion resulted in a . g/dl increase, and the second unit was only . g/dl. the last transfusion of units increased by only . g/dl. less than three weeks later, the rhc antigen typing was microscopic/mixed field only after extended incubation, indicating the removal of r r units was nearly complete. in a case from , ohto and kariyone (transf. ; vol , no. ) reported a cr Ásurvival study of f rbcs in a patient with anti-f. they showed that the initital survival of f cells was fairly normal, however, after days, there was a sudden increase of red cell destruction, and by day all f cells were cleared from the circulation. it is not unusual to find auto-anti-f as many have been reported, however, it is unusual to find the auto-antibody has caused the clearance of three units of f-negative blood. this patient will be monitored to see if the autoantibody recurs and determine if it still has anti-f specificity. background/case studies: use of dithiothreitol (dtt) treated reagent red cells (rrbc) is increasing in blood banks as an effective way to negate the interfering panreactivity caused by daratumumab, an anti-cd drug for treatment of multiple myeloma. daily preparation of dtt-treated rrbc for testing of individual patients is burdensome for the laboratory and may delay patient care. we evaluated the effectiveness of batch-prepared dtt-treated rrbc, stored up to days after treatment, in antibody detection tests. study design/methods: in-date rrbc (ortho clinical diagnostics, raritan nj) were selected based on phenotype to match the antisera to be tested. rrbc were treated with . m dtt (sigma-aldrich, st. louis mo) and stored in reagent red cell diluent. rrbc were tested with commercial antisera (ortho clinical diagnostics, raritan nj and immucor, norcross ga) per the manufacturer's instructions for specificities from the rh, duffy, kidd and mns blood groups (see table ). patient source antibodies (anti-d, anti-c) were also tested. testing was performed before dtt treatment, on the day of dtt treatment and up to days following the dtt treatment of rrbc. reactions were graded using standard serological grading of (negative) to (positive) reaction strength. stored dtt-treated rrbc were also observed for hemolysis during the storage period. results/findings: see table for a summary of results. commercial monoclonal and human source antisera, and patient source antibody, were reactive with the dtt-treated rrbc throughout the storage period. reactivity decreased by less than one reaction grade for all antisera and patient source antibodies tested. mild to moderate hemolysis was noted in the dtttreated rrbc's during the storage period. conclusion: dtt-treated rrbc showed adequate reactivity with various red cell antisera after storage for up to days. this suggests that dtttreated reagent red cells can be stored for at least days and used for the detection of alloantibodies with minimal effect on detection ability. batch preparation and storage of dtt-treated rrbc can increase testing efficiency and decrease turn-around-time when performing pre-transfusion testing for patients receiving anti-cd therapy. interference: more than just kell? marilyn stewart*, angela treml and geoffrey wool. university of chicago background/case studies: daratumumab (dara) is an anti-myeloma and anti-lymphoma agent that is known to interfere with routine blood bank antibody screening tests. dara is an igg monoclonal antibody that binds cd that is present on the red cell surface. at the university of chicago blood bank, we have seen many patients treated with dara and were showing this interfering reactivity. it has been well described that cd is a disulfidelinked molecule and its immune epitopes are disrupted by reducing agents such as dtt. we performed a validation of dtt-treatment of reagent rbc to abrogate dara interference. study design/methods: the validation was done to prove that dtt treated red cells could be used to screen patients receiving dara and still detect clinically significant allo-antibodies. screening cells and panel cells selected for dtt treatment were those rbc homozygous for clinically significant antigens, therefore allowing rule-outs of clinically significant antibodies in patient plasma. several patients that had received the dara drug protocol were selected for testing as well as many patients that had allo-and auto-antibodies (but not dara treatment). reagent screening cells and panel cells were treated with . m dtt prepared using the sop from judd's methods in immunohematology and the aabb technical manual. the treated cells were preserved between testing episodes using alsever's solution, stored at abstract - c, and observed for hemolysis (none was seen) for up to days. all immunohematology testing using dtt-treated cells was performed using gel methodology. untreated and dtt treated cells were tested with anti k before any patient testing was done. the untreated cells reacted - with the anti k, and the treated cells were negative. these controls were run and tested each time dtt treatment was done. thirty eight patient samples, including six dara patient samples were tested. results/finding: of the six patients who had dara interference in their untreated antibody screens, all samples had negative reactions with the dtt treated cells except one patient, which had weak reactions in one cell. this specimen was repeated three times and all repeats had weak positive reactions in the same cell. this sample was sent to the arc reference lab for dtt treatment and all clinically significant antibodies were ruled out. patients with allo-antibodies present in their plasma did react with the dtt treated cells as would be expected based on the underlying alloantibody, with the exception of newly formed anti-e antibodies in patients. plasma from these four patients with a nascent anti -e all showed no reactivity with dtt treated cells. plasma from fourteen patients with a long history of anti-e (greater than months) did react with the dtt treated cells. conclusion: dtt treatment eliminates dara interference as previously described, but also unexpectedly lessens the ability of treated cells to react with nascent anti-e. because of the negative testing with some of the alloanti e antibodies, dara-treated patients at ucm will be given both kell and e negative blood if they have immunohematology testing performed using dtt reagent cells. mahboubeh rahmani* , monique scott , garcia curtis , ellice wong , alexa j siddon and christopher a tormey . yale-new haven hospital, va connecticut healthcare background/case studies: benign ethnic neutropenia (ben) seen in approximately % to % of persons of african descent is characterized by neutrophil count of < . x /l with no obvious cause and no increased susceptibility to infection or any other adverse effect. at present, there is no laboratory assay used to identify this condition and it is generally diagnosed on a clinical basis. in this study, we investigated whether duffy (fy) blood group phenotyping would be a potentially useful modality to help identify patients with ben; such testing could potentially be used as a surrogate test to prevent unnecessary further work up including bone marrow biopsy in the correct ethnic and clinical setting. study design/method: cases included patients clinically diagnosed with ben; and controls were chosen randomly from the pools of patients from whom a cbc and type and screen were checked for any other reason. cases and controls were tested for the rbc antigens fy a and fy b phenotype using serologic methods. the fy phenotype, absolute neutrophil count (anc), white blood cell (wbc), hemoglobin level, platelet count, and medical diagnoses were extracted from the medical record. where appropriate, data were compared statistically using the mann-whitney u test with significance set at p< . . results/finding: subjects who were clinically identified as having probable ben included patients (mean age . ; all self-identified as african-american; / were male) and controls included patients (mean age . ; self-identified as african american; ( / male). all of the cases ( %) diagnosed with ben had fy(a-b-) phenotype. mean anc ( . x /ul) and wbc counts ( . x /ul) were significantly lower in the cases with ben and fy(a-b-) phenotype (p . and . , respectively) compared with controls (mean anc . x /ul ; mean wbc count . x /ul). there was no significant difference in mean platelet counts ( x /ul vs x /ul; p . ) or mean hemoglobin levels ( . g/dl vs . g/dl; p . ) between the two groups. none of the patients with ben had an accompanying marrow-suppressive hematologic disorder based on record review; however, subjects in the control group had accompanying conditions that were potentially marrow-suppressive including hepatocellular carcinoma, acute myeloid leukemia, and myelodysplastic syndrome. conclusion: testing for fy phenotype could potentially be used as a surrogate test in patients with chronic neutropenia in a correct ethnic and clinical setting for the diagnosis of ben. further studies regarding fy phenotyping comparing controls with neutropenia for any reason to our ben population are in progress to better determine the positive predictive value. these tests were compared to the provue for concordance. additional samples tested with anti-igg,-c d were correlated against tube testing for the dat and antigen typing for: c, c, e, e, and k. results/findings: the ih- had % concordance for all blood grouping assays. for ahg assays, the ih- detected an anti-jka e, anti-fya warm antibody, antibody to a high incidence antigen and a warm antibody that were missed by the provue. the ih- identified one additional anti-e not identified on the provue. discrepancies were also noted with the non-cord dat results. five samples were positive on the ih- with anti-igg,-c d vs. tube testing; reflecting the increased sensitivity of gel methodology over tube. the table below summarizes the results. conclusion: this study demonstrated that the ih- analyzer and associated ih-system tm gel cards are equivalent to the ortho provue. with random access capability, minimal operator touchpoints, broad test menu and excellent assay performance, the ih- is an ideal immunohematology system for the hospital transfusion service environment. chris elliott*, susan barnes, fiona lisle, debra smith and whitehouse natalie. background/case studies: the erytra eflexisv r (grifols) is a new fully automated, mid-size analyzer that performs pre-transfusion compatibility testing using dg gelv r technology. erytra eflexisv r analyzer performance, usability and adaptability to different workflows was evaluated in the routine environment of a large uk acute hospital transfusion laboratory. study design/methods: a comparison study was performed between the erytra eflexisv r and erytra (our routine system providing the reference platform). a total of tests were performed on , adult patient samples and donor red cell units. erytrav r eflexis performance was evaluated according to a series of scenarios designed to simulate routine workload using the system in different configurations. concordance between systems was assessed and discrepancies analyzed. time to first result (ttfr), overall turn-around time (tat) total workload from first result to last result (throughput, results/h), manual "hands-on" time and walk-away time were all recorded. for ease of use evaluation, we ranked usability features with number of steps and timing of activities including sample sort and loading, routine testing, post-run procedures, consumables used, and space requirements. fault recognition and messaging was assessed by simulating failures e.g. reagent absence. results/findings: blood grouping, antibody screening, antibody identification (using panels), direct antiglobulin test, red cell phenotyping and serological crossmatching were successfully tested. concordant results between the erytra eflexisv r analyzer and reference method were obtained in . % of samples tested. there were discrepancies, all antibody screening ( false positives, failure to detect a very weak prophylactic anti d and positive reaction not detected on the erytra but panels on both systems suggested a genuine anti cw). ttfr and tat depended significantly on a number of factors including; number and variety of tests requested and whether the stat functions were activated. the analyser seemed to prioritise antibody screening prioritization of the group, especially for stat samples, was considered preferable the laboratory team found the software easy to use with some improvements over existing ertyra software. physical design of the analyser was considered good with easy access to almost all areas. probe changing was quick and simple. while the analyser successfully flagged all error scenarios some messages were considered misleading and could be better phrased. conclusion: results showed the erytra eflexisv r offered a robust automated solution for routine transfusion testing. the device could comfortably deal with a medium laboratory (processing - group and screens per day). it is very flexible being able to deliver grouping, antibody screening and identification, dat, phenotyping and serological crossmatching ,compensating for its' single probe and wash station by clever use of incubators, centrifuges and design features. this allows a compact design with maximum flexibility without compromising on turnaround times cp evaluation of two monoclonal anti-e as reagents for the detection of the rh e antigen and its variants gregory a. denomme* , kathleen bensing , michael schanen , cindy piefer , randall w. velliquette , christine lomas-francis and connie m. westhoff . immunohematology reference laboratory, versiti/bloodcenter of wisconsin, immunohematology and genomics laboratory, new york blood center background/case studies: monoclonal antibodies are used as reagents for automated and manual phenotyping. false negative phenotypings have implications for variant antigens; e.g. altered c antigen mistyped as a cblood unit stimulating anti-c in a c-recipient. the development of new / / / cecf / / / rhce*ce or rhce*ce compound heterozygotes ce g ce g or ce c, g or ces or ceti / / / ce g ce c, g or c, g / / / ce g ces or cemo or ceek or ceek(var) or cern / / / ce c, g ce c, g or cemo or ceti / / / ce c, g/ce g/ g; ces/ceti; cear/ceek; ceek/cejal; cemo/cebi / / / total / / / a transfusion vol. supplement s reagents should include an evaluation of antigen variants to confirm fidelity. we evaluated two monoclonal anti-e reagents with comparator reagent using a large panel of molecular confirmed rh e variants. study design/method: two monoclonal anti-e clones, rd / and rd / , and a licensed comparator anti-e (p gd ms ), all from diagast (loos, france), were evaluated. rbc samples were either recovered from frozen storage (n ) or edta blood from donors (n ) and were tested using a manual tube method or on a pk automated platform. a score ( ) or greater was deemed acceptable for manual tube and a positive call for automated testing. results were tabulated by complexity of rhce*ce alleles (table ) . results/finding: the specificity of the monoclonal anti-e were confirmed using common rhce haplotypes: r r , r r , r r, and rr. twenty-one different rhce*ce alleles were included in the extensive panel: were rhce*ce that were in trans to rhce*ce; were various rhce*ce plus rhce*ce c compound heterozygotes; were rhce*ce or rhce*ce homozygotes; were various rhce*ce and rhce*ce compound heterozygotes. the comparator reagent was negative or unacceptably weak for rhce*ce alleles in trans to rhce*ce (rhce*cear, rhce*cemo, rhce*-cejal, rhce*cehar), with rhce*cear/rhce*ce c compound heterozygote, and with rhce*cecf homozygote. rhce*cear, rhce*cemo, rhce*cejal, and of rhce*cecf homozygotes were detected using the comparator reagent. rd / and rd / failed with and e variants, respectively (table ) . failure to detect the e variants was observed using both manual tube and automated methods for the comparator and the rd / clone. none of the reagents detected e antigen variant expressed on example of rhce*cehar/rhce*ce. conclusion: rd / and rd / anti-e reacted with more e variants than the comparator reagent. the e antigen encoded by rhce*jal and rhce*ar is not always detected when in trans to rhce*ce. however, double-dose expression was detected suggesting that the monoclonal reagents bind weakly to the respective altered e antigen epitopes. the e antigen encoded by rhce*cehar continues to be a challenge to detect. meihong liu*, teresita mercado, orieji illoh, maria rios and zhugong liu. obrr, cber, fda background/case studies: extended molecular typing of a large number of blood donors can increase the likelihood of identifying donor red blood cells (rbcs) that match those of the recipient. this is especially important in the management of chronically-transfused patients and patients with rbc alloantibodies. several high-throughput multiplex blood group molecular typing platforms have been developed to determine blood group antigen phenotypes. targeted next-generation sequencing (ngs) provides comprehensive sequence information focusing on specified genomic regions, and allows the simultaneous detection of genetic variants from multiple genes in a large number of samples. we developed and evaluated targeted ngs assays using two different target enrichment platforms for extended blood group genotyping. study design/method: two custom design platforms sureselect and halo-plex were used independently for preparation of probes that target the entire genes of blood group genes associated with the expression of blood group antigens from blood group systems. we used the illumina's hiseq / system to perform next generation sequencing first on sureselect-enriched genes from dna reference samples with average target design coverage of . %, and then on haloplex-enriched genes from dna reference samples with average target design coverage above . %. twelve samples were enriched and sequenced in both methods to allow a direct comparison. all reference samples were previously characterized for blood group genetic variants in these genes using taqman snp assay and sanger sequencing assay. serological data were also available for these samples. the ngs data were analyzed by clc genomic workbench. sequencing variants were detected and annotated using dbsnp database. blood group genotype calls by the two targeted ngs methods were compared with the reference results. results/finding: for the two targeted ngs methods, we evaluated and compared the target enrichment efficiency, off-target enrichment, quality of ngs, sequencing coverage, and genotype concordance. a higher percentage of the haloplex reads ( . %) were mapped to the target regions relative to the sureselect reads ( . %). the mean sequence coverage depth of the targeted bases was around x for sureselect method and x for haloplex method. some exons, such as rhd exons and , , rhce exon , ermap exons and , cd exons and , cr gene (most exons) and gypb exon , are consistently covered with less than x coverage by both sureselect and haloplex targeted ngs methods. both methods detected rhd gene deletion in a few representative samples. the genotype call concordance on blood group genetic variants was assessed by comparing ngs results to taqman genotyping and sanger sequencing results, and more than % concordance was obtained for both targeted ngs methods. incorrect calls were restricted to four complex blood group genes: mns, rhd, rhce and abo, and involved mainly heterozygous variants and indels. conclusion: using two targeted ngs methods, we have correctly detected more than % blood group genetic variants in selected genes. evidence rhce*cehar does not encode for rh (hr b ) antigen debra j bailey* , trina horn , paul mansfield , najmi qazi , pamela nickle , jessica keller , margaret a keller and jan r hamilton . background/case studies: the rhce gene has many variant forms, yet for many, the phenotypes encoded by these variant alleles is unknown or incomplete. new information can be elucidated when two altered alleles or haplotypes are expressed in an individual with subsequent alloantibody formation. the rhce*cehar allele was first described in and has a phenotype of c e c e w f w , g , hr w , hr , hr s , rh: , rh: with a partial d antigen expression. we describe new information regarding an rh haplotype that includes an rhce*cehar allele and its apparent rh: (hr b ) expression. study design/method: rbc typing was performed by standard tube methods with polyclonal and monoclonal antisera. antibody identification studies were performed by standard tube hemagglutination methods by published techniques. molecular immunohematology testing was performed on genomic dna extracted from whole blood and included hea, rhd and rhce beadchips (immucor) and pcr-rflp analysis for rhce c. c>g and rhd c. c>t. results/finding: a sample from an african american female with a history of an anti-e and anti-k was evaluated for unexpected antibodies. her red cell serologic rh phenotype on an untransfused sample was d c e c e . her plasma contained an alloanti-s and an antibody that reacted strongly with all random e k s reagent red cells except her own. the unidentified reactivity persisted following ficin and dtt pretreatment of reagent red cells. only d and dc red cells were non-reactive in initial tests. differential adsorption studies excluded antibodies to all other common antigens and hr b except e, s and k. when subsequent examples of e s k red cells homozygous for the rhd*diiia-ce( - )-d, rhce*-ce c, g, t haplotype (i.e., r' s /r' s ) and rhd*diiia, rhce*-ce c, g, t/ rhd*diiia-ce( - )-d, rhce*ce c, g, t (i.e., bastiaan genotype) were found to be non-reactive with the patient's plasma, the antibody specificity was determined to be anti-hr b . the patient's red cell antigen genotype identified the following probable rh haplotypes: rhd* , rhce*cehar and rhd*diiia-ce( - )-d, rhce*ce c, g, t. additional antigen typing of the patient's red cells with unlicensed antisera indicated an hr ( of sources) and hr b ( of sources) phenotype. conclusion: the rhd*diiia-ce( - )-d, rhce*ce c, g, t haplotype is one of the rh haplotypes expressed by the original hr b individual bastiaan. the hr b antigen status of red cells of individuals with the rhce*-cehar allele has not been described. we report an individual with the probable rhd* , rhce*cehar and rhd*diiia-ce( - )-d, rhce*ce c, g, t rh haplotypes and production of alloanti-hr b . the specificity of the alloantibody produced and the red cell hr b serologic antigen type supports the conclusion the variant allele rhce*cehar does not encode for the hr b antigen. excluding clinically significant alloantibodies in the presence of interfering antibodies with high-titer, low-avidity characteristics. background/case studies: high-titer, low-avidity antibodies (htla) are a group of clinically insignificant antibodies (ab) directed against highprevalence red cell antigens. they interfere with the exclusion of clinically significant red cell ab and crossmatch testing, leading to long work-ups and potential transfusion delays. we often use automated solid phase red cell adherence assay antibody panels (sp) when htla interference is seen by other methods, and undertook this study to determine its efficacy. study design/methods: a search of the laboratory information system database was conducted for patients with htla between / / and / / . all patient samples with available records of the full serological investigation were reviewed for testing method and results, with specific attention to the value of a given test method in permitting exclusion of clinically significant ab (rule out). results/findings: over approximately years, patients had htla established at least once by titration studies. serological investigations on a total of samples using a combination of gel, sp, and peg and liss tube methods, and occasional dtt and ficin panels, found that htla interference noted most frequently in gel (primary method) was, indeed, less often seen with sp. however, the proportion of cases achieving rule out on sp was no greater than that with peg testing (table) . for samples where rule out could not be performed with a combination of methods, patients were assigned to phenotype-matched transfusions, or testing was referred to a reference laboratory. reference testing on samples was successful in rule out in % of cases. in an additional patient samples, with negative antibody screens, htla were identified upon work-up for incompatible crossmatches. conclusion: sp is useful in avoiding interference from htla, but this conclusion is limited because sp was performed in only % of samples, and the inability to use select cell panels with sp made it difficult to complete rule out on samples containing multiple ab. peg testing was available for % of samples, and was at least as effective. further, manual testing allowed flexibility in selecting test cells when other ab were present. both sp and peg testing may be used alone or in combination to avoid interference due to htla, and can potentially decrease the number of patients requiring phenotype-matched units due to incomplete serological evaluations. background/case studies: the dau family of rhd alleles is characterized by c. c>t (p.thr met). the dau allele harbors only this change, is not associated with depressed or altered d antigen expression, and is the ancestral allele from which other dau alleles are purported to have evolved. srivastava et al (transfusion , : ) recently summarized serologic characteristics and associated anti-d alloimmunization for dau family alleles. we investigated two samples with the c. c>t change referred with weak d antigen expression. study design/method: serologic testing was performed by standard tube methods using licensed anti-d reagents and the albaclone partial rhd typing kit. genomic dna was isolated from wbcs and used in manual and array assays and for amplification and sequencing rhd. results/finding: sample was from a yo multiracial female. her rbcs reacted s at immediate spin (is) and in iat with immucor gammaclone and series and , and mi at is and in iat with ortho bioclone anti-d. rbcs did not react with of (lhm / & / ) anti-d in the partial d typing kit. this pattern did not match any of the defined partial d epitope patterns. rhd beadchip found no changes but rflp detected c. c>t characteristic of dau. rhd sequencing confirmed c. c>t and identified two adjacent changes, c. g>t and c. g>t (c. _ delinstt), in exon encoding p.gly leu. sample rbcs reacted w at iat with both ortho bioclone and quotient albaclone delta, but were non-reactive with immucor gamma-clone, series and , and quotient albaclone blend and alpha anti-d. papain treated rbcs were s in iat with ortho bioclone. these results suggested a d el like phenotype. rhd beadchip found no changes but rflp detected c. c>t. sequencing confirmed c. c>t and found a new c. c>t change (p.ser -leu) in exon . the c. t has not been reported, but c. g encodes a stop codon (p.ser stop) in japanese (vox sang , : ). conclusion: we report two new alleles: rhd with c. _ delinstt (p.gly leu) and rhd with c. c>t (p.ser leu), both also carrying the c. c>t (p.thr met) characteristic of the african dau cluster. d antigen associated with p. leu is a partial d antigen with a novel epitope pattern. the p. leu change is associated with a del-like phenotype, the first observed to our knowledge for a dau allele, and d antigen on the rbcs is not detected in iat by / commercial anti-d. the rhd nucleotide changes reported here are not in dbsnp database. this study brings the dau family of alleles to . the number and diversity of alleles in the dau cluster supports that the c. c>t change is a major ancestral african background allele (wagner et al, blood , : ). tae eun kim*. krc btri background/case studies: there have been the cases of anti-d alloimmunization caused by the transfusion of serologically d negative blood component. by analysis of genotype of the blood component, all of them were confirmed as asian type del. for that reason, the application of genetic analysis for the blood donor has been required in addition to serological assay. we established the algorithm for the genetic analysis of rhdin blood donors. in this study, we would introduce the experience of the application of the algorithm and the results in the preliminary test. study design/method: from september to present day we got samples of repeated blood donors who are known to be d negative, c positive and/or e negative from blood centers. we obtained the consent for the test from all of the donors who provided samples. as a genetic analysis, we accomplished polymerase chain reaction with sequence-specific primers (pcr-ssp) for the region of promoter, exon , exon and exon in rhd gene. based on the results of pcr-ssp, we discriminated the results into total rhd deletion, rhd-ce-d hybrid and rhd variant. when the results were discriminated to be rhd variant, we additionally analyzed the sequence of exon to confirm the existence of c. g>a and c. t>a variations. for the sample with indeterminate results, we performed sequencing for the full region of exon. when the result was confirmed to be rhd deletion or rhd-ce-d hybrid, the blood components were regarded as rhd negative. when the result was confirmed to be rhdvariant, the blood components were regarded as rhd positive. blood components were not supplied until the final results were obtained. results/finding: for the sample, we identified cases ( . %) of total rhd deletion, cases ( . %) of rhd-ce-d hybrid, and cases ( . %) of rhd variant. of rhd variant were determined to be asian type del with c. g>a variation. cases of rhdvariant were regarded to be unknown variation. conclusion: the frequency of rhd variant in this study was % higher than that of the general d negative donors not considering rhce phenotype in a previous study. for that reason, we considered that the genetic analysis of rhd targeting the donors of d negative, c positive and/or e negative is more efficient approach to identify rhd variant and better way to improve blood safety in the transfusion medicine related with rhd negative blood donors. lei fang tsai*, ping chun wu, shu hui feng, yi wen tsai, ming hung chen and shun chung pai. taipei blood center, taiwan blood services foundation background/case studies: certain abo subgroups or physiologic conditions may lead to mixed-field agglutination on abo typing among blood donors. the b phenotype was found to be the most common subgroup in taiwanese. however, it is hard to distinguish the b phenotype from other b subtypes also with mixed-field agglutination using routine serology without the genotype. this study aimed to evaluate if flow cytometric method could alternatively differentiate different b subtypes with mixed-field agglutination rather than using molecular genotyping. study design/method: blood samples from taiwanese blood donors exhibiting known common abo phenotypes were included to establish normal flow cytometric patterns and genotyped. blood samples (n ) from b subtype donors with mixed-field agglutination by routine serology (tube method and gel card) were further analyzed by flow cytometry and genotyping. flow cytometric method was performed by facscalibur flow cytometry using the gamma-clone anti-a and -b. for genotyping, exon and exon of the abo gene were amplified and sequenced. the abo*b . allele was confirmed by pcr-rflp analysis. results/finding: among subjects with b or ab phenotypes, were genotyped as abo*b . . the abo*b . group performed similar characteristic flow cytometric pattern and the profile was reproducible over time. the pattern showed the main population of cells expressed no b antigen, while a percentage ( . . ) of the rbcs exhibited b antigen levels diminishing with increasing of fluorescence. other subjects with b or ab subjects, genotyped as abo*b . (n ), abo*bw. (n ), abo*bw. (n ), abo*bw. (n ) and abo*bw. (n ), displayed flow patterns differed from the abo*b . group. the abo*bw. , abo*bw. and abo*b . subjects also showed a main population of cells expressed no b antigen and, however, less percentage of rbcs exhibited b antigen levels (< % in abo*bw. and abo*bw. subjects and < % in abo*b . subject). both abo*bw. and abo*bw. displayed a wedge-shaped pattern. conclusion: the flow cytometric method for the detection of b antigens on rbc might be useful in discriminating between b subtypes with mixed-field agglutination, especially abo*b . genotype. this approach could assist the serological abo subgrouping in clinical reference laboratory. frequencies and specificities of "solid-phase only" detected erythrocyte antibodies: is solid phase testing worth the headache? karen finegan*, karen gray, jill adamski, theresa kinard and qun lu. background/case studies: an effort to re-evaluate automated testing platforms (automated solid-phase red blood cell adherence vs automated gel column agglutination) was recently initiated due to the perception of excessive equivocal reactions from the solid-phase resulting in "unnecessary" workup at one site of a hospital system. the data available from parallel testing on solid-phase, gel, and peg performed at another cite of the same hospital system was collected and evaluated to determine the frequencies and specificities of "solid-phase only" detected erythrocyte antibodies and to see if solid-phase only antibody workup is necessary for patient care. study design/methods: throughout , the transfusion service used automated solid-phase red blood cell (rbc) adherence as the primary method for antibody screening and identification. all solid-phase antibody screen positive samples were re-tested using both gel column agglutination and peg method manually in order to determine which method should be used for antiglobulin phase crossmatch of rbc products. all antibody screen results on three methods and final antibody identification results were transcribed into a spread sheet and analyzed. results/findings: a total of patients were positive on solid-phase antibody screen and re-tested on gel and peg antibody screen. in % (n ) patients antibody reactivity observed in solid phase only and the concurrent gel and peg testing were completely negative. of them clinically significant rbc alloantibodies, warm autoantibodies, clinically insignificant antibodies were identified in % (n ), % (n ), and % (n ) of the cases, respectively. rbc alloantibodies identified in solid-phase only included anti-e (n ), anti-jka (n ), anti-k (n ), anti-jkb (n ), both anti-e and anti-c (n ) (see table ). conclusion: solid-phase only rbc antibodies are clinically important in a significant portion of cases (roughly in cases). workup for solid-phase only antibodies is not "unnecessary" workload. transfusion of corresponding antigen negative rbcs to these patients prevented possible hemolytic transfusion reactions. full-length nucleotide sequence of ackr alleles encoding duffy (fy) antigens in africans of ethiopia qinan yin*, kshitij srivastava, addisalem taye-makuria and willy a flegel. background/case studies: the human ackr gene (previously known as darc), comprising two exons and a single intron, encodes a multi-pass trans-membrane glycoprotein expressing the duffy blood group antigens (fy). the duffy protein acts as a chemokine receptor for various proinflammatory cytokines and for the malaria parasites plasmodium vivax and p. knowlesi. the study of fy variants in the low altitude and tropical gambela region is important, as malaria is endemic and the endogenous population is living in this region for a long time. in the present study, we determined the full length nucleotide sequence of the ackr gene encoding fy antigens in donors from ethiopia's southwestern gambela region. study design/method: edta-anticoagulated whole blood was collected from study volunteers in the gambela region (nct ). the whole ackr gene was amplified in one reaction covering , base pairs (bp). this primary amplicon was re-amplified using nested primers covering nucleotides. nucleotide sequence was obtained by sequencing reactions and manually annotated using ncbi refseq ng_ . . the sequencing covered bp of both exons, bp of intron, bp of '-flanking region, bp of '-utr, bp of '-utr and bp of '-flanking region and encompassed all the variations present in dbsnp and nhlbi esp databases. results/finding: among the samples, a total of snps, including one novel snp in '-utr were observed. snps occurred in the exons, in 'and 'flanking region, in '-utr and in the intron. all individuals carried the snp indicative of the common fy: phenotype; while individuals were homozygous and was heterozygous for the gata box mutation. no splice site mutation was detected. as individuals were observed as being homozygous or heterozygous for snp, we could unambiguously assign distinct alleles. in the remaining individuals with or more heterozygous snps, allele specific pcr is required to identify the alleles. conclusion: we sequenced more than . kb of the ackr gene and identified at least different alleles. the present study found that the vast majority of alleles ( / ) in the gambela population as defined by snps, were similar to the clinically relevant fy* n. allele, which in turn is defined by only snps at positions c. - t>c and c. g>a. out of the remaining alleles, were similar to fy* with the fy(b ) phenotype and was similar to fy* w. with the fy x phenotype. the high frequency of fy* n. ( %) in this study is similar to other studies conducted in western, central and south-eastern regions from gambia to mozambique ( %- %). a more detailed analysis, including other regions of ethiopia, will be useful to support transfusion care in the us for ethiopian-americans, the majority of whom may be of mixed ethiopian ethnical background. judith aeschlimann*, sunitha vege, christine lomas-francis and connie m. westhoff. immunohematology and genomics laboratory, new york blood center background/case studies: the homology, proximity, and inverted orientation of rhd and rhce on the chromosome favor gene conversion events. regions of rhd are transferred into rhce and conversely, resulting in hybrid alleles that encode novel or the absence of high prevalence antigens. rhd*diiia-ce( - )-d is the most common hybrid and is found in african blacks. it arose by conversion of exons - of rhce*ces into rhd*diiia and no longer encodes d antigen, rather (somewhat confusingly) encodes partial c antigen from the rhd locus. this hybrid allele is in cis to rhce*-ces, together known as the r's haplotype. we investigated atypical rh genotyping results in three samples; two associated with weak d typing and one patient with sickle cell disease (scd). study design/method: serologic testing was by standard methods. genomic dna was isolated from wbcs. all samples were investigated by hea precisetype, rhd and rhce beadchip, rflp, and rh-cdna sequencing. snp-specific sequencing was used to establish linkage/phasing. results/finding: sample (male) and sample (multiracial female), both c c e e (presumed r r ), presented with weaker than expected d typing; is and / at iat. rhd beadchip identified the common african rhd*diiia-ce( - )-d hybrid encoding partial c antigen with apparent conventional rhd in trans. these results did not provide an explanation for weak d antigen. hea indicated rhce*ce /ce, concordant with the rh phenotype, but c. c>g and c. g>t (heterozygous) was also detected. as rhce*ce with g and t has not been reported, rh-cdna analysis was done. transcripts from the rhce locus included one conventional rhce*ce in trans to rhce*ces with exons and replaced with rhd*diiia, and from the rhd locus, one conventional rhd and the hybrid rhd* diiia-ce( - )-d were found in both samples. sample (scd male), d c e c e , by rh beadchip had one conventional rhd and rhd*diii type , and rhce*ce g/ces. as rhd*diiia type has never been found with either of these rhce alleles, rh-cdna analysis was performed. transcripts representing a unique conversion event at the rhd locus, specifically rhce*ce( c) exons and had replaced those exons in the common hybrid rhd*diiia-ce( - )-d and expression of partial c antigen was lost these unique hybrid alleles have been deposited as genbank#: ky and ky . we report two different and novel complex rh rearrangements: two samples thought to be r r had a unique rhce locus representing a gene conversion into rhce*ces, designated rhce*ces-diiia( - )-ce. in kind, a sample genotyped as diii type rather had a novel rhd locus representing a gene conversion into the common hybrid, designated as rhd*ce c( - )-diiia( )-ces( - )-d . these represent novel events on the r's haplotype that can confound rh genotyping interpretations. interestingly, samples and have weaker than expected d antigen typing, despite the presence of a conventional rhd with rhce*ce [r haplotype (dce)]. it is important to further investigate samples with unconventional results when interpreting rh genotypes. high-frequency antibodies anti-lu(b-) and anti-yt(a-) in a multi-transfused patient: a case study nadia baillargeon*, carole ethier, cynthia parent, jessica constanzo-yanez, maryse st-louis, marie-claire chevrier and andre lebrun. hema-quebec background/case studies: a -year-old caucasian female was referred to our immunohematology reference laboratory (irl) for serological investigation. she was diagnosed with anemia, renal failure and cardiac history. her hemoglobin level was recorded at g/l. her pregnancy history was not provided. she had received units of packed red blood cells (rbcs) in the past including unit within the last months. none of the transfused unit was phenotypically-matched. the referring hospital obtained panreactivity in gel with liss-suspended rbcs and ficin-treated rbcs and negative direct antiglobulin test (dat) and autocontrol (at). study design/methods: abo/rh, dat and antibody identification were performed by h ema-qu ebec's irl according to approved techniques. in addition to liss-suspended rbcs and papain-treated rbcs, trypsin-treated and chemical-treated reagent rbcs such as dithiothreitol (dtt) were tested. alloadsorption were done using papain-treated allogeneic rbcs (r r , r r , rr). id core xt platform (progenika biopharm / grifols, vizcaya, spain) was used to analyse polymorphisms which determine antigens including carthright and lutheran blood groups. pcr-ssp (sequence specific primer) and pcr-rflp (restriction fragment length polymorphism) were also performed to verify the absence of the high frequency antigens yt a and lu b . sibling samples were also requested to conduct a family study. results/findings: initial serologic testing showed strongly reactive panels in gel with liss suspended rbcs, papain-treated rbcs as well as trypsintreated rbcs and dtt-treated rbcs but negative at in each media leading to a probable antibody directed against high-frequency antigen. alloadsorption procedure allowed the identification of an anti-jk a . a select panel of high frequency antigens absent in caucasian population was tested. the patient's sera react weakly with one jk(a-), lu(a-b-) reagent cell. in the meantime, genotyping results confirm the probable phenotype of the patient as jk(a-) lu(b-) yt(a-). additional testing in gel using trypsin and dtt differential effects on antigens lu(b) and yt(a) were performed to confirm antibody specificities. no rbcs unit jk(a-) lu(b-) yt(a-) were available for transfusion. selected units were jk(a-) and lu(b-) as alloanti-yt a are known to cause none to moderate transfusion reactions. her daughter' sample were types as yt(a ) and lu(b ). conclusion: serological study showed the presence of an anti-jk a in addition to two antibodies directed against high prevalence antigen namely anti-lu b and anti-yt a . the association of various selected serologic procedures combined with ethnic clues and genotyping results serves to solve this uncommon antibody combination. background/case studies: the kel blood group system, consisting of antigens encoded by the kel gene, is organized into exons. there are approximately kel alleles associated with a kell null phenotype (k ) in which no kell antigens are expressed, and alleles associated with a kell mod phenotype (k mod ). individuals with the k mod phenotype express very weak amounts of antigen on the surface of the rbc, and expression levels vary based on the allele present. here we describe the molecular and serologic testing that was performed in the case of a year-old hispanic male blood donor whose rbcs phenotyped k-k-js(b-) kp(b-). study design/method: the blood donor was phenotyped for k, k, kp b and js b antigens using standard tube agglutination methods. adsorption and elution studies of the donor red cells were performed using commercial anti-k antisera (american national red cross). genomic dna (gdna) was isolated from an edta blood tube using standard techniques. dna was genotyped for human erythrocyte antigens using the precisetype tm hea molecular beadchip (immucor). exons , , , and and flanking intron sequences were amplified and sequenced. total rna was extracted using rneasy lipid tissue mini kit (qiagen) and kel cdna was amplified and the resulting pcr product was subjected to sanger sequence analysis and aligned using sequencher (genecodes). results/finding: precisetype tm hea molecular beadchip testing predicted the sample to be k-k kp(a-b ) js(a-b ). kel-cdna sequence analysis was performed and detected a single transcript species with c. c, c. c, t, and missing the sequences corresponding to exons , and . amplification of the exons from gdna did not identify any nucleotide changes when compared to the reference sequence and the splice sites were intact. cdna analysis was repeated and the same aberrant transcript was detected. adsorption and elution studies of the k antigen demonstrated weak anti-k reactive after c incubation at the peg-igg-agt phase. conclusion: here we describe a donor homozygous for a novel kel* allele. this donor was presumed to have a k phenotype based on serology, but after molecular testing, has been reclassified as a k mod phenotype with extremely weak expression of k. the discovery of the aberrant transcript led to adsorption and elution studies to confirm the presence of weakly expressed k antigen on the red cells. the variant alleles reported to date (http://www.isbtweb.org/working-parties/red-cell-immunogenetics-and-bloodgroup-terminology/) are associated with missense mutations. in contrast, the allele reported here is associated aberrant mrna transcript. we propose that this allele be named kel* m. . here we report a case of a possible novel b subgroup observed in a pregnant black female. the patient specimen was referred to our reference laboratory to investigate a possible abo discrepancy. the referring facility reported the patient's red blood cells were nonreactive with reagent anti-a and anti-b and the patient's plasma was reactive with a cells, but nonreactive with b cells using automated gel methodology. study design/methods: serological testing of the patient's red blood cells was performed using routine and enhancement methods. molecular testing by pcr-rflp was performed to determine the patient's genetic abo typing and predicted abo phenotype. results/findings: serological testing of the patient's red blood cells is summarized in table ; similar results were obtained with multiple sources of antisera. enzyme treatment failed to enhance reactivity. patient sera strongly agglutinated a and a cells, but failed to agglutinate multiple sources of b cells at all phases of testing. molecular testing by pcr-rflp resulted in an uncommon banding pattern and indicated the presence of c. deleted g, characteristic of o alleles, c. t, characteristic of a and some uncommon o alleles, and c. a and c. a, characteristic of b alleles. genomic sequencing of exons and confirmed the presence of an o allele, abo*o del g, t, t), and the presence of a b allele ( g, g, t, a, a, c, and a), but did not reveal any changes associated with previously reported weak subgroups of b. conclusion: while serologic abnormalities in pregnancy have been reported due to decreased antigen expression, the unusually weak reactions observed when testing this patient are unlikely due to pregnancy alone. additional abo gene sequencing is required to determine the specific allele mutation responsible for this weakened antigen expression. carine arnoni* , tatiane vendrame , janaína muniz , diana gazito , afonso cortez , lilian castilho and flavia latini . associa, associac¸ão beneficente de coleta de sangue, hemocentro de são jos e do rio preto, hemocentro unicamp background/case studies: after the elucidation of the molecular basis of vel, molecular tools have been used to explain the reduced expression of vel antigen in different populations. negative or weak reactions are generally related to the -bp deletion in smim in homozygous or heterozygous status. however, other nucleotide changes have been described to reduce the vel expression, as for example, the major a allele of the snp rs located in the second intron of the gene, a regulatory region in erythroblasts. this study aimed to characterize the genetic changes related to atypical vel expression in a brazilian population. study design/method: a total of blood donor samples from the southeast region of brazil were typed for vel with an anti-vel serum from our inventory in gel-iat. samples typed as vel-negative were further analyzed by adsorption-elution. molecular study was performed in samples with negative results, in samples reacting and in samples with reactivity of . dna was isolated from peripheral blood and smim was sequenced. results/finding: from donor samples studied, were serologically vel negative by gel-iat but positive by adsorption-elution, presented a reaction and the remained samples showed a reactivity of . genotyping results showed that the samples with negative results and of samples that presented reaction were heterozygous for the bp deletion and presented the a allele rs in homozygous status. from the of remaining samples with reactivity of , ( %) had the a allele of rs and ( . %) had the a allele of rs . in contrast, in the samples with stronger reactions we found the a allele of rs in ( . %) samples and the a allele of rs in ( . %) samples. conclusion: the molecular changes rs and rs are located in intron distancing nucleotides. this study reinforces the association of the a allele of rs with reduction of vel expression and suggests the involvement of a new rs change in vel expression. in conclusion, the several patterns of vel expression found in different populations can be influenced by different molecular changes. background/case studies: the d antigen is the most immunogenic antigen after abo. consequences of misclassification of the d-antigen in patients or donors can be severe. some persons inherit mutations resulting in quantitative reductions of d antigen on the cell surface (weak d), some inherit rh haplotypes that result in biochemical effects that reduce the availability of the d antigens to reagent anti-sera (ceppellini effect), and others inherit d genes which are qualitatively different than wild type d. these latter individuals are often not identified until after they have formed anti-d. we hypothesize that some of these persons at risk of forming anti-d might be uncovered if they have weak and/or disparate d typing results with reagents that recognize different epitopes of the d antigen. study design/methods: all testing was performed using microtiter-well direct agglutination on the galileoneo or galileoecho (immucor, norcross,ga). any specimen that did not react as (rh negative), or ! on the neo or ! on the echo (rh positive) for both series and series anti-d antisera were included. patients with discrepant historical types also were evaluated. any specimens meeting the inclusion criteria were tested on the neo, echo, and by saline tube method using series and series anti-d antisera. genotyping was performed from whole blood samples sent to immucor genotyping laboratory in warren, nj using an algorithm of: rhd beadchip, rhdxp (prototype assay), rhd zygosity, and rhce beadchip. results/findings: patients met inclusion criteria for molecular testing for the d antigen. weak or rhd variants were identified in of ( . %) of the samples. ceppellini effect (i.e. c in trans to rhd) resulting in weak d reactivity was seen in of ( %) of samples. of ( . %) of the samples that resulted in weak or discrepant reactivity had some type of genetic cause that was resolved by using our algorithm. of ( %) of tested samples had results indicating weak/variant d proteins with the potential to cause alloimmunization to the d antigen. the remaining of ( . %) samples did not have identified genetic cause for the weak and/or discrepant d test results and were presumptively classified as wild type d. conclusion: transfusion services that use the galileoneo or galileoecho to perform rh typing should consider molecular testing of patients whose rh typing results are discrepant, or positive but < on the galileoneo or positive but < on the galileoecho, as about half of these patients can develop anti-d. this is particularly relevant for females of child-bearing potential where avoidance of d-positive transfusions and administration of rhig during pregnancy is prudent until their d typing can be confirmed by molecular testing. carine arnoni* , tatiane vendrame , janaína muniz , rosangela person , lilian castilho , afonso cortez and flavia latini . associa, associac¸ão beneficente de coleta de sangue, hemocentro de são jos e do rio preto, hemocentro unicamp background/case studies: rhd and rhce, are major protein constituents of red blood cell membrane, composing a complex together with rhag. many variant rh proteins have been described and most of them affect the integration of rh proteins in the membrane. d antigen expression can be affected by several molecular changes and also by the rhcehaplotypes. the present study investigated the score of reactivity of samples presenting a strong reduction in d expression. study design/method: a total of samples were included in the study, being previously genotyped as rhd*dar . , rhd*dar . and rhd*dau . the samples were phenotyped in neov r (immucor) to d, c/c and e/e antigens by direct agglutinationin microplate. results obtained in neov r were expressed in a score from - corresponding to the reaction intensity. zygosity assay was performed by a multiplex real-time quantitative pcr using a set of rhd-specific primers in rhd exon . rhce genotyping was performed by pcr-rflp and ssp-pcr. the presence of a d-cehybridexon was identified by amultiplex pcr. sequencing and identification of rhce variants were also performed when necessary. results/finding: zygosity results showed that of samples ( dar . , dar . and dau ) had rhd genes, were phenotyped as c e-c e and genotyped as rhce*ce/rhcece. rhd and rhce genotyping in these samples showed the presence of the d-ce-d s hybrid gene. rhce variants investigated in dar . samples showed the rhce*-cear/ce s genotype, in dar . samples the rhce*cevs. /ce s genotype and in the dau sample the rhce*ce s /ce genotype. table describes the differences found in the reactivity of d among the samples carrying the (c)ce s allele and in the samples homozygous for rhce*ce. the results showed that the presence of rhce*(c)ce s significantly reduces the expression of d antigen, probably due to the expression of the partial c partial antigen in trans to rhd. additionally, the samples with reduction on d expression carrying rhce variant alelles phenotype can be useful to provide compatible blood to some patients with rarerh variant alleles. background/case studies: drugs are known to interfere with routine blood bank testing. a novel monoclonal humanized f antibody (hu f -g ) that binds human cd has been entered into clinical trials for patients with acute myeloid leukemia, non-hodgkin lymphoma and solid tumors. we describe two cases of patients treated with hu f -g (anti-cd ) who had abo discrepancy with extra-reactivity in the reverse typing and a panaggutinin in the plasma. study design/method: this is a retrospective review of two cases with immunohematology work-up showing abo discrepancy and plasma panagglutinin. the first case is of a year old female with progressive follicular lymphoma who was enrolled in phase b/ trial of hu f g in combination with rituximab designed for patients with relapsed/refractory b cell nhl. she had no prior transfusion history and her historical blood type was not known. two rbc units were requested in anticipation for a surgical procedure. the second case is of a year old male with refractory diffuse large b cell lymphoma enrolled in hu f -g clinical trial. his historical blood type was a rh d positive with a negative antibody screen. he received three rbc units within the past month prior to testing and receiving the anti-cd therapy. results/finding: the abo typing in the first case showed a discrepancy between the forward typing ( with anti-a, non-reactive with anti-b) and the reverse typing ( with both a cells and b cells). rhd typing was positive. the extended reagent rbc panel tested with the patient's serum reacted with all cells tested at the immediate spin (is) phase ( to ), at liss- c ( to ), at liss-polyspecific ahg (m ), and at peg-anti-igg (m to ). plasma reactivity at is persisted with dtt or ficin treated red cells and was not removed by cold autoadsorption, cold alloadsorption, or rest adsorption. repeat testing, which avoided the is and c readings, was non-reactive in the antihuman globulin (ahg) phase using both liss and peg enhancements, ruling out clinically significant alloantibodies directed toward common red blood cell antigens. the direct antiglobulin test (dat) and autocontrol were negative. the rbc units issued to the patient were crossmatch compatible at o c ahg phase. the abo typing of the second case performed after anti-cd administration showed a discrepancy between the forward ( with anti-a) and the reverse ( with both a and b cells). rhd typing was positive. the antibody screen performed in solid phase technology was positive with all reagent red cells. his plasma reacted with all reagent red cells at is ( ), at c in liss ( ), and liss-polyspecific ahg (m ). the dat and autocontrol were negative. his genotype was determined to be a /o and full rbc phenotype by dna analysis was obtained. repeat testing which avoided the is phase did not show reactivity at peg-ahg excluding all alloantibodies directed toward common red blood cell antigens. conclusion: anti-cd therapy interferes with blood bank testing by causing abo discrepancies and panagglutinin reactivity in the plasma at is, c liss, but not at ahg phase using gamma-clone anti-igg, unlike the anti-cd interference. knowledge of patient's blood type and phenotype before starting this therapy is critical for providing safe blood. background/case studies: a middle-aged male with discrepant abo typing results was investigated. initial forward typing was group o but no anti-b was seen in the reverse typing. an unexpected reaction was noted with an anti-a,b reagent. genotyping surprisingly showed abo*o. . / o. . , consistent with group o. after initial testing at the referring center, samples were sent for extended analysis. study design/method: standard serological methods and flow cytometry were used. a panel of abo reagents (n ) and lectins were tested with both native and papain-treated red blood cells (rbcs). lewis phenotyping was performed, as was genetic testing for abo, gbgt and a galt. papain-treated patient rbcs were used to screen donor plasmas (n ) and two reactive plasmas were dtt-treated. results/finding: positive reactions were obtained with polyclonal anti-a,b and a monoclonal anti-b (clone g / ) when tested with the patient's papain-treated rbcs. a panel of lectins gave negative results. genetic testing confirmed the predicted group o and ruled out the presence of fors or nor antigens. the patient was le(a-b ) and thus a secretor. a positive crossmatch was seen with % of group o plasmas, while no reactivity was obtained with a or b plasmas. dtt treatment of crossmatchpositive plasmas indicated the antibody to be mainly of igg type. this was confirmed by positive flow cytometry cross match using anti-human igg secondary antibodies. reactivity remained after b-zyme treatment, thus excluding the normal (type or ) b antigen to be the underlying reason. inhibition with lewis substance significantly decreased reactivity. enzyme activity assay showed the patient's plasma to contain a fully functional b glycosyltransferase. on the suspicion that the patient had non-erythroid cells producing breactive type chains, a sample from a hematopoietic stem cell transplant (hsct) patient (group b secretor receiving group o donor cells) was included as a control and gave the same type of reactions. conclusion: the medical history of the patient was queried and he had indeed undergone an hsct $ years earlier. the reactions are likely due to uptake of recipient-derived ble b (type ) antigen (isbt no. ), which is the dominant lewis antigen in the recipient's original blood group, b le(a-b ). interestingly, b-zyme did not affect ble b . anti-ble b is not simply anti-b plus anti-le b but an inseparable and rarely reported specificity, which appears to be common among group o donors. the phenomenon reported here has unknown clinical implications but highlights the complexities of carbohydrate blood groups. background/case studies: the provuev r and visionv r (ortho scientific, raritan new jersey) automated analyzer use mts-gel tm card technology to perform immunohematology testing. benefits of automated testing include improved efficiency and enhanced reliability. after eight years of using the provuev r our transfusion medicine service switched to the ortho visionv r analyzer in january of . shortly after implementation, technologists reported increased time spent performing manual resolution of indeterminate (designated as "?") results. additionally, some test columns were noted to be visually negative but called positive ( ) by the analyzer. the objective of this study was to investigate the cause of "?" and apparent false positive results on visionv r three-cell antibody screens. study design/methods: with assistance from ortho diagnostics, analyzer archives were queried to identify the number of gel card columns used for screens, the number of columns with "?" results, and the gel card lot numbers used for testing from / / to / / . reactivity was determined to be false positive based on supervisory review of digital images and antibody panel results. investigation also included review of daily qc records, instrument maintenance, instrument diagnostics, and camera calibration. results/findings: of , columns run as part of antibody screens, , ( . %) columns generated "?" results. assuming seconds of technologist time per "?", we estimate that . hours were needed to resolve and update these results. among all potential causes investigated, only the gel card lot number was associated with the number of "?" generated (table ) . in cases, all three columns were visually negative but the analyzer reported reactivity with of cells. all cases had mts-gel tm antibody identification panels performed, of also had a mts-gel tm ficin panel. the yield for the panels performed was two routine panels with weak reactivity against hla cells, and four ficin panels with weak reactivity with no apparent specificity. fourteen patients coincidentally had a subsequent type and screen; were negative. one patient newly demonstrated anti-jka. fifty percent ( / ) of visually negative but analyzer positive samples were tested with gel card lot number , % ( / ) with lot , and % ( / ) with lot . conclusion: the incidence of "?" and visually negative analyzer positive results is dependent on the specific lot of mts-gel tm cards used. the difference between the lots is being investigated by ortho diagnostics, and remains to be explained. to avoid unnecessary waste of technologist time and other resources, with assistance from ortho diagnostics, we have results/finding: of the methods evaluated, the dtt method proved the most useful for mitigating dara interference. cord cells were effective but in limited supply and alloadsorption was ineffective. of the three different dtt methods evaluated, the tube method initially failed which led to re-evaluation with the addition of liss (passed). the gc method was the most sensitive method. following release of dara, samples from patients ( cross-match samples, units issued) were tested using both liss tube and gc iat methods. despite dtt treatment, the gc method remained positive by iat in / patients. further testing was performed in / . eight were tested for the presence of antibodies at c and confirmed in / . rouleaux formation was observed in / patients, / had reactivity detectable at c. no transfusion reactions have been reported to date nor has alloantibody formation been observed to date. conclusion: as previously reported, the dtt method was the most useful for mitigating dara interference. the observed interference seems to be due to rouleaux and/or cold reactive antibodies -seen least in the liss tube iat. this may be due to the washing phase in this technique which dissipates rouleaux formation. reactivity due to cold reactive antibodies can be eliminated by performance at strict c. our practice is now to use both dtt iat methods on initial patient referral, if residual reactivity in gc is observed use liss tube in preference thereafter in these patients. a further observation is that investigation of pan-agglutination could include the use of cord cells to confirm/exclude dara use if suspected. wendy beres* , sandra nance , david moolten and p. dayand borge . ( ): - ). our laboratory tested random allogeneic blood donors, and autologous donors (which were intended to represent a hospitalized patient population), to determine a mean and range of "normal" levels. this . year retrospective study was performed to assess levels of rbc-bound igg, iga and igm in normal donors. study design/method: residual edta-anticoagulated aliquots from random allogeneic and autologous blood donors were sequestered and tested per institutional review board approved protocol. the samples were tested by fc with fluorescein isothiocyanate (fitc)-labeled anti-human igg and iga (jackson immunoresearch lab, west grove, pa) or fitc-labeled anti-human igm (life technologies, carlsbad, ca) at optimized dilutions in dulbecco's pbs containing . % bsa. the becton dickinson facscalibur tm or facscan tm (san jose, ca) fc analyzed k rbcs from each sample. edta-anticoagulated samples and ig coated control rbcs were tested to determine fc settings and control for validity and cross-reactivity. controls reacted as expected. there were less autologous donors tested and with a mean age of these donors could have been older than the allogeneic donors, but the mean age of the allogeneic donors was not captured. despite the relatively small number of samples tested there was a higher than expected instance of allogeneic donors having elevated rbc-bound iga, igg, and igm levels. this emphasizes the need to include testing of normal donor populations in establishing expected reactivity, thus normal and abnormal ranges for flow cytometric testing. long range pcr reveals the genetic basis of an antibody in pregnancy to a high prevalence mns antigen judith aeschlimann* , anna burgos , virginia lew , sunitha vege , susan veneman , christopher j gresens , jonathan hughes and connie m. westhoff . immunohematology and genomics laboratory, new york blood center, blood centers of the pacific, bloodsource background/case studies: recombination events have generated many gypa and gypb hybrids giving rise to glycophorin (gp) variants that express low-prevalence antigens (e.g. mia, miny, mur). in rare individuals who are homozygous these alleles are associated with lack of highprevalence antigens (e.g. enkt, eneh, enav). complex hybrid recombination events can make it challenging to elucidate specific alleles present in samples, particularly heterozygotes. we investigated samples from a pregnant asian (hmong) woman with an antibody to an unidentified highprevalence mns antigen, and samples from her sister. study design/method: standard methods were used for rbc typing with licensed and unlicensed reagents and for antibody identification. dna was isolated from wbcs, and hea precisetype, exon-specific amplification and sequencing gypb exons - , and long range sequencing of exon - ( . kb amplicon) were performed. snp-specific primers were used to associate changes (phasing) to specific alleles. results/finding: rbcs of the pregnant proband typed s-s-(gammaclone anti-s), and the plasma reactivity was consistent with an antibody to a high background/case studies: the rhd antigen is clinically significant and immunogenic and therefore individuals who develop anti-d are at risk of haemolytic transfusion reaction. rhd polymorphism shows substantial ethnic variability and at least rhd variants associated with weak d alleles have been reported. in this study, we report two new rhd alleles in brazilian blood donors associated with weak d antigen expression. study design/method: the d status was evaluated with commercially available monoclonal anti-d reagents: blended igm/igg (clones th- / ms- ), igm (clones ms and p x ) and igg (ms ) in tube and on gel cards. c, c, e and e phenotyping were performed in gel. most common weak d and partial d alleles were investigated by allele specific (as) pcr and with the rhd beadchip platform from immucor. direct automated sequencing of the rhd exons and flanking intron regions was performed by the sanger dideoxy method. in order to determine rhd allelic combinations, we also performed rh-cdna cloning and sequencing. background/case studies: donors negative for multiple common antigens or lacking a high prevalence antigen are efficiently identified using a red blood cell (rbc) genotyping panel. when serology is used to confirm antigen negative status, discrepancies are identified, albeit rarely. investigation of the discrepancy often leads to identification of variant antigens. it is known that the set of gyp variants associated with expression of the st a antigen can also be associated with n typing discrepancies in m n-individuals (meyer et al. br j haematol. ; : - ) . the st a allele, also described as gyp* , is a hybrid gypb-gypatranscript with the crossover in intron . we sought to investigate five n typing discrepancies for which alternative genotyping methodology was performed and found to be concordant with the initial panel. background/case studies: a sample from a years old pregnant, african american female g p was sent to the blood bank for abo/rh and antibody screen. the sample was analyzed using the provue analyzer (ortho diagnostics). the patient was typed as o pos with no reverse type discrepancy. a retype of the same sample was performed using tube method with biorad reagents per hospital policy due to no previous abo/rh history on file. the retype showed that the patient was a subgroup with anti-a antibody present in the plasma. the sample was referred for genotyping, with the suspicion of a like phenotype. genetic testing did not support the serological findings of a subgroup and a new abo allele, abo* c that has never been reported in correlation with an a like subgroup was detected study design/method: the patient rbcs were typed with anti-a (immucor) and anti-a,b (biorad and grifols dg gel). an anti-a antibody work up was performed using three different lots of a cells and three lots of a cells, as well as a type o screening cell and auto control . the tubes were read at is and also incubated at rt and c for min. the patient 's initial antibody screen using ortho gel was negative. conclusion: the patient delivered a healthy baby boy at weeks of gestation. the baby cord was sent to the laboratory. the baby serological type showed an a b phenotype and it was referred for genetic testing. the baby rbcs showed the same abo* c found in the mother. the previously reported abo* a allele encoded an aspartic acid to asparagine change at position p. consistent with an a weak phenotype. also, at least five other alleles encoding an a phenoytpe consisted of polymorphisms at positions c. through c. , giving special characteristics to this new and unreported abo allele. from the data collected, it can be concluded that this a / aweak phenotype is encoded by the variant allele abo* c. this highlights the clinical relevance of confirming the serology of abo subgroups by molecular methods. philip berardi*, jacqueline cote, gwen clarke, vito scalia, robert liwski and mindy goldman. canadian blood services background/case studies: elucidation of the molecular basis of blood group expression has led to the development of high throughput molecular methods for predicting blood group antigens. the commonly used single nucleotide polymorphism (snp) arrays require nucleic acid isolation which is typically achieved by extracting genomic dna from whole blood. this method requires venipuncture and may not be an ideal approach for severely anemic patients or potential donors that are unable to provide a sample of whole blood due to their remote location. dna extracted from buccal swab samples offers a noninvasive alternative to venipuncture and may provide a safe and efficient means of transporting samples from remote locations to reference laboratories for extended blood type prediction. canadian blood services (cbs) has performed large scale dna extraction and hla genotyping for the onematch stem cell and marrow registry using buccal swabs since ; buccal swabs are also used by other unrelated donor stem cell registries, such as the us nmpd. we sought to assess the accuracy and reliability of using dna extracted from buccal swabs in predicting blood group antigen expression. study design/method: we performed parallel red cell genotyping on an automated typing platform, the progenika/grifols idcorext assay (progenika biopharma-grifols, bizkaia, spain) using dna extracted from blood and buccal tissue from volunteers. for antigen systems with available serologic reagents, we also compared results with serologic typing. we evaluated three different methods of dna extraction and performed testing regardless of dna yield or purity. two buccal swabs (puritan medical products, guilford, maine) were used for each test. swabs were stored at room temperature, and dna extraction was performed within six days of collection. in the initial phase of the study, buccal swab samples (n ) were processed with the automated biorobot m robot using the magattract dna mini m extraction method (qiagen, venlo, the netherlands). extracted dna had a mean concentration and purity of . ng/ml and . respectively. in the second phase of the study (n ), dna extractions from buccal swabs were performed using methods available in our national red cell immunohematology reference laboratory: the qiaamp dna mini kit, using either manual or an automated qiacube robotic workstation (qiagen, venlo, the netherlands). results/finding: the manufacturer's recommended analytical range for dna concentration was - ng/ll and the recommended purity was an absorbance ratio of . - . (a / ) for use of the id corext platform. dna extraction from buccal swab samples did not meet these specifications in several cases. however, in most cases, a lower concentration of dna was adequate for prediction of phenotype. the dombrock system was the most susceptible to failure of interpretation in the samples with a low dna concentration, with "no call" results reported. there was % concordance in genotyping results when source dna was extracted from whole blood or buccal tissue; there was also % concordance between predicted phenotype and serologic testing results. conclusion: this study supports the use of genomic dna extracted from buccal tissue on the id corext for predicting rbc phenotype with high accuracy. extraction methods may require optimization to achieve dna yields within the recommended analytical range of the assay. performance evaluation of id rhd xt, a genotyping assay for the detection of high-prevalence rhd negative and weak d types araitz molano , izaskun apraiz , maría azcarate , miguel angel vesga , montserrat rubia , mercedes piedrabuena , fernando puente , barbera veldhuisen , ellen van der schoot and m onica l opez* . progenika biopharma, a grifols company, centro vasco de transfusi on y tejidos humanos, banco de sangre y tejidos de arag on, sanquin blood supply research background/case studies: it is well established that weak d , and phenotypes are not at risk for forming allo-anti-d, whereas a few weak d and all partial d and negative phenotypes are. routine serologic d typing does not distinguish among them, consequently rhd genotyping is recommended, especially in patients. id rhd xt (progenika, grifols) is a qualitative, pcr/luminex v r xmap hybridization-based genotyping test for the identification of the following rhd gene allelic variants: rhd*weak d type , rhd*weak d type , rhd*weak d type , rhd deletion, rhd*pseudogene and rhd*diiia-ce( - )-d and itgb gene: hpa a and hpa b, in genomic dna extracted from whole blood specimens. in this study the performance of id rhd xt genotyping assay was evaluated in terms of whole system failure rate, call rate and accuracy for rh and hpa- blood group typing. study design/method: a cohort of previously serotyped samples for d antigen obtained from three european blood centers were analyzed with id rhd xt at progenika. samples were distributed as recommended by the annex of the common technical specifications / /ce for a ivd product of list a (! % clinical samples, > % neonatal specimens and ! % weak d donors). for the intended use of the product, weak d serotyped donors were enriched (n , %). commercial serology tests for d antigen predicted phenotype and bi-directional-sequencing (bds) for weak d type confirmation and hpa- predicted phenotype were used for comparison. transfusion results/finding: no system failure, % call rate and no inconclusive results were obtained. discrepancies were found for d antigen between serology and id rhd xt predicted phenotype results, although a % concordance was obtained when analyzed by bds, considering id rhd xt result correct. concordance between id rhd xt and bds results for the weak d type was %. the following id rhd xt predicted phenotype results were obtained: d negative (n ), no amplification variant (n ), weak d type (n ), weak d type heterozygous (n ), weak d type (n ), weak d type heterozygous (n ), weak d type (n ), weak d type heterozygous (n ), weak d types , or not detected (n ). regarding hpa- blood group, the predicted phenotype results obtained by id rhd xt were % concordant with bds results: hpa- a positive (n ) and hpa- a negative (n ), hpa- b positive (n ) and hpa- b negative (n ). conclusion: id rhd xt genotyping assay performed as a reliable and accurate method for predicting the genotype and phenotype of high prevalence rhd negative and weak d types ( % specificity and % sensitivity for d antigen, hpa- a and hpa- b antigens). that makes it a useful tool for the implementation of the rhdgenotyping recommendation on patient blood transfusion and anti-d prophylaxis. background/case studies: scd patients form red blood cell (rbc) antibodies at higher rates than other transfused populations. multiple predictors of alloimmunization have been reported but not well replicated in large scd cohorts. we investigated the clinical, laboratory and genetic predictors of alloimmunization. study design/method: a large scd cohort was established in brazil to investigate disease outcomes. at participating sites, patients are currently transfused with abo/d/cc/ee/kell matched rbcs prophylactically and extended phenotypically matched rbc after first antibody forms. policies for matching are center-specific and evolved to increased levels of matching over the exposure period included in this study. transfused subjects with rbc alloantibody of defined specificity within the cohort were compared to transfused antibody negative subjects using chi squared to compare categorical variables and t-test or wilcoxon rank-sum tests as appropriate to compare continuous variables. backward elimination multivariable logistic modeling was used to generate odds ratios (or) and identify independent predictors of alloimmunization using results of univariate analyses. all subjects had peripheral blood whole genome snp typing performed using an affymetrix array, which included enhanced content for blood related snps. genome wide association (gwa) analyses were conducted using a logistic model to identify additive genetic effects associated with alloimmunization. a p value < . (clinical analysis) or < x - (gwa) was considered statistically significant. results/finding: of the cohort patients, ( . %) transfused subjects were included with alloimmunized children < years ( . % of ) and alloimmunized adults ( . % of ). in multivariable logistic regression models, age (or . , p . , for age compared to - ), gender (or . , p . , for female compared to male), transfusion history (or . , p< . , for transfusions compared to - ), site, hemolysis (or . , p . , for log transformed lactate dehydrogenase) and presence of autoimmune disorders (or . , p< . ) were independent predictors of alloimmunization. gwa identified a single snp of unclear biologic significance associated with alloimmunization (eefsec gene responsible for incorporation of selenocysteine into proteins). conclusion: rbc alloimmunization is primarily driven by transfusion burden in this scd cohort. hemolysis remained significantly associated with alloimmunization after controlling for transfusions. presence of an autoimmune disease was also associated with rbc alloimmunization, indicating more systemic immune dysregulation may be present in scd patients who develop rbc alloantibodies. however, the gwa did not identify snps in immunoregulatory genes significantly associated with rbc antibody formation in the study population. background/case studies: physiologic anemia is more severe in preterm infants and worsened by the blood loss required for laboratory tests. to reduce iatrogenic anemia, placental blood, which otherwise would be discarded, can be used for laboratory testing. mother and infant blood are mixed in the placenta during delivery and pre-transfusion test results potentially can be altered due to fetal-maternal hemorrhage. there has been no published study to show if pre-transfusion test results of placental blood give the same result as the heel stick samples, which is the standard of practice. study design/methods: transfusion service tested sample pairs from newborns less than , gr birth weight. one of the samples was collected from the newborn as a heel stick sample, the other from the placenta. the following tests were performed on the sample pairs: abo, rh, antibody screen and direct antiglobulin test with igg (dat). results/findings: abo, rh and dat tests were performed on sample pairs. dat test was negative on sample pairs and two were positive. there was % concordance with the abo, rh and dat tests performed on these sample pairs. antibody screen was performed on placental blood samples and heel stick samples. twenty eight sample pairs were negative with the antibody screening test. there was one positive heel stick sample, which was also positive using the placental sample. one heel stick sample was negative for the presence of an antibody but found to be positive with the placental blood sample. this antibody which was detected only in the placental sample was a passive anti-d mother received during pregnancy. this discrepant result indicates that the placental blood sample was more sensitive to detect a weak antibody. conclusion: this study shows that placental blood sample is not inferior to heel stick sample regarding abo, rh and dat testing. based on this comparison study placental blood can be used for pre-transfusion testing for < , g birth weight newborns. o-( . %), ab ( . %), b-( . %), and a-( . %). among the tested donors, . % were d positive with r r being the most common rh phenotype. in the kell blood group system, . % of the donors were k positive, while the k antigen was found to be . %. the most common phenotype in the duffy blood group system was fy(a-b-), while the fy(a b ) was found at a higher frequency compared to what has been reported in the black population. (table) the commonest phenotypes for the kidd and mns blood group systems were jk(a b ) and m n-s s at % and . % respectively. the le a and le b alleles were seen in . % and . % of donors respectively, while lu b -phenotype was found in . % of the donors. the frequencies of the rare phenotypes jk(a-b-), le(a b ) and lu(a-b-) were . % , . % and . % respectively, while the m n-s-s-and m-n s-s-phenotypes were not found. the frequency of the p antigen was found to be at . % similar to what has been reported in caucasians. conclusion: this is the first study to examine the frequencies of rbc blood group phenotypes among the omani blood donors. the results show higher frequencies of the rare null phenotypes fy(a-b-), jk(a-b-) and lu(a-b-) compared to what has been reported in caucasians. the frequencies of the duffy blood group system resemble what has been reported in the black population. this data is helpful in understanding the influence of the arab ethnic background on the rbc blood group systems and warrants large genotype-phenotype studies in the region. quantitation of anti-d in serum using flow cytometry amanda whitelonis*, izekial butler, karen leighton, scott jones and anand srinivasan. qualtex laboratories background/case studies: rh(d) antibodies (anti-d) are developed in rhnegative individuals when exposed to d antigens. this scenario is commonly observed in alloimmunized antenatal and volunteer immunized patients. quantitation of anti-d in serum is important in the clinical setting to predict the risk of hemolytic disease of the newborn. quantitation of anti-d is also performed in quality control operations of organ procurement organizations and plasma fractionators. it is a common practice to report the strength of anti-d in serum as antibody titer values but quality control operations require a quantitative value. we have developed a screening assay using flow cytometry to quantitate anti-d in serum. study design/method: we have developed a method to quantitate anti-d in serum using flow cytometry, by modifying the protocols of christensson et al., and hilden et al.. red blood cells from rh-positive blood samples were washed three times in phosphate-buffered saline (pbs) at ph . and the supernatant was discharged. a dilution buffer containing % human serum albumin (v/v) in phosphate buffered saline was prepared. serum samples or who anti-d standards, suspended in dilution buffer were mixed with ll of washed red cells. the cell suspensions were incubated for min at c. following incubation, fitc-labeled anti-igg diluted in buffer was added and the mixture was incubated for an additional min at c. the samples were then analyzed by flow cytometry using gates for a typical red cell based on the forward and side-scatter signals. green fluorescence was collected using a band-pass filter set for - nm. events were recorded at a frequency of cells. results/finding: multiple dilutions of who anti-d reference standard were tested against rh-positive red blood cells from five different donors. the reproducibility of the assay was determined by measuring the change in coefficient of variance due to dilution procedure, machine variation and sample storage condition. after optimizing these factors, a linear regression was calculated to establish the standard curve. the fluorescent intensity emitted by probes demonstrated a linear correlation with the concentration of rh(d) antigens in reference standard. serum from thirty rh(d)-immunized volunteers were analyzed for concentration of anti-d and the results were benchmarked with antibody titer values. conclusion: based on our study, we conclude that the quantitation of rh antigens by flow cytometry can be used as a reliable assay to measure the concentration of anti-d antibodies in serum. the method is reproducible and advantageous over reporting antibody titer values. the operations of this platform can be translated to a well-plate based high-throughput flow cytometry. sarah k harm* , mark yazer , nancy m. dunbar and biomedical excellence for safer transfusion (best) collaborative . university of vermont medical center, university of pittsburgh, dartmouth-hitchcock medical center background/case studies: the use of emergency issued group a plasma and uncrossmatched group o whole blood (wb) in patients without a valid abo group is becoming increasingly common in the usa. it is unclear if low titer products should be provided in this situation and indeed a universally agreed upon threshold that would qualify as "low titer" has not been established. this study was designed to determine the rate of high titer donors using a titer threshold of . study design/methods: three academic hospitals that routinely issue group a plasma units for emergency issue participated in this study. before issuing this plasma to patients, a : dilution of the donor's plasma in saline was produced and added to group b reagent red blood cells (rbc). if any degree of macroscopic agglutination after immediate spin was observed, the unit was considered high titer and it would only have been issued to group a or o recipients. at these three centers no temperature, plasma volume or time enhancements were performed in the titer procedure, and anti-human globulin was not added. at one center samples were taken from the plasma of group o wb units and the same procedure was followed using a and b reagent rbcs; if at least one antibody demonstrated macroscopic agglutination after immediate spin, the wb unit was considered high titer and it was then centrifuged into an rbc unit for transfusion while the plasma and platelet components were discarded. two centers provided plasma testing data for a -year and -year period, respectively. one center provided plasma and wb testing data for a -year period. results/findings: in total there were group a plasma units tested and ( . %) had a high titer anti-b. the range of high titer group a plasma units between these three centers was . %- . %. of the wb units tested, ( . %) units had a high titer; / ( . %) of the units had a high titer anti-a, / ( . %) had a high titer anti-b, and / ( %) had high titers of both anti-a and anti-b. background/case studies: dithiothreiol (dtt) is a sulfhydryl reagent that denatures selective blood group antigens. reagent red blood cells (rbcs) treated with . m dtt is used as a tool in identifying antibodies to high frequency antigens. recently, dtt has become widely used in destroying cd on reagent rbcs and render them free from plasma anti-cd drug interference. procedures for the preparation of . m dtt has been published advocating the use of buffered saline at different ph levels. in this study, an effect of ph on . m dtt treatment time is investigated. study design/methods: non-buffered saline (nbs, thermo fischer scientific inc, middletown, va), used in the preparation of . m dtt, was adjusted to ph . , ph . , ph . using sodium phosphate dibasis (sigma aldrich, saint louis, mo). reagent rbcs (immucor, norcross, ga)(n ) were treated with the . m dtt solutions in parallel by mixing : ratio of packed rbcs to . m dtt solution followed by incubation at c. for up to minutes during treatment, the expression of k antigens was measured every minutes by tube method using different sources of anti-k. to assure uniformity, all reactions were graded by the same investigator. each reaction grade (in each rbc and each antiserum) is converted into a semiquantitive score and an average score was calculated every minutes for each ph level. the reduction in average scores between different phs were also calculated at every minutes to measure the impact of . m dtt reagent ph on the rate of k antigen destruction. results/findings: the expression of k antigen, measured by agglutination grades with two different k antisera, is significantly weakened (by ! ) after minutes of dtt treatment at ph . ; minutes at ph . and minutes at ph . . complete loss of k expression was seen after minutes of dtt treatment at ph . ; minutes at ph . and minutes at ph . . the reactivity patterns of k antigen tested with sources of anti-k correlate with each other. the reductions in average scores were seen between to minutes range of dtt treatment time when ph . was raised to ph . ; to minutes range when ph . was raised to ph . ; and to minutes range when ph . was raised to ph . . conclusion: the use of higher ph buffered saline may shorten the treatment time it takes to weaken or destroy k antigen. based on the comparison of reaction scores between different ph levels, the ph levels did not have an impact on dtt treatment up to minutes and/or beyond minutes of incubation. the ph of the . m dtt reagent relative to the treatment time is a factor to consider during the validation of dtt-treatment process and qualification of . m dtt reagent in a laboratory. background/case studies: data on the characteristics and frequencies of clinically significant red cell antibodies within the prenatal population have not been well established in the united states. the aim of this study was to determine if frequencies of red cell antibodies differed between geographically distinct regions within the continental united states. study design/ method: the aim of this retrospective study was to evaluate a cohort of prenatal patients (n , ) drawn between july , and june , . these patients were divided into united states census bureau regional and divisional categories according to their place of residence. prenatal blood work was collected which included an abo, rh(d) and a screen for unexpected alloantibodies. samples found to be positive for red cell antibodies were sent to one of nine regional laboratories for identification. results/ finding: in total, , patients were found to possess clinically significant red cell antibodies for an overall incidence of . percent. the three most commonly encountered antibodies were anti-d (n ) . %, anti-m (n ) . %, and anti-e (n ) with a frequency of . %. a total of ( . %) prenatal women were found to possess two or more antibodies. in general, the combination of anti-d and anti-c proved to be the most common, with instances ( . %) followed by anti-e and anti-c with ( . %), and anti-c, anti-e with ( . %). of the multiple antibodies identified, ( . %) included at least one antibody from the rh blood group. the south region had the largest number of antibodies identified with or . % of the total. the west had ( . %), the midwest ( . %) and the northeast with ( . %). a contingency table, using the two-sided fisher's exact test, was performed comparing the northeast, south, midwest and west regions. the p value of anti-d was calculated to determine nonrandom associations and values of . and below was deemed significant from a region-to-region perspective. with regard to anti-d, the pacific division comprised of california, oregon, washington, and alaska, had p values below the . thresholds when compared against seven of the eight other divisions. the west south central division (texas, oklahoma, arkansas, and louisiana) did not show statistically significant results when compared against the pacific division (p . ). conclusion: depending upon the antibody, statistically significant variations between geographical regions and divisions within the united states were observed. this relationship between antibody and locality requires further investigation but may be attributed to the presence or absence of red cell antigens among different racial and ethical populations. reduction in repeat testing using gel technology amy mata* , lindsy rich , sherry stern , sharon wangen and camille van buskirk . mayo clinic, mayo clinic rochester background/case studies: our institution currently uses the immucor neo (immucor, inc., norcross, georgia) to perform abo/rh and antibody screen (absc) testing utilizing solid phase technology. when results are unable to be obtained from the immucor neo, testing is repeated on the manual testing bench using tube agglutination. this repeat testing can lead to significant expenses including reagents, supplies, and technologist time. it was decided by leadership in our laboratory that it would be beneficial to observe how other methodologies perform in this regard. a side-by-side evaluation was performed between the immucor neo and the ortho vision (ortho clinical diagnostics, rochester ny) to determine if there was a significant difference in the amount of repeat manual tube testing that needed to be performed. the evaluation looked at abo/rh and absc testing as those are the only tests that are currently automated in our laboratory. study design/method: thirty specimens that were processed on the immucor neo and resulted in no type determined (ntd) for abo/rh testing were selected to be tested on the ortho vision. twenty-three specimens that were processed on the immucor neo and produced positive results for absc testing were selected to be tested on the ortho vision. all specimens were edta tubes and were collected within the previous days. the timeframe between when the specimen was tested on the immucor neo and the ortho vision was to days. results/finding: of the ntd specimens from the immucor neo, resulted in valid abo/rh typings on the ortho vision. three results were flagged indicating possible extra reactivity. upon performing a visual review of all results, it was determined that there was no reactivity and a valid result was present. the other samples required manual tube testing to interpret the abo/rh and were due to mixed field, weak isoagglutinins, unexplained extra reactivity, and hemolysis. of the absc specimens that were resulted out as positive on the immucor neo, specimens produced a negative result on the ortho vision and were confirmed to be negative with manual tube testing using peg as the enhancement media. one specimen was flagged for fibrin, but upon performing a visual review, was determined to be negative. nine specimens that were positive on the immucor neo were also positive on the ortho vision. one specimen proved to be an anti-m that was seen in gel but not in tube and one specimen displayed unexplained reactivity in gel as it was negative in tube and all clinically significant antibodies were ruled out. all showed discrepant results with monoclonal anti-c reagents, with a similar pattern of reactivity: - with ms (n ), - s with ms (n ), no reaction with ms , dgc , p x (n ). samples tested with a polyclonal anti-c showed a - reactivity. d c e c e cases tested with a polyclonal and monoclonal anti-e (ms , ms , ms , ms ) showed no weakened reactivity. rhce sequencing (genomic dna or cdna) showed a c. g>a mutation in exon , predicted to encode the p.gly ser substitution. for apparent r r donors, a f-negative type allowed the prediction of a rhce*ce a/rhce*ce genotype. altogether, our results are consistent with the presence of a very likely rhce*ce a allele (c and e in cis) in all samples. d c e c e individuals were reactive s with the original source of anti-rh , slightly weaker when compared to rhce*ce a/rhce*ce rbc samples available from our cryobank ( ). conclusion: our results confirm that the c. g>a mutation alters the conformational properties of the rhce protein, either on a ce or ce background, and encodes the low-prevalence locr antigen (rh ). the locr reactivity appears to be rather similar when coded by rhce*ce a or rhce*ce a alleles. this was quite an unexpected finding, since the p.gly ser substitution is close to the critical amino-acid for c/c expression (p.pro ser). none of our cases made anti-c and/or anti-e but few were subject to a potential alloimmunization background. however, as rhce*-ce a was reported to code for a partial c (rh:- ), we consider that rhce*ce a likely encodes partial c and e, this being also supported by the predicted localization of the p.gly ser change on the second extracellular loop of the rhce protein. background/case studies: weak d genotyping is recommended for transfusion recipients, pregnant women, and newborns who had a rhd typing discrepancy, or a serological weak d phenotype, to determine if they carried the weak d genotypes , or . the purpose of this study was to analyze the underlying rhd genotypes of the patient samples received for weak d genotyping since published recommendations, in particular those found to not carry the weak d , , or genotypes. study design/methods: between / and / samples were received for weak d genotyping. testing was performed using pcr-rflp targeting the sequence variants in the rhd gene that have been previously defined. samples that did not have weak d types , , or genotypes, but a transfusion vol. supplement s had evidence of rhd genetic sequences in exon and/or intron in preliminary testing were evaluated by sanger sequencing for rhd and rhce exons - to determine the underlying rh genotype. when provided, the patient's ethnicity and presence of anti-d was recorded. results/findings: the majority of the samples were from obstetrical patients ( %) followed by transfusion patients ( %); % had no clinical indication provided. samples ( %) were found to be weak d type , , or ( , , and samples, respectively). samples ( %) appear to be genetically rhd negative. genetic sequencing was performed on samples; had rhd genetic variants that were not weak d types , , or (table) . all of these variant rhd samples also showed some variation in the rhce gene. two samples ( %) had wild type rhd alleles; further evaluation is ongoing. conclusion: most samples tested by weak d genotyping were found to be weak d types - . of the samples that had evidence of an rhd gene and did not carry the known weak d types - polymorphisms, ( %) of were found to have other rhd variants, and ( %) did not have underlying genetic variation detected in the rhd gene. the majority of the non weak d types - variants were dar alleles, which are often associated with anti-d production. background/case studies: rhd genotyping has been recommended to guide transfusion of d-negative rbcs and administration of rh immunoglobulin to patients with discordant or weaker than expected d typing, particularly for females and ob patients . the recommendation is based on observational evidence, primarily from europe (flegel , curr opin hematol : ) , that individuals with weak d types , , and are not at risk for clinically significant anti-d. the implications and utility of this approach for the diverse u.s. population are not yet clear. here we report months experience with rhd genotyping on samples referred with discrepant or weak d typing investigated from january to april . study design/method: serologic testing was performed by standard tube agglutination with licensed reagents. dna isolated from wbcs was used in manual rflp and rhd beadchip assays and rhd sequencing for some. ethnicity was known for samples ( . % caucasian, . % african american/african, . % multiracial, . % hispanic, % asian, and . % other). results/finding: rhd genotyping identified weak d types , , and in / ( %) and alleles known to encode partial d phenotypes in / ( . %) (table) . uncommon or rare weak d alleles including types , , , , , , (n ), , , , , and were found in ( . %). the partial d alleles found were diverse, but the largest number included partial rhd*d . (n ) and *dar ( conclusion: in a multiracial cohort of individuals with weaker than expected d typing % were due to weak d types , , or and would not be considered at risk of clinical significant anti-d, but for % there is potential or unknown risk. these studies are important to gain insight into the prevalence of specific alleles in the u.s. multiethnic population and to continue to evaluate and refine rhd genotyping for clinical practice. cp rhd* . allele causes discrepant genotyping results for rhce small c sabine scholz* , sandra schneider , sabrina k€ onig , susanne helmig and vicky van sandt . inno-train diagnostik gmbh, rode kruis vlaanderen background/case studies: in the human rh blood group system the c, c, e, e and d antigens are expressed by the two highly homologous genes rhce and rhd. after d, c is the most immunogenic rh antigen. the difference between c ( c) and c ( t) is caused by the snp on position on the rhce gene. the rhd* . allele (also known as rhd cat vii type ) carries the snp t>c on the rhd gene and additionally the snp t>c. this rhd* . allele has been described to partially express rhc on the d polypeptide (faas, transfusion, ) . aims: genotyping was performed to clarify the cause of the weak c expression. serology of a patient sample (male, ) indicated a partial c phenotype with a cde. study design/method: rhd and rhce phenotyping was done by accredited routine protocols (monoclonal ab id card: diaclon rh subgroups, seraclone anti-c). genotyping was performed with a taqman probe assay (rbc-fluogene veryfy, inno-train diagnostik gmbh), sso (rbc-lifecodes, gen-probe inc.), in-house ssp-pcr (hila, rode kruis-vlaanderen) and commercial ssp-pcr (rbc-ready gene cde, inno-train diagnostik gmbh). sanger sequencing of the rhd gene was performed using an inhouse method (inno-train diagnostik gmbh). results/finding: discrepant genotyping results were generated by different test systems: the taqman probe based assay showed in repetition a ccee genotype, while the sso system rbc-lifecodes predicted in repetition a ccee phenotype. in ssp-pcr the sample showed a weak c band with the in-house method, while there was no band visible with the commercial test kit. the parallel analysis of the rhd gene with rbc-ready gene cde test system revealed a variant d cat vii rhd allele. sequencing of the dna sample identified two snps on one of the rhd alleles ( t>c, t>c) confirming a rhd* . and one rhd* allele. hispanic female in preparation for surgery resulted in variable reactivity and weakly positive d reactions when using microtiter-well agglutination versus tube testing. determination of whether the d antigen expression represented a weak d or a variant d could not be resolved by serologic testing alone. here we report the characterization of a novel rhd gene mutation identified by rhd gene sequencing. study design/method: serologic typing was initially performed by microtiter-well agglutination by automated analyzer platforms galileo neo and galileo echo (immucor, norcross,ga) and by standard tube testing using the immucor series and anti-d reagents. further immunohematologic evaluation was performed by standard tube testing (immediate spin -is, and indirect antiglobulin -iat) using orthobioclone, immucor gammaclone, immucor series and series , and albaclone anti-d reagents. dna isolated from wbcs was used in manual rflp and rhd beadchip assay (immucor, bioarray) and rhd sequencing. results/finding: rbc reactivity is summarized in the table. dna testing detected a hybrid rhesus box associated with the rhd gene deletion, indicating the patient was hemizygous for rhd. rflp assay and rhd beadchip did not identify any changes. rhd gene sequencing identified a new c. a>g change in exon encoding an amino acid change p.met val. the predicted location of this change is within the fourth transmembrane segment of the rhd protein. conclusion: we identified a novel rhd allele with c. a>g (p.met val) change in exon . several snps, deletions, and insertions have been reported with changes in exon . phenotypes of these genetic variations result in rh negative, weak d types, and variant d. since this change has not been previously identified, we are unable to determine if this confers a risk of anti-d alloimmunization, but the rhd c. a>g snp results in serologically weak phenotypic expression of d antigen when tested by microtiterwell agglutination on the neo/echo platforms. in this case the combination of microtiter-well agglutination and dna sequencing helped identify a new allele which would be missed by standard tube serologic testing and the current commercially available array assays. serologic and molecular detection of an antibody to a high incidence antigen in patient with history of chronic transfusions georgia spanos* , juan merayo-rodriguez , christopher lough and nancy eckert . lifesouth community blood centers, life south community blood centers, lifesouth community blood center-headquarters background/case studies: the jo a antigen is one of three high incidence antigens in the dombrock system. the prevalence of this antigen is % in most populations and greater than % in the black population. the jo a antigen can be resistant or enhanced with enzyme treatment (ficin/papain) and typically variable with dithiothreitol (dtt), w.a.r.m. tm (immucor) and zzap treatment. anti-jo a is an igg antibody that demonstrates at ahg phase. hemolytic transfusion reactions to the jo a antigen vary from none to moderate/severe. hemolytic disease of the fetus and newborn (hdfn) has not been observed with any antibody associated in the dombrock system. there are two common phenotypes present in the black population:hy negative/ jo a negative and hy weakly expressed/jo a negative. study design/methods: an antibody identification and red blood cell (rbc) units were requested for an o positive, year old, african-american female with a history of sickle cell disease and no history of pregnancy. the patient was not recently transfused, however, had a history of chronic transfusions. last reported transfusion was three years prior to the current specimen. there were no known rbc antibodies at the time of the request. facility reports that the patient's hemoglobin(g/dl)/hematocrit(%) (hgb/hct) is . / . and does not appear to be in sickle cell crisis. a request for phenotypically matched units, as per hospital policy, for c, e, k and s was received by our immunohematology reference laboratory (irl). results/findings: anti-jo a was detected in patient plasma reacting with liss and peg (tube method) and manual gel-iat. the antibody was resistant when tested with dtt treated red cells. in-house frozen reagent rbcs negative for the jo a antigen (positive for hy) were used to serologically prove the presence of the antibody to this high incidence antigen. an allogenic peg adsorption was performed to rule out other common clinically significant antibodies. anti-kp a was identified using this adsorbed plasma. further testing with molecular genotyping (grifols idcore xt ) confirmed the patient's genotyping as antigen negative for the jo a , kp a and positive for hy. conclusion: molecular testing is frequently performed on patients and retained donor samples from our local community donor pool throughout florida, georgia and alabama. staff is able to search our database for any combination of antigen negative phenotypes using the internal (k) blood establishment computer software (becs) integrated blood bank information system (ibbis). this enabled us to locate one refrigerated and three cryogenically preserved jo a negative rbc units. we found eligible blood donors that could be recruited via an automatically generated call list. the request for rbcs was cancelled. patient's clinical symptoms improved without transfusion and repeat hgb/hct increased to . / . the patient's sibling is historically negative for the jo a antigen and should future transfusions be required, it was recommended that a directed donation be made on the patient's behalf. in order to continue having blood components available to meet all our patient's needs, irl staff is consistently screening and searching our inventory for blood components that are negative for rare antigens to retain for patients needing antigen negative units in a timely fashion. rbcs of two females whose samples were referred for rhd genotyping with previously reported alleles for which serologic reactivity had never been investigated. study design/method: serologic testing was performed by automated analyzer, galileo echo and neo (immucor, norcross, ga), and by standard tube testing with licensed anti-d reagents and the albaclone advanced partial rhd typing kit. genomic dna isolated from wbcs was used for immucor rhd beadchip assay, pcr-rflp, and rhd sequencing. results/finding: patient was a yo female, c e c e , whose rbcs reacted by echo and by neo with anti-d , and '?' with anti-d . testing with d and d by tube gave and w on initial spin (is) respectively and by indirect antiglobulin test (iat). rbcs were non-reactive at is with ortho bioclone and biorad seraclone, and w with immucor gammaclone anti-d, and all were at iat. rbcs did not react with two (lhm / & / ) of anti-d in the alba partial d kit. this pattern did not match any partial d identified by these clones. rhd beadchip detected an inactive rhd pseudogene in trans to rhd. gene sequencing confirmed the presence of the pseudogene, but rhd had a c. c>a change encoding p.his gln. patient was a yo pregnant female, c e c e , whose rbc were w at is and at iat with immucor series and and gammaclone, and moderately reactive, is and iat, with alba alpha, alba blend and delta anti-d. rbcs did not react with two (lhm / & / ) anti-d in the partial d kit with no known partial d pattern. dna testing predicted she was rhd hemizygous and rhd beadchip detected markers for rhd*dar but exon gave low signal (ls). sequencing found a hybrid dar with ce-specific nucleotides in exon from c. to c. encoding amino acid changes p.ile leu and ser asn. conclusion: we found two previously reported rare alleles: rhd with a c. c>a (p.his gln), previously found in france (lefloch et al. genbank ku ), and rhd*dar with part of exon replaced by rhce, reported in sub-sahara africa (granier et al. transfusion : ) designated rhd*dar(ce :v v-s n) with an allele frequency of . to . . blood samples were not available to test for alterations in d expression for either allele. we provide serologic evidence that these alleles, found in two females evaluated by rhd genotyping, inform transfusion and rh immune globulin prophylaxis, as they encode partial d phenotypes with novel epitope expression patterns, meaning these patients are at risk of forming allo anti-d. background/case studies: hu f -g is a human monoclonal igg antibody recognizing cd that is in clinical trials to treat hematologic or solid malignancies. cd is a transmembrane glycoprotein that binds to signalregulatory protein a (sirpa) on macrophages and functions to regulate phagocytosis. blocking cd is thought to enhance phagocytosis and promote anti-tumor responses. cd is also highly expressed on rbcs, and the purpose of this study was to evaluate anti-cd drug interference in blood bank testing. study design/method: serologic testing was performed by standard methods. serial samples (n ) from patients were tested over the course of month treatment. plasma was tested at immediate spin (is) and by iat with r r , rr, d--, rh mod and rh null rbcs, as cd expression levels vary depending on rh phenotype. dtt and enzyme treated rbcs were also tested. both immucor gamma-clone anti-igg (does not detect igg ) and ortho bioclone anti-igg (total igg) were used. for titration plasma was diluted in pbs. allo-adsorptions were performed with papain treated rr rbcs and eluates were made using gamma elu-kit ii. results/finding: anti-cd was observed in plasma as soon as hour post infusion. plasma reacted to at is and with all panel cells in peg iat using ortho anti-igg. d--, rh mod and rh null rbcs were nonreactive at is and weaker ( and ) in peg iat with ortho reagent. reactivity with all panel cells by ortho igg gel card was . in contrast, iat reactivity using gamma-clone anti-igg was only w to , and this reactivity was confirmed to be carry-over agglutination. d--, rh mod and rh null were non-reactive in peg iat using gamma-clone anti-igg . the anti-cd titer was at is and peg iat with gamma-clone anti-igg, but was ! with ortho anti-igg. plasma reacted with dtt, trypsin, papain, a-chymotrypsin or w.a.r.m. treated rbcs. somewhat unexpected, autocontrols were negative and dats were non-reactive or microscopic only. acid eluates (n ) were reactive with ortho, and non-reactive with gamma-clone anti-igg. plasma reactivity was removed after x allo-adsorption with papain treated rr cells, but in some samples low level (micro- ) reactivity remained. peg adsorption was invalid due to precipitation/complexing of antibody. robust plasma reactivity interferring in abo reverse typing was observed, and weak spontaneous agglutination of the rbcs in the abo forward and rh typing. conclusion: hu f -g anti-cd therapy interferes with routine pretransfusion testing, not only antibody screening and crossmatch, but abo and rh typing. high levels of cd expression on rbcs results in plasma agglutination at is, mimicking reactivity observed with igm antibodies although hu f -g is igg . reactivity was observed in all phases and with all test methods. cd is not cleaved from rbcs by dtt, trypsin, papain/ ficin, dtt with ficin (w.a.r.m.) or a-chymotrypsin, and treatment of rbcs with these does not mitigate interference. numerous adsorptions with papain treated rr rbcs were required to remove anti-cd reactivity from plasma. use of immucor gamma-clone anti-igg, which does not detect igg , can mitigate interference in iat although carryover reactivity may be observed. due to blocking by anti-cd on the patient rbcs, dat and autocontrols were weak or non-reactive; however eluates prepared from the dat rbcs were strong and pan-reactive using ortho anti-igg. background/case studies: a caucasian woman with history of a caesarean section and a rbc tx in . in august , she was admitted to hospital for trauma surgery, ab screening was negative and two units were transfused without transfusion reactions. five days later she was referred to a tertiary care trauma center due to a severe postop infection and need for a reoperation. ab screening was now positive, with an antibody reacting with all panel cells detected. because of the urgent need for rbc tx, two weakly cross-match positive rh k matched units were transfused with a warning of possible alloantibodies. the patient got acute hemolysis. study design/method: a gel technique was used in the hospital transfusion laboratory. in addition, various antibody identification panels and special serological and genotyping methods were used in the reference laboratory. kel sequencing was done by the international immunohematology center. results/finding: the hospital transfusion laboratory results were o rhd neg, dat neg, and the ab identification was with untreated and with enzyme-treated cells, with weakly positive autocontrols. a sample was submitted to the reference laboratory for additional investigation. dat was weakly positive, while ab identification results were similar to the hospital results. different pheno-and genotyping methods were used in addition to several identification panels to exclude rare blood groups. after pk, vel neg, jk:- etc. had been excluded, k-phenotyping revealed a k -phenotype. a total of silencing mutations are known for the kel gene and the genotyping kits used did not recognize these. the anti-ku antibody reacts with all cells apart from the k -phenotype. the presence of dtt-sensitive anti-ku was confirmed with dtt-treated panel cells. anti-ku may cause immediate and delayed hemolytic transfusion reactions. samples were taken from the patient's two siblings and daughter. kel sequencing revealed kel* n. with c. t encoding p. ter (reported in an individual from austria in ). there are two known k -patients in our country, both homozygous for c. t. the daughter was a c. t heterozygote, while the siblings did not have this variant. a new operation is necessary but no k -donors are available in our country. with the help of the isbt rare donor working party, a k o rhd neg donor was found in japan and one unit was delivered to us for use in the next operation. conclusion: an alloantibody should always be suspected when autocontrol is weaker than panel cell reactions, even if the direct coombs is positive. a combined serological and genotyping approach offers the best solution for problematic antibody cases. compatible blood is not always available in rare blood group cases, but international co-operation may be of help in finding a suitable donor. transfusion strategy for the serologic weak d phenotype in tunisia based on rhd alleles and rh haplotypes mouna ouchari* , kshitij srivastava , houda romdhane , saloua jemni yacoub and willy albert flegel . nih, dtm/cc/nih, regional blood transfusion center sousse, regional blood transfusion center sousse, tunisia background/case studies: d antigen variants have been studied molecularly in many arab populations, including gaza, tunisia, egypt and libya, a transfusion vol. supplement s since . the tunisian population has the largest known prevalence of weak d type . alleles, occurring in of rh haplotypes, compared to in , or less in europe. a systematic study was missing for samples with the serologic weak d phenotype routinely found in blood donor and patient testing in tunisia. the study was designed to obtain data on weak d type . in a population known to harbor the greatest prevalence of such allele worldwide. study design/methods: a total of , random blood donors were serologically screened for the d antigen using routine techniques. samples with weak reactivity were tested with a panel of monoclonal anti-d (partial rhd-typing set) to identify partial d phenotypes. the rhd gene was sequenced in all samples with serologic weak d phenotype. the rhce gene was also tested molecularly by either direct sequencing or using the rhce beadchip kit to ascertain the rhce allele linked to the rhd allele. results/findings: a total of discrepant samples ( . %) were observed and expressed the serologic weak d phenotype. among them, carried an allele of the weak d type cluster ( . %), of which samples ( . %) showed the weak d type . allele. only sample each was found for the weak d types , and and the dvii, while samples showed the consensus rhd sequence. no mutation in any of the rhd exons was detected in another samples. the molecular analysis of the rhce gene showed that out of samples with serologic weak d phenotype ( . %) had a variant rhce allele and the most common associations were: weak d type . linked to rhce*cevs. . ; weak d type . . with cear; and weak d type . to rhce*cevs. , while the other rhd alleles were linked to one of the common rhce alleles. conclusion: almost % of the weak d phenotypes in tunisia were caused by alleles of the weak d type cluster, of which % represented the weak d type . allele. based on established rh haplotypes for variant rhd and rhce alleles and the lack of adverse clinical reports in tunisia, we recommend d positive transfusions for patients and no rhig administration for pregnant women with weak d type . in tunisia. we propose this strategy as a pragmatic clinical decision, even if eventually a rare allo-anti-d immunization would occur in tunisia associated with weak d type . phenotype. there is a possibility that the rhce*cevs. . allele, typically associated in tunisian individuals, may protect from allo-anti-d immunizaton and other rhce alleles, such as rhce*ce more often associated in individuals of other ethnic groups, may not. however, we conclude that this conjecture has not much evidence in support at this time and would need corroboration by experimental and clinical data, before used to guide clinical recommendations. martha rae combs* , heather simmons , christine lomas-francis , gayane shakarian , sunitha vege , lauren hutelmyer , sandra nance , jessica poisson , nicholas bandarenko and connie m. westhoff . duke university hospital, immunohematology and genomics laboratory, new york blood center, arc pennjersey, american red cross, immunohematology reference laboratory, biomedical services background/case studies: plasma from a transfused, a , year old white female, post liver transplant with rbc aplasia, reacted at rt and in peg iat with all rbc samples tested except her own. study design/method: standard hemagglutination methods were used for antibody id and antigen typing. acid eluates were prepared using gamma elu-kit ii (immucor). genomic dna was isolated from wbcs and used for hea precisetype array and kel and sc gene sequencing. samples from the proband and her mother were tested, as applicable. results/finding: the patient's dat was negative. her plasma reacted with . m dtt-treated and papain-treated rbcs, all available rbc samples lacking high-prevalence antigens, and with phenotypically similar rbc samples [c , k , fy(a ),s ]. reactivity was detected to a titer of ; it was not removed by prewarm technique or by x peg alloadsorption. the adsorbed plasma reacted with . m dtt-treated rbcs. extensive rbc phenotype results were unremarkable except for the following: k , k , js(b ), kp(a b ) and sc: , . her plasma reacted with k o , mcleod, sc: , rbc samples and dtt-treated sc: rbcs at rt and peg iat but her diluted plasma and pretransfusion eluate showed relative kp b specificity. the patient was transfused aliquots of crossmatch incompatible kp(b ), s rbcs. her post-transfusion dat was with anti-igg, with anti-c d. the eluate reacted with all rbc samples except kp(b ) sample. she tolerated additional aliquots from phenotypically similar rbcs untested for high-prevalence kell or scianna antigens. the hea precise-type predicted k , k , kp(a b ), js(a b ) and sc: , , discordant with her rbc phenotype. kel gene sequencing identified a homozygous change, c. a>t (p.glu val) (kel* . ) encoding the low prevalence antigen, ul a , but no changes associated with lack of kell system antigens; however, her rbcs typed ul(a ). sc sequencing found heterozygosity for a '- g>a change (rs , to % prevalence) and conventional sc* , predicting sc: , , . kel and sc results on the mother were kel* /kel* . , heterozygous for the sc change '- g>a, and her rbcs typed k k kp(a b ), sc , ula , consistent with dna predictions. plasma collected months later was nonreactive at rt and in peg iat. her rbcs were dat and now typed k , kp(a b ), ul(a ) sc and sc ,concordant with predicted kell and sc phenotypes. conclusion: we report an example of kell and scianna antigen suppression or blocking in the presence of autoantibody or an alloantibody in the kel system. to our knowledge, this is the first report of a ul a kel* . homozygote. the rbcs may lack a high-prevalence antigen antithetical to ul a . without dna testing and gene sequencing, the patient would be presumed to have kell null and sc null phenotypes, a search for k o , and/or sc: , rbc units would be performed and we would not have been prompted to re-type her rbcs when the dat was negative. background/case studies: anti-jka is a common antibody identified in the blood bank and providing phenotypically characterized red cells lacking this antigen is important in avoiding an acute or delayed hemolytic transfusion reaction. in nearly all cases, this antibody is identified in the context of a phenotypically homozygous jkb patient, jk(a-b ). other scenarios are quite rare. we present two cases of anti-jka in which this phenotype was not observed. study design/method: patient a is a -year-old multiparous female with no known transfusion history. her blood typed as o positive with a positive antibody screen, negative dat, and a clearly identified anti-jka in plasma. the patient phenotyped as jk(a-b-). genotyping revealed the presence of the jk*b allele, but not the jk*a allele. complete sequencing of the jk gene showed an intron polymorphism in homozygosity. specifically, the patient showed a jk*b(ivs - a)genotype, associated with a jkb null phenotype. anti jk was not identified. the conclusion was an allo-anti-jka in a jk null patient. the patient did not receive any transfusions. patient b is a multiply transfused old female. her blood typed as a positive with a positive dat and antibody screen. both the plasma and eluate revealed an anti-jka. despite the recent transfusion, the patient phenotyped as jk(a ) and jk(b ) . genotyping showed the presence of both jk*a and jk*balleles. whole gene sequencing was not performed. there was no hematologic or biochemical evidence of hemolysis. the patient was considered to have an auto-anti-jka and jka negative cells used for transfusion. results/findings: patients a and b both developed anti-jka while having uncommon phenotypes/genotypes. conclusion: it is common for jk null patients to develop anti-jk . however, we speculate that expression of the kidd glycoprotein with the jkb epitope was below the threshold of serological detection, but enough to prevent the formation of anti-jk or anti-jkb. auto-anti-jka is usually reported in the context of an active hemolytic process, but patient b illustrates an auto-anti-jka without hemolysis which is more commonly observed with autoantibodies exhibiting specificity for rh epitopes. these rare cases of anti-jka require phenotypic and genetic analysis for the jkb epitope and jk*b allele respectively, and in more complex cases whole gene sequencing. background/case studies: donor genotyping for red blood cell antigens has become common practice in many blood bank laboratories. package inserts for commercial assays indicate false negative results may be generated when unexpected rare mutations affect primer or probe binding and cause allele dropout or failed amplification. these outcomes may go unrecognized unless serological results are available for comparison. study design/method: a routine blood donor, self-identified as african american, was selected for red blood cell genotyping. dna was extracted and genotyping was performed using two commercial platforms a transfusion vol. supplement s (precisetype, bioarray, warren nj; idcore xt , grifols, emeryville, ca). genotype results were compared to historical serological results. discrepancies were resolved by sanger sequencing (grifols ih, san marcos, tx). results/finding: genotyping results showed variants in both the duffy (fy) and kell (kel) blood group systems. the donor's genotype was concordant on both platforms, fy*a/fy*b_gata, kp*a/kp*a, for a predicted phenotype: fy(a b-); kp(a b-). when genotype results were compared to historical serology, it was noted that the donor previously typed fy(a-) on separate donations. no previous kpa or kpb serotyping was available. sequencing of fy exon revealed a g>a mutation, fy *n. , known to silence fya. sequencing of kel exons - exposed a silent polymorphism in exon , g>c. this polymorphism causes a dropout artifact yielding a false negative kpb interpretation. conclusion: the discrepant fy*a result, as well as the unlikely kp(b-) type prompted the request for sequencing. the rare fy *n. mutation has been reported in people of caucasian descent. this is the first example of this fy mutation identified in this regional population. the kpb antigen is present in nearly % of all populations. however, kp(b-) is most frequently seen in people of caucasian descent. to date, self-identified african american donors have been genotyped as kp*a/kp*b at this blood center. given the diversity of regional heterogeneity, it is feasible to identify a kp(b-) donor, self-reporting as african american. red blood cell genotyping offers an abundance of information, but cannot replace serology as the sole means of red cell antigen characterization. donor ethnicity continues to play a key role in selection for genotyping and the search for rare and unusual red cell types. in this case, a donor selected for genotyping based on ethnicity was initially thought to have genetic variants not previously reported in those of african descent. only was proven to be present. this case acts as a reminder that genotype limitations must be considered even when using licensed methodologies. this case report presents two group o pediatric patients who had been on enteral feeds and had absent/weak anti-b that became strong over time in patient . study design/methods: patient was a year-old male born prematurely with short gut syndrome who underwent a small bowel and liver transplant at years of age. anti-b changed from undetectable/weak to strong at the age of years. patient was a month-old female with a metabolic urea cycle disorder who underwent a liver transplant. anti-b was / . both patients were on total parenteral nutrition (tpn) since birth and had strong anti-a and normal immunoglobulin testing. abo typing with enhancing techniques is presented in table . results/findings: both patients typed as group o on forward typing. anti-a was strong in both patients. anti-b varied in strength in patient with - reactions up to years of age. thereafter, abo typing showed mainly strong anti-b. patient had / anti-b. conclusion: intestinal bacteria stimulates production of anti-a and -b. unexpected changes in anti-b that caused abo discrepancies are reported here for children on long-term tpn. patient had absent/weak anti-b since birth up to years of age, then developed strong anti-b with no change in feeding regiment and medications. patient had consistently strong anti-a and absent/weak anti-b. these findings support the notion that normal colonization of the gut is important in the development of anti-a and -b and suggests that microflora of the gut in patients on prolonged tpn is different leading to the delayed formation of these antibodies compared to individuals on normal enteral diet. difference in strength of anti-a and-b could be due to stronger a than b antigen expression on gut bacteria. results/finding: a daratumumab protocol was established that incorporated use of the cord panel. multiple myeloma patients selected as candidates for daratumumab treatment were baseline tested for blood type and antibody screen, dat and genotype. after daratumumab infusion, a two unit crossmatch was order as a precaution in the event the patient developed a reaction to the medication. repeat of the antibody screen demonstrated panagglutination which served as a positive control for the medication. the cord panel ruled out underlying alloantibodies. selected red cell units were crossmatched at immediate spin phase to avoid expected indirect antiglobulin reactivity. conclusion: the cord panel was used times over a five month period to rule out underlying alloantibodies. tests for the daratumumab protocol consisted of a routing antibody screen followed by a cord panel for resolution. the daratumumab protocol significantly reduced testing time and allowed for the provision of compatible blood products in an efficient and cost effective manner. teresa gorey* and elizabeth hart . brigham and women's faulkner hospital, university of massachusetts-dartmouth background/case studies: the purpose of performing a pre-transfusion antibody screen is to detect clinically significant unexpected antibodies and to decrease the probability of detecting clinically insignificant antibodies. several antibody detection methods (polyethylene glycol (peg), liss, and albumin) are routinely used in small transfusion services. the utility of peg is to enhance the sensitivity of detecting clinically significant antibodies by the indirect antiglobulin procedure. the code of federal regulations, title , cfr part . (a), states the manufacturer's instructions are followed when testing for unexpected antibodies. the package insert for gamma peg tm (immucor inc., norcross, ga), states that negative reactions may be examined with an optical aid. based on these directions, our institutional policy is to confirm all negative reactions using the microscope. study design/method: a one-year retrospective document review was performed on all patient samples in which a positive antibody screen (absc) triggered the antibody identification (abid) to be performed in . a total of samples were evaluated. each abid was subcategorized; ( ) as being a new antibody for our facility or in the patient's shared electronic health record within the partnersv r healthcare system and ( ) whether a microscopic absc result triggered the abid. also, patients with known antibodies were grouped according to a microscopic absc result. a comparison of the new patients and the previously known antibody patients with microscopic results were reviewed to determine if the antibodies were clinically significant. results/finding: a total of abids were performed on new patient samples. of the new abid samples, ( %) had microscopic absc results. for the previously known antibody patients, there were which accounted for % of the total abids performed. when reviewing the total abid workups, a total of ( %) of the abscs had microscopic results which resulted in an abid being performed. the antibodies identified in the new antibody samples were: conclusion: a total of % of the new antibodies identified based on a microscopic absc were clinically insignificant. the manufacturer's directions were followed but they do not state that an optical aid is required to confirm all negative results. due to the results of this study, a decision will be made to: ( ) discontinue the use of the microscope, ( ) switch to a peg manufacturer whose directions indicate to observe macroscopically for agglutination, or ( ) define the use of the agglutination viewer as the optical aid. decreasing the number of abids will save time and money while providing potential rbcs for transfusion in a timely and efficient manner. anton") has a prevalence greater than % in all populations. hereditary absence of anwj has only been described once (in a single family). however, red cell expression of anwj may be markedly decreased to near undetectable levels in blood donors of the in(lu) (or "dominant lutheran inhibitor") phenotype. similarly, anti-anwj antibody formation is rare, with only cases reported in the literature. the antibody developed in the context of hereditary absence of anwj (i.e., a true alloantibody) in only one of the cases. in the other nine cases, the antibody occurred in the context of autoimmune or lymphoproliferative disease, where, in this context, it is believed to have developed secondary to transient anwj antigen suppression. most of the reported cases lacked clinical or laboratory evidence of hemolysis. however, in the most recently reported case, involving a -yearold woman with aplastic anemia, the antibody was associated with acute hemolytic reactions after rbc transfusions, necessitating transfusion support with anwj-negative and in(lu) rbcs. the case was also unique in that the anti-anwj resulted in a direct antiglobulin test (dat) that was positive for complement only, rather than igg like all previous cases in which the dat was performed and was positive. study design/method: a -year-old woman with severe aplastic anemia experienced acute hemolytic transfusion reactions (ahtr) with development of a panagglutinin on indirect antiglobulin test (iat) screens. prior to identifying the specificity of the panreactive antibody, the patient received rbc transfusions and showed signs of hemolysis with six of them. the first three transfusions were prior to her positive iat and were electronically crossmatched. the next seven transfusions were incompatible by antihuman globulin (ahg) phase crossmatch, but were extended phenomatched for clinically significant antigens. the patient's ahtr signs and symptoms included fever, rigors, nausea, vomiting, dark urine, flank pain and "impending doom" anxiety; while her laboratory findings included hemoglobin decreasing below pre-transfusion levels, and increased total bilirubin and ldh. the dat, while initially negative during the immediate posttransfusion workup of the transfusion reactions, eventually became positive for igg only ( - ), and negative with anti-c b, c d reagent. the antibody showed a peak gel-igg iat titer of . results/finding: the antibody was identified as having anwj specificity. the patient's pre-transfusion sample showed weak anwj expression (w ), altogether suggesting an auto-anti-anwj. monocyte monolayer assay testing using the patient's plasma and rbcs from the ahtr-implicated units yielded monocyte indices ranging from to %, consistent with the clinical hemolysis observed. given the patient's group o, rh d negative blood type and continuing transfusion dependence, in order to avoid further ahtrs, international collaboration was necessary in order to procure and provision group o, rh d negative rbcs that were also serologically negative for anwj. the patient was successfully transfused three such units without further incident. conclusion: this is the second documented case of anti-anwj in a patient with aplastic anemia and, overall, the third anti-anwj case associated with ahtr. this case also underscores the importance of international collaboration. cold auto-antibody anti-p anti-m anti-sd a anti-le b anti-jk a anti-k anti-e anti-c results/finding: three hundred and ninety weak d genotypes have been determined to this day with frequencies of % (type ), % (type ), % (type ), % (type ) and % other than , , or . further investigation was conducted to determine the molecular identity of the «others». out of samples, ( %) were confirmed to be legitimate serological weak or partial d, mainly deletions of exon or both exons and . a surprising amount of samples were discovered to be normal rhd. conclusion: along with sandler et al. ( ) data, our findings highlight the difficulties hospitals face in interpreting serological weak d. trend analysis was conducted regarding the reagents and technologies used by each hospital, the origin of the request and the ethnicity of the concerned patient, but no significant correlation could be identified at this point. altogether, our findings allow to share the frequency of weak d types , , and obtained in serological weak d, years old quebec's women, and also highlight the need for further investigation of standard practices amongst hospitals regarding the management and interpretation of atypical d typing. were classified as fnhtr. taco incidence was , %. no trali happened in the period. prophylaxis were used in % of patients. conclusion: fnhtr is described as the most common adverse event related to transfusion, but our data showed a higher incidence of allergic reactions. fnhtr occurred times less than allergic reactions. this might be explained by universal leukoreduction and universal prophylaxis adopted at our institution. further studies are necessary to evaluated the benefit of this approach. that cannot be associated with a specific rbc unit or were deemed unrelated to transfusion, rbc transfusion aes were analyzed. chi-square test and logistic regression were used to compare the ae incidences among transfusion groups. results/finding: univariate and multivariate logistic analyses showed that irradiated rbcs were associated with a significantly increased incidence of transfusion-related aes (p < . ). there was a significant difference in febrile non-hemolytic transfusion reaction (fnhtr) ( . % vs . %, p < . ) or aes with a non-allergic type inflammation etiology ( . % vs . %, p < . ) including transfusion-related acute lung injury, transfusion-associated dyspnea, but not transfusion-associated circulatory overload, infections or hemolytic transfusion reactions, between irradiated rbcs and non-irradiated rbcs. in contrast, the incidences of allergic aes ( . % vs . %, p . ) were similar between these two groups. the incidences of inflammation aes after transfusion of irradiated rbcs that were stored for , , , and weeks were . %, . %, . % and . %, respectively (p . , logistic regression) but there was a significant difference in the incidence of inflammation aes caused by irradiated rbcs stored for a week ( . %) and longer than a week ( . %) (p < . ). conclusion: irradiated rbcs associated with a higher incidence of transfusion inflammation aes compared to non-irradiated rbcs and this risk increased when rbcs were stored longer than week after irradiation. while it is likely the patient population is a factor in ae caused by irradiated rbcs, it is also possible that rbc radiation damage, as shown in previous studies, contributed to this increased ae incidence. a list of patients with one of these icd codes was generated. the emr was searched to find the clinical scenario in which trali was mentioned. these patients' records were then searched within our laboratory information system (copath), to determine if they had a transfusion reaction reported to our transfusion medicine service. results/finding: the search of our electronic medical record found patients from - , who had trali mentioned in their chart as a diagnosis or possible/likely diagnosis. one patient was excluded from our study because trali was mentioned as a past medical history from an outside hospital. only the patients who had trali listed as a diagnosis or possible diagnosis were included in this study. these patients had clinical scenarios in which a transfusion of a blood product occurred which was followed by various forms of respiratory distress. the clinical teams caring for these patients were either giving a diagnosis of trali or considering trali as a possible diagnosis. of these cases, only of them were reported to our transfusion medicine service as transfusion reactions. of the reported cases, one was determined to be trali and the other one was consistent with taco. eight out of those cases were never reported. background/case studies: despite diligent efforts to transfuse the safest product available to patients, undetected alloantibodies may cause delayed hemolytic transfusion reactions (dhtr). this transfusion reaction is seen in as many as out of transfused products. therapeutic plasma exchange (tpe) may be employed to mitigate ongoing immune mediated hemolysis, but few reports in the literature describe tpe for clinical management after profound hemolysis. study design/method: case review of a patient was performed after diagnosis and treatment of severe dhtr. results/finding: a man with a history of gastrointestinal bleeding presented to the emergency room with shortness of breath and "hematuria". he had a known history of anti-d and anti-c, and was transfused two units of crossmatch compatible rbcs seven days prior during a previous admission. readmission hemoglobin (hb) was . g/dl but declined to . g/dl the next day. an antibody screen was consistent with anti-d, anti-c, and direct antiglobulin test (dat) was negative. he received three units of crossmatch compatible rbcs over days and with poor responses. on day , routine labs could not be reported due to marked hemolysis, he had "worsening hematuria", creatinine rose from . mg/dl to . mg/dl (reference . - . mg/dl), and lactate dehydrogenase was above reportable linearity, > u/l (reference - u/l). testing revealed additional anti-e, anti-jkb, dat c , plasma free hb . mg/dl (reference - . mg/dl), and hemoglobinuria. four of five transfused rbc units were jk(b ), one of which was also e . one volume tpe was performed to remove free hb on days , , and using fresh frozen plasma as replacement fluid for haptoglobin supplementation. creatinine peaked at . mg/dl on day , decreased to . mg/dl before discharge on day results/findings: twenty three cases were identified, of which had medical records available for analysis. ten ( %) patients were male, the mean age was . years (range - years), ( %) had an underlying hematologic malignancy or bone marrow disorder, and ( %) had a history of coronary artery disease (cad). the implicated units included ( %) red blood cells and ( %) platelets; ( %) patients received a single unit, and ( %) received two or more within the previous hours; the mean volume transfused was . ml (range - ml). the mean time to onset of chest pain was . minutes (sd minutes), with % of patients presenting within . hours and % within hours of starting the transfusion. chest pain was present as the only symptom in % of the cases, and for the other cases the accompanying symptoms included dyspnea ( %), fever ( %), back pain ( %), and hypo-and hypertension ( %). a post-transfusion chest x-ray was performed in % of cases, and all showed no evidence of pulmonary edema to suggest possible volume overload/transfusion associated circulatory overload (taco). electrocardiogram was performed in % of cases and showed no findings to suggest acute ischemia. three ( %) patients had a minimal increase in their troponin levels, although had a history of chronically elevated troponin due to stress cardiomyopathy. fourteen ( %) patients received some form of treatment, including increased oxygen supplementation, metoprolol, acetaminophen, morphine, and oral calcium carbonate; the pain resolved after more than minutes in the majority of patients ( %). no cases resulted in new admission to the icu or procedure cancelation. conclusion: chest pain associated with transfusion was infrequent, but several such cases were identified during the review period. this symptom is not a diagnostic criterion for any of the other hemovigilance categories and merits further characterization to determine whether blood product transfusion could be the cause of the chest pain. larger observational studies to power clinical characterization could help to further inform hypotheses regarding a transfusion-related mechanism, which could be interrogated by translational research studies. background/case studies: thrombotic microangiopathy (tma) in children is most commonly seen in the form of hemolytic uremic syndrome (hus). however, tma may be seen in the presence of streptococcus pneumoniae (spn). the action of bacterial neuraminidase of spn results in exposure of the normally "hidden" thomsen-freidenreich antigen (t-antigen) found on erythrocytes and other tissues. ultimately, this may lead to spn induced hemolytic uremic syndrome (phus) with subsequent hemolysis and end organ damage by naturally occurring anti-t antibodies against the exposed t antigen. specific lectins or anti-sera can confirm exposure of the t antigens in phus. alternatively, phus can be identified by minor crossmatch incompatibility resulting from agglutination of exposed t antigens on recipient's erythrocytes to anti-t antibodies in the plasma portion of blood products. we present a case of suspected phus that resulted in a compatible minor crossmatch leading to concern and eventually diagnosis of atypical hus (ahus). study design/method: a months old boy presented with respiratory failure. he was found to have blood cultures positive for spn as well as hemolytic anemia, thrombocytopenia, and acute renal failure. he was shiga toxin negative and had normal levels of adamts . based on the findings, the clinical team was concerned for phus. therefore, he received washed erythrocytes. for his thrombocytopenia, our institution does not routinely provide washed platelets due to decrease quality of the platelet product. as a result, a minor crossmatching was suggested and performed to determine if t activation was present. results/finding: minor crossmatch was performed with patient's erythrocytes and plasma of abo-identical platelets to be transfused. no agglutination was seen at immediate spin, degree, or anti-human globulin phase. check cells were found to be . these findings were conveyed to the clinical team and platelets were issued without washing. due to the lack of identification of t activation by minor crossmatching and poor clinical response despite appropriate antibiotic treatment, additional studies were performed by the primary team for complement mutations and found to be consistent with ahus. the patient was then treated with eculizumab with clinical and laboratory improvement. we present a case clinically consistent with phus. confirmation of this diagnosis is done with lectins or anti-sera that are not readily available. an alternative means of identifying phus is by minor crossmatch incompatibility. by demonstrating minor crossmatch compatibility, we further elucidated a definitive diagnosis of ahus with appropriate management. background/case studies: orthotopic liver transplantation (olt) is a complex and technically challenging procedure that can be complicated by severe intraoperative bleeding. we report a case of massive transfusion in an olt patient necessitating an abo blood group switch (from o to a ) to sustain transfusion support and minimum group o rbc inventories. study design/methods: type & screen (ts, gel) and anti-a titers (tube) were performed using routine methods. a chart review was performed for pertinent medical and laboratory findings. results/findings: the patient was a -year-old o man with cirrhosis secondary to nonalcoholic steatohepatitis and alpha- antitrypsin deficiency who presented for olt (donor o ). during olt, the patient endured substantial bleeding from retroperitoneal collateral vessels complicated by post-transplant coagulopathy. he required rapid high volume rbc and plasma support, which strained hospital inventories. after receiving units of o rbcs and units of o plasma with ongoing severe hemorrhage, he was switched to group a products. ten units of a plasma were transfused to wash out anti-a antibody prior to transfusion of a rbcs. due to difficulty controlling the bleeding, biliary reconstruction and fascial closure were delayed for hours post-transplant. the patient's total estimated blood loss was > l. he received a total of units of rbcs (including a ), units of plasma (including a ), units of cryoprecipitate, and units of platelets. towards the end of the second procedure, the patient's hemorrhage was stabilized and the final two rbc units he received were o . on postoperative day (pod) , a ts showed predominantly a rbcs with trace o rbcs, as well as very low anti-a igm and igg titers (table ) . he received two additional o rbc units ( each on pod and pod ) with increasing o rbcs on ts and rising anti-a titers. his blood type was unequivocally o by pod . the patient showed recovery of liver synthetic function on pod (factor activity %) complicated by cholestasis. conclusion: this study shows successful switching of a group o patient to group a in the setting of rapid hemorrhage and massive transfusion. by pod , the patient had reverted to o with recovery of anti-a titers. at months post-olt, the patient is alive with signs of improving biliary graft function. a new rfid transfusion safety system anna millan* , alfred mingo , maria isabel gonzalez , antoni mena and juan pedro benitez . bst, at-biotech background/case studies: a new transfusion safety system (tss), based on processes and technologies, especially, identification by radio frequency (rfid), is currently implemented in two hospitals, a general one (h ) and an oncology center (h ). the tss is fully effective in protecting against incidents, and specifically offers mechanisms to detect near misses (nm) by using procedural and physical barriers, assuring that the pretransfusional sample extraction (pse) and the blood components administration (bca) only take place at bed side, using a location control and interacting with the clinical and transfusion information systems (tis). the tss allows to analyze the transfusional activity information in real time to project organizational changes in both transfusion services and hospital units, and to create a new classification of nm. study design/method: retrospective analysis of transfusion activity in both h and h shows pse and bca, out of and respectively, since the tss deployment in . retrospective analysis and classification of security events has been done. results/finding: activity results for both hospitals are shown in the table below. the safety events have been classified in pretransfusion sample extraction (pse), blood component assignment (bcas) in the transfusion service and blood component administration (bca) near misses (nm). for h , nm related to pse accounted for . % of all, being the mistake in concordance between patient identification and prescription order the most frequent ( . %). the nm detected in bcas were . % of all and mostly ( . %) occur when the patient information in the tis does not match the one registered in the tss. the nm detected in bca are . % of all and mostly ( %) the systems detects a not assigned bracelet. for h , nm related to pse accounted for the . % of all, being the error in concordance between the transfusion security number in the bracelet and in pretransfusion sample the most frequent ( . %). the nm detected in bcas accounts for . % of all and in . % occurs when the patient information in the tis does not match with the one registered in the tss. the nm detected in bca are . % of all and in . % of them the blood components were assigned to another patient. ( , , , , , , , , , , , , , ) were analyzed via a commercially available elisa. comparison of adequate response to ppv , defined as ! mcg/ml for > serotypes, was perform based on alloimmunization status. statistical significance was determined by comparing means of subgroups using paired and non-paired t-tests. results/findings: pre-vaccine sp titers were available in patients (alloimmunized, ); pre-and post-vaccine titers were available for patients (alloimmunized, ). of the patients, were on chronic transfusions, were on hydroxyurea, were surgical splenectomized, patients had no history of surgical splenectomy or status was unknown. forty-four patients had a previous history of ppv in the previous years; / also reported previous history -valent sp conjugate vaccine within the last years. baseline pre-vaccination titers (n ) showed no difference between alloimmunized and non-alloimmunized patients (all p-values > . ). in the group with pre-and post-vaccination (n ) titers available, out of ( %) non alloimmunized patients had an adequate response versus out of in the alloimmunized group ( %, p ns background/case studies: blood transfusion is the most common procedure performed in the hospital setting and the transfusion process is monitored to ensure regulatory compliance. to safeguard safety, efficacy and regulatory compliance, transfusion services actively benchmark transfusionrelated errors (tres) as they occur from "vein-to-vein", i.e. from collection of pre-transfusion sample to final infusion of product -with the goal of ensuring that the right product/dose goes to the right patient at the right time. multiple over-lapping error documentation processes are needed to capture and report tres from within and outside of blood bank (bb). we present a comprehensive error management program along with data on five years of benchmarking tres at a large academic medical center. study design/method: tres were detected by capturing and reporting of sample suitability, testing variances and biologic product deviations. in addition, tres as observed and reported by providers and clinical staff (i.e. blood delays/undertransfusions, transfusions without consent, infusions with wrong fluids) were reported to the bb and hospital quality through the veritas system, a hospital based reporting system that enables reporting any occurrence with potential for causing patient harm. all serious errors were reviewed daily and summation of tres was discussed on a monthly basis. mapping tres within the "vein-to-vein" was performed by reviewing the fiveyear of transfusion medicine quality records (from to ). patient harm events recorded within the veritas system from january to july were investigated in depth. transfusion reactions were excluded in this analysis. results/finding: an average of tres per month and per year were found over five years. % of tres are associated with pre-bb activities, % occur within bb, and % are post-bb events. sample collection and handling represent % of total tres. most tres ( %) were reported by bb staff, % were reported by non-bb staff. patient harm analysis revealed an average of four level (near miss), three level (no known harm), and . level (patient harm) per month. no deaths related to tre were detected over the seven month january to july period. patient harm was associated with tres occurring in the bb ( %) and post-bb ( %). these events were reported externally ( %) and by bb staff ( %). conclusion: although most tres were detected in the pre-bb phase, no patient harm was associated with these events indicating an efficient capture prior to causing patient harm. the tres causing patient harm, including near miss events, were mostly reported externally and they occurred entirely in the post-bb and bb phases. these results suggest that significant opportunities for quality improvement may be achieved in two areas: the pre-bb phase aimed at reduction of waste associated with sample collection and handling, and the post-bb and bb phases aimed at improving tre detection and decreasing patient harm. background/case studies: uncrossmatched red blood cells (rbc) and emergency issued platelets (plt), plasma and cryoprecipitate (cryo) are lifesaving in a bleeding patient without a valid type and screen. collectively termed "emergently issued products" they are issued as a bridge until pretransfusion testing is completed. this study evaluated the utilization and wastage rates of blood products during pregnancy-related hemorrhage where the first products issued were emergently issued. study design/methods: a list of patients on whom blood products had been emergently issued between january , and march , was obtained from the blood bank at a regional maternity care hospital. patients who were not experiencing a pregnancy-related bleed (e.g., postpartum hemorrhage or bleeding relating to a complication of pregnancy such as a ruptured ectopic pregnancy or bleeding post spontaneous or therapeutic abortion) were excluded. the total number of products (emergently issued plus crossmatched or non-emergently issued products) that were transfused, returned back into the blood bank's inventory, and wasted within hours of the first emergently issued products were enumerated. apheresis plt units were multiplied by and added to the number of individual whole blood plts; apheresis plasma units were multiplied by and added to the number of whole blood plasma units. results/findings: seventy women who received emergently issued blood products during a pregnancy-related hemorrhage were identified. average age was . the majority of these patients with pregnancy-related hemorrhage who received at least one unit of emergently issued blood products received at least one unit of the product that was issued to them and few units were wasted. that plt wastage was higher than the other products was likely due to the -hour post-pooling room temperature shelf life. keeping wastage rates low while meeting the clinical needs of these patients is the ideal situation for the blood bank. . patient blood platelets were higher before prophylactic than therapeutic transfusions ( [ /l vs. [ /l, p . ). there were no significant differences in the frequency of effective therapeutic ( % vs. %, p . ) and prophylactic ( % vs. %, p . ) transfusions between the prcs and gypcs. we did not find significant differences between prcs and gypcs in cci after prophylactic ( . . vs. . . ) and therapeutic ( . . vs. . . ) transfusions, in cc after prophylactic ( . . vs. . . ) and therapeutic ( . . vs. . .) transfusions. there were no significant differences between prcs and gypcs also in ma after prophylactic ( . . vs. . , p . ) and therapeutic ( . . vs. . . , p . ) pc transfusions. reduction of the severity of bleeds was obtained in ( %) of the cases after prpc transfusions and in ( %) of cases after gypc transfusions. there were no significant differences in the frequency of adverse post-transfusion reactions between the groups (respectively, and cases). background/case studies: an 'end-to-end' electronic transfusion management process including a bedside administration system was developed and implemented in this large multi-site academic center in . it enables the safe administration of blood components at the patient bedside and provides an audit trail for all blood components. an error was identified in the electronic bedside transfusion process which was reported to our national hemovigilance scheme in under the category 'errors relating to information technology'. this error was the incorrect use of the emergency transfusion process for non-emergency transfusions. the standard (non-emergency) process requires a scan of the barcode on the patient's wristband containing their identification details which is verified against the same details from the barcode on the compatibility label attached to the blood bag. the emergency transfusion option is only intended for use with 'emergency group o rhd negative blood units' which, unlike non-emergency units allocated to specific patients, do not have a compatibility label. the emergency transfusion option skips the compatibility label barcode scan as the emergency units can be transfused to any patient needing urgent transfusion. it was found that the emergency blood option was being misused for non-emergency transfusions, leading to blood units not being checked to ensure they were for the correct patient. study design/method: this center worked with the software supplier to develop a solution which corrects the weakness in the process. the revised process involves providing a universal compatibility label for emergency units so that all units (emergency and non-emergency) require a scan of the compatibility label on the blood bag and the patient's wristband at the bedside before transfusion. the use of the emergency process was audited pre and post implementation of the new process to determine whether it was being used correctly or not. results/finding: there were units administered using the emergency transfusion process in the months before the change was implemented. it was found that / ( %) units were non-emergency units administered incorrectly without a bedside compatibility check. following the implementation of the change there were no instances of incorrect administration of non-emergency units in the next month ( components administered), / ( %) were emergency units which were administered correctly. users of the system reported the revised process was quicker, safer and unified with other functions on the device. conclusion: the improved process for the administration of blood in an emergency now prevents users from following the incorrect procedure for non-emergency transfusions and missing the essential final bedside electronic check. this report indicates the need for continued vigilance of the functionality of electronic transfusion processes, and the correction of any weaknesses compromising patient safety. background/case studies: recent recommendations indicate one red blood cell (rbc) unit should be transfused at a time with reassessment after each transfusion to determine the need for more. however, the practices of canadian transfusion medicine (tm) experts and what constitutes a reassessment are unknown. therefore, we conducted a survey of tm experts across canada to gather information on their practices and criteria for reassessment. study design/method: tm experts were identified and contact information obtained from the canadian national advisory committee (nac) and from contacting least one tm expert per province. each respondent was assigned a unique study id after consenting to the survey, allowing for anonymity on analysis. the survey contained demographics, general practice questions, and questions regarding transfusion in: ) a stable anemic inpatient, ) a stable anemic inpatient to be discharged, and ) an asymptomatic post-operative inpatient. results/finding: we identified canadian tm experts: ( . %) provided a response and most had a primary place of practice in a laboratory setting ( / ; . %). for a stable, non-bleeding, anemic inpatient, . % of respondents recommended transfusing one rbc unit, then reassessing. recommendations were more variable in outpatient settings, with . % generally recommending transfusing two rbc units then reassessing. recommendations for reassessment were mainly functional status/symptoms and vitals within a short time period ( - hours), a repeat hemoglobin > hours later dependent on the clinical scenario, and a search for an underlying cause of anemia in outpatient settings. lab practitioners emphasized volume status, cardiac examination, and transfusion at lower hemoglobin thresholds. with an asymptomatic patient to be discharged, fewer respondents chose to transfuse ( . %) compared to an inpatient potentially symptomatic due to anemia ( . %). none of the respondents suggested transfusion in an asymptomatic post-operative patient who had a hemoglobin trending down. conclusion: tm experts generally recommend transfusing one unit at a time in stable inpatients. assessment for transfusion should focus on patient symptoms, pertinent physical exam, hemoglobin levels, and an underlying cause. "top-up" transfusions were not recommended. these recommendations may help guide clinicians, but further research is needed to generate higher quality evidence around the clinical benefits and cost effectiveness of these practices. background/case studies: current evaluation of red blood cell (rbc) post transfusion recovery is based on ex vivo labeling of stored rbcs with radioactive chromium- ( cr). this method has several limitations including the risks associated with radioactivity, and the inability to evaluate multiple rbc populations in the recipient. rbc labeling with s-nhs-biotin (bio-rbcs) overcomes many of these limitations and offers safe and longitudinal tracking of multiple transfused rbcs in vivo. the purpose of this study was to scale up and optimize the biotinylation procedures to the current good manufacturing practice (gmp) environment. study design/method: packed rbc units (n ) were divided into two ml aliquots, which were labeled with selected concentrations of s-nhsbiotin ( and lg/ml) in a cgmp closed system (average bio-rbcs hematocrit of . . %). optimization of labeling efficacy was determined by flow cytometric analysis of bio-rbcs using fluorochrome-conjugated streptavidin (sa). approximately million rbcs were measured in triplicate. quantum simply cellular beads were used to quantify fluorochrome (molecules of equivalent soluble fluorescence, mesf) and infer number of biotin molecules per rbc. the lower limit of detection was determined for rbc labeled with varying amounts of biotin. product quality and safety were evaluated by endotoxin and sterility testing, and by determining the levels of spontaneous hemolysis before and after rbc biotin labeling. results/finding: investigation of different fluorochromes, laser excitation wavelengths and laser power to maximize the signal to noise ratio of labeled and unlabeled rbcs revealed that nm excitation of phycoerythrin (pe)-sa and high laser power ( mw) provided the best separation between the two bio-rbc populations, and between labeled and unlabeled rbcs. labeling with lg/ml of biotin resulted in $ , mesf/rbc, and were detectable among unlabeled rbc at a lower limit of detection (lld, % ci) of in , ( . %). the lld for rbc labeled with biotin at lg/ ml was $ in million ( . %). biotinylation was not associated with increased levels of hemolysis ( . . % before labeling versus . . % after labeling; p . ) or bacterial contamination. conclusion: the resulting manufacturing process produces large volumes ( ml/transfusion) of bio-rbcs with low risk of contamination or hemolysis. the flow cytometry assay can detect bio-rbc in unlabeled blood at very low frequency. we plan to use this technology to study the impact of donor characteristic on rbc storage stability and post-transfusion survival. background/case studies: blood products offer resuscitation benefits in trauma over crystalloid/colloid volume expanders (which provide no hemostatic benefit or oxygen delivery), but usage is often hampered by supply or storage needs. hemoglobin-based oxygen carriers (hbocs) are not red cell replacements but may supplement oxygen delivery and expand volume during transport until blood is available. since hemostasis is critical in resuscitation, this study evaluated bovine hemoglobin glutamer- (hboc- ) effects on coagulation parameters alongside freeze-dried plasma (fdp) in an in vitro model of hemorrhage/resuscitation. study design/method: whole blood (wb) was collected from healthy donors under an approved institutional standard operating procedure. in the first study (limited resuscitation), samples were: ( ) wb, ( ) wb % hboc volume (model of two units in an adult), ( ) wb % fdp, and ( ) wb % hboc % fdp. samples ( )-( ) simulated autoresuscitation by adding % plasmalyte to - . susceptibility to lysis was tested with ng/ml tissue plasminogen activator (tpa). follow-up studies were performed with severe resuscitation simulations of %, %, %, and % volume replacement with hboc and/or fdp, with or without prior % plasmalyte dilution. coagulation parameters were obtained with a coagulation analyzer and thromboelastography (teg). rbcs/hemoglobin were measured on a hematology analyzer. thrombin generation was quantified by thrombogram. platelet aggregation was measured in multiplate and adhesion to collagen under shear in bioflux. viscosity was evaluated by rheology. results/finding: a limited resuscitation model with hboc and/or fdp had no effects on fibrinogen, pt, aptt, ph, hct, or hemoglobin. in teg, wb, wb hboc, and wb hboc fdp had reduced clot strength with dilution and tpa. there was increased susceptibility to tpa-induced lysis between wb and wb hboc in autodilution simulation (mean lysis . % vs. . %; p<. ). hboc and fdp had no statistically significant impact on thrombin generation. no effects on platelet aggregation were observed; no significant differences within diluted v. undiluted groups were seen in platelet adhesion under flow. hboc ( %) did not significantly change viscosity. severe resuscitation simulations had increased pt/ptt and reduced clot strength, particularly in hboc-only resuscitation; however, even % hboc volume replacement produced clots with acceptable teg parameters. conclusion: in a limited resuscitation model with hboc- , there were no significant in vitro effects on hemostatic parameters (except increased susceptibility to lysis); more severe resuscitations impacted coagulation parameters but did not prevent clotting. considering the large impact healthy platelets have on coagulation function, further in vitro studies with impaired platelets are warranted alongside in vivo studies of hboc plasma as initial resuscitation of hemorrhagic shock. therapy in patients with acute major bleeding. while published literature has largely focused on the efficacy and safety of pcc, actual usage practices are less characterized. our aim was to describe the pcc usage practices within a tertiary care center. study design/method: we conducted a retrospective review of the electronic medical records of patients who received pcc between its addition to our institution's formulary in / and / . we compiled information about the usage of pcc in these patients. descriptive statistics were generated with microsoft excel. results/finding: of patients, were on warfarin. pcc was most frequently prescribed for hemorrhage due to surgery ( %). pcc was given for warfarin reversal in % of cases. a subset of patients received plasma within hours prior to pcc ( %) or hours after ( %). pcc was most frequently ordered in the or/perioperative service ( %). conclusion: the majority of pcc usage was "off-label" in terms of being prescribed for indications other than warfarin reversal. the most frequent indication was hemorrhage due to surgery, and pcc was most often ordered in the or/perioperative service. although guidelines recommend the use of pcc as a plasma alternative, plasma was administered within hours of pcc in a notable subset of patients. background/case studies: in emergent situations, when a patient's life may be jeopardized by delaying transfusion, a physician may decide to transfuse blood emergently. however, in some cases, poor communication and lack of clear expectations between the blood bank and patient care areas can lead to frustration and delays in the timely provision of blood products. an incident prompted an appraisal of our emergency release protocol (erp), which revealed gaps in communications and expectations by both the blood bank and nursing personnel. thus, it is imperative that there is a standardized er protocol with clear communications for both the blood bank and nursing personnel. reported here is the outcome of a process improvement that resulted in improved communication, expectations, and turnaround (tat) for our er protocol. study design/method: in , several meetings were conducted with stakeholders (critical care units (icu), emergency department (ed), internal medicine, interventional radiology (ir) etc.) in an effort to identify process gaps, improve communications, and expectations for er episodes. the goals was to design a process for emergent blood product request and release in life threatening situations that will; ) simplify and expedite the process; ) improve communication and expectations to decrease tat; ) improve patient safety and meet compliance. in order to achieve these goals, a series of activities were conducted. these included meetings with all stakeholders to ensure process improvement meet the needs intended. a series of training sessions with nurse educators in icu, ed, ir and surgery managers were conducted. during the meetings, communication goals, and expectations were defined and agreed upon. training sessions included powerpoint presentations to educate staff members and performance of dry runs, to identify weaknesses and strengths with the process flow. the impact on the current process was analyzed and, as a result, led to the revision of the current sop, addition of pre-labeled emergency pack blood ( units of o neg rbc's) and implementation of an electronic emergency blood order set. results/finding: in the ten months post implementation of our improved, standardized er pack protocol, a total of er episodes were received. the average tat from order to delivery at the bedside was reduced by % ( . minutes compared to minutes previously), while the compliance rate for er orders and physician documentation was % ( / ), with no current wastage of blood products. conclusion: the implementation of the improved standardized er protocol significantly improved communications and expectations, decreased tat and delays in transfusions while ensuring patient safety and compliance to regulatory requirements. background/case studies: massive transfusion (mt) in the trauma setting has been extensively studied. yet, the literature in non-trauma areas, especially oncology is rather sparse. the following study was conducted to understand the background and outcomes of mt in cancer patients. study design/methods: this was a single center retrospective study performed at a large cancer center between february -february . mt was defined as the transfusion of ! rbc units in a -hour period. the following data were collected included: age, gender, primary diagnosis, surgery or acute care type, amount and type of blood components transfused, whether or not a massive transfusion protocol (mtp) was activated, and survival at days. results/findings: thirty mts occurred during a one year period. a total of , blood products were transfused during that time period. gender distribution was / ( %) males, and the average age of all patients was with a range of to years of age. surgical patients accounted for / ( . %) mts, and / ( . %) were critical care patients. tumor categories included carcinomas ( / ), sarcomas ( / ), leukemias ( / ) and lymphoma ( / ). resection of tumor followed by complex reconstruction was the cause of the majority of mts. metastatic renal cancer ( / ) was the most common disease seen followed by sacral chordoma ( / ). mtps were activated in only / ( . %) cases. thirty-day survival was seen in / ( . %) patients. only of mortalities was a surgical case (peritoneal mesothelioma), and the remainder were caused by gi hemorrhage ( / ) or perisplenic hematoma ( / ). the overall ratio of rbc:ffp in the entire patients ( background/case studies: plasma is a straw-colored supernatant of blood that is used for type and screen (t&s) and crossmatch. in the analytic phase of testing, plasma is examined prior to processing. plasma occasionally becomes discolored, interfering with crossmatch procedures. timely identification of the etiology allows for corrective actions and minimizes delay in transfusion. study design/method: during the analytic phase of blood bank testing, samples were evaluated for t&s and crossmatch; this identified three samples with discolored plasma. we present a series of cases that illustrate the testing process. results/finding: a -year-old woman diagnosed with breast cancer presented for mastectomy with sentinel lymph node biopsy. a preoperative t&s specimen contained bright green plasma. review of her preoperative case revealed exposure to intravenous methylene blue. this dye is known to alter the color of urine, tears, and blood with no known pharmacologic effects. alternative causes of green plasma include other dyes used to locate sentinel nodes and oral contraceptive use. although not ideal, this sample could be used for crossmatch by tube method, but not automated gel technique. a specimen drawn one week later contained clear plasma. a -year-old woman diagnosed with a warm autoimmune hemolytic anemia was refractory to blood transfusions secondary to alloantibodies. administration of a synthetic blood product resulted in dark maroon colored plasma. the most common cause of a dark red color is hemolysis of the sample, which is usually discarded. in this instance, the hemoglobin color was due to the infused product, an experimental bovine pegylated carboxyhemoglobin that affects colorimetric evaluation of blood samples. with this in mind, the sample was not discarded and testing was completed by tube method. a -year-old woman admitted with acute stroke was treated with a thrombolytic. her t&s revealed cloudy white plasma that could not be used for the crossmatch procedure. common causes of white plasma include purulence, hypertriglyceridemia, and sampling of blood drawn proximal to administration of radiopaque agents such as propofol. although an etiology could not be identified a repeat specimen drawn several hours later was clear. conclusion: these cases highlight the importance of an appropriate evaluation of discolored plasma. once a discolored sample is identified, a repeat sample is required to confirm the change in color. in the first two cases, the discoloration persisted, prompting further clinical investigation. once the etiology was identified, need for further testing and eligibility for further transfusion was determined. testing by tube method could be performed in two cases. in the third case, repeated sampling revealed a clear sample and the transfusion process continued without delay. decisions regarding the analytic phase of testing must include reevaluation of the sample, identification of the etiology, and comprehension regarding how to proceed when discoloration persists. caleb wei-shin cheng* , , rebecca ross , christopher a tormey , , and amit gokhale , . yale university school of medicine, yale-new haven hospital, va connecticut healthcare background/case studies: daratumumab (dara) is a igg monoclonal antibody therapy that specifically targets cd , a glycoprotein highly expressed on plasma cells, where it has been successfully used in patients with refractory or relapsed multiple myeloma. dara interferes with blood bank testing as it binds to cd expressed on red blood cells, causing pan reactivity. the dara interference can be overcome with the use of dithiothreitol (dtt) treated reagent red blood cells. to minimize alloimmunization and to provide crossmatch compatible blood to treated patients, we instituted a dara protocol in our blood bank. the purpose of this retrospective study was to identify the outcomes of our protocol, with a particular focus on the development of de novo alloantibodies during dara treatment at our institution. study design/method: all dara patients' antibody workups were completed using dtt pre-treated reagent red blood cells. if the antibody screen was negative, k antigen negative rbc products are provided. if an antibody is identified, k negative along with that particular antigen negative blood is provided. our electronic medical record (emr) was searched for patients who received dara over the past eight months. study subjects were examined to see if they had pre-existing alloantibodies before dara treatment and whether they formed new alloantibodies during dara treatment. the age, gender, type and screen pre-dara treatment, type and screen post-dara, intervening blood transfusions, and the date of first dara treatment was recorded. results/finding: overall, subjects were identified for analysis. their mean age was . years, with male and female subjects; all were diagnosed with multiple myeloma. we found an alloimmunization rate of % ( / ) prior to administration of dara. of these patients, were transfused with red blood cells (rbcs) after initiation of dara therapy. following our testing/matching protocol, none of these ( %; / ) patients formed a confirmed, new alloantibody during dara treatment; each of these patients underwent at least one follow-up screen after their first rbc unit. we also found no complications in providing crossmatch compatible units to any of the patients. conclusion: to our knowledge, this is the largest case series reporting on results of overcoming dara interference with blood bank type and screen testing. the protocol implemented in our laboratory appears to be successful in providing compatible units and preventing alloimmunization in patients receiving dara therapy. it is possible that the drug, targeting antibody forming cells, may have an immunosuppressive effect on the humoral response; further studies of this effect may be warranted. background/case studies:a multi-facility transfusion service began stocking liquid plasma in september of for use in massive transfusion and trauma situations. due to the infrequent occurrence of these incidents, the liquid plasma would outdate before use. a policy to use liquid plasma in nonemergent situations when the units were nearing their expiration dates was implemented. this study evaluated the effects of that policy on inr values of plasma recipients. study design/methods:a retrospective analysis was developed to compare the effectiveness of fresh frozen plasma (ffp) and liquid plasma (lqp) in changing inr values of recipients. all plasma units transfused within the facility from september , through april , were identified. the following data was obtained from the hospital and laboratory information systems for each unit: the recipient, primary reason for transfusion of plasma, number of plasma units transfused, type of plasma transfused, preand post-transfusion inr values, and whether or not vitamin k was administered. patients were divided into groups based on the type of plasma units transfused and were evaluated based on primary reason for transfusion, number of units transfused, and administration of vitamin k. the change in inr for each recipient was calculated, along with the average change in inr for each group. background/case studies: in gynaecological settings, most but not all relatively young anaemic women are iron deficient due to blood loss associated with menstruation. transfusion could generally be avoided in those without haemodynamic instability. the oral antifibrinolytic drug tranexamic acid is an effective and well tolerated treatment for menorrhagia. besides, iron replacement is often necessary for a prolonged period of time after normalization of haemoglobin (hb). the present study attempted to look into transfusion appropriateness and the use of iron and tranexamic acid in transfused women in hong kong. study design/methods: anonymous data of gynaeological patients age was retrieved from a central database of public hospitals which included age, number of units of red cell transfused, pre-and posttransfusion hb, the use of iron and/or tranexamic acid during hospitalization and upon discharge. all transfusion episodes associated with surgical operations during same admission are excluded. results/findings: in , , unique women receiving a total of , units of red cells (rc) in , transfusion episodes were identified. their median age was (range - ). the distribution of pre-and post-transfusion hb and units of rc transfused were summarized below: in this cohort, pre-and post-transfusion hb were absent in ( . %) and ( . %). ( . %) transfusion episodes were associated with the use of units or more rc. as a result, ( . %) episodes resulted in a post transfusion hb ! g/dl. parenteral iron or tranexamic acid was uncommon during hospitalization and was given (< . %) and ( . %) women respectively. upon discharge, ( . %), ( . %) and , ( . %) women were prescribed with oral iron alone, oral tranexamic acid alone or both respectively. however, neither were given to ( . %) women. conclusion: in the present study, it is observed that . % transfusion episodes were given at hb ! g/dl. a substantial number of episodes ( . %) were transfused with multiple units and resulted in almost half having a post transfusion hb level (! g/dl). for iron replenishment and bleeding control, up to . % transfused women were not given iron or tranexamic acid at discharge. the results indicate that awareness of both transfusion appropriateness and iron deficiency anaemia management have to be improved. it is recommended that in-depth education and training should be provided for a better gynaecological patient blood management. background/case studies: granulocyte transfusions may be utilized to boost the immune response in patients with life-threatening neutropenia or neutrophil dysfunction and evidence of treatment-refractory bacterial or fungal infection. however, granulocytes are rarely administered due to uncertainty regarding efficacy, difficulty in collection, and increased propensity for adverse reactions. we report a case of granulocyte transfusion therapy following chimeric antigen receptor t-cell (car-t) therapy in a patient with severe neutropenia and multiple infections in the context of relapsed b-cell acute lymphoblastic leukemia (b-all). study design/method: granulocytes ( . - . x per unit) were collected from abo-identical unstimulated donors at a regional blood center. each unit was irradiated with gy and transfused over - hours within hours after the time of collection. the patient's response and laboratory data were reviewed in the medical record. conclusion: this data suggests that a diagnosis of aml is associated with anti-hla antibodies. an increased frequency of blood group a in patients with aml has been reported, but here no statistically significant difference between abo blood group frequencies was found in any category except the patient's with hla antibodies. blood group b has a significant association with hla alloimmunization in the studied patients. it has been reported in a large study of female blood donors that no difference in hla antibody frequency was observed based on abo blood group at centers using the flow-based assay. although the reasons for the higher rate of group b blood type among patients with anti-hla antibodies and hematologic malignancies is unknown, this could be due to variation in immunizing events (pregnancy vs transfusion) or immune dysregulation related to the hematologic malignancy, especially aml. females with aml who are blood group b appear to be most likely to have hla alloimmunization among patients with hematologic malignancies. implementation of electronic solution to reduce risk of mistransfusion in a regional transfusion service debra lane* , lee grabner , brenda herdman , robert fallis , amin kabani and charles musuka . canadian blood services, kenora rainy-river regional laboratory program, diagnostic services manitoba background/case studies: patient misidentification and improper sample labeling has been an ongoing risk for the safety of blood transfusion. the rate of mistransfusion has remained unchanged in over years. attempts have been made to reduce mistransfusion including barrier devices, barcoding and rfid. within a regional background/case studies: the role of donor age and sex on hemoglobin content and susceptibility to hemolysis during storage of red blood cell (rbc) units is receiving increased attention. however, the impact of donor characteristics on efficacy of rbc transfusion has not been studied in largescale donor-recipient outcomes databases. study design/methods: we conducted an analysis using blood donor data routinely collected by a blood center and transfusion recipient data from a large community hospital network between and before patient blood management initiatives. linkage was performed between blood donor characteristics and hospitalized rbc transfusion recipients who received a single rbc unit. studied exposures for this analysis were blood donor sex and age in addition to rbc storage age. the wilcoxon test was used to examine changes in hemoglobin level following rbc transfusion, and , and % were male. recipients of rbc's from male and female donors had similar pre-transfusion hemoglobin levels ( . g/dl; p . ); however, transfusion recipients of male donor rbc units had higher post-transfusion hemoglobin levels and larger increments in hemoglobin compared to those of female rbc units ( . vs . g/dl; . vs. . g/dl; both p . ). female recipients had a larger rise in hemoglobin per rbc unit compared to male recipients ( . g/dl vs. . g/dl; p< . ). female sex of the recipient remained a significant predictor of change in hemoglobin after accounting for recipient age and estimated circulating blood volume in multivariable analysis (p . ). rbc storage age and the age of the donor were not significant factors in changes in hemoglobin levels in multivariable analysis, p . and p . , respectively. conclusion: rbc units from male donors resulted in a larger rise in hemoglobin levels compared to those from female donors, and these changes were more apparent in female recipients even after accounting for effective circulating blood volumes. this suggests that the dose of hemoglobin is lower in female than male rbc units. this analysis demonstrates the feasibility of using this approach to study the association between donor characteristics and rbc efficacy, hemolysis and other donor-component-recipient interactions. background/case studies: people who identify as jehovah's witnesses (jw) comprise less than % of the population of the united states. however, as a group they can present a special challenge in medicine due to a religious aversion to blood products, based on biblical readings. the degree of this religious refusal can vary from individual to individual, but as institutional policy, a conservative approach is warranted. however, in large institutions where multiple teams manage a single patient, blood refusal information can be lost or poorly communicated from provider to provider. as such, a system to alert providers of patient blood refusal was recently implemented through the electronic medical record in a large west-coast institution. study design/method: the electronic medical record (emr) utilized in this study in an institutionally modified version of epic ea best practices alert (bpa) was designed to trigger each time an end user attempted to place orders related to blood transfusion, transfusion-related lab testing, or human-derived pharmacy items on patients with blood refusal codes in their history, problem list, or religion (jehovah's witness) discrete data fields. the alert constitutes a "soft-stop" in which the ordering provided is prompted to either cancel the triggering orders or acknowledge the blood refusal/religious history and override the warning with an option to select a reason for the override. data on the triggers are automatically collected through the emr systems and generated into a report by informatics personnel. results/finding: the available data covers triggers in the two month postimplementation of the bpa. the bpa triggered times in total, affecting patients and users. stratified by location, the majority of triggers occurred in the perioperative areas ( times) and the liver icu ( times) with a minority occurring on the regular hospital floors and emergency department. nurses, attendings, residents, pharmacists, and nurse anesthesiologists were the users affected. orders that triggered the bpa included type & screens, human albumin % iv solution, human albumin % iv solution, immune globulin (human) solution. conclusion: despite the limited and very preliminary data, the user action findings seem to indicate that the bpa is effective in halting up to half of the contraindicated orders for blood-derived products and type & screens orders. given the limited types of orders that the bpa is triggering on, the pattern suggests that the bpa is potentially alerting some previously unaware providers of the patient's religious status and/or the fact that certain pharmacy items are blood-derived, and therefore unacceptable to many jw patients. despite these positive initial findings, this is an ongoing study to track the efficacy of the bpa and more data needs to be collected for better metrics of the institutional sensitivity to patient blood refusal. intervention to address inappropriate cryoprecipitate-ahf orders at a tertiary medical center sirisha kundrapu* , , mahmut akgul , , hollie m reeves , , robert w maitta , , marcie pokorny , anne capetillo and katharine a downes , . background/case studies: although introduced for the management of hemophilia a, now cryoprecipitate is primarily indicated for low fibrinogen levels. at our institution the transfusion medicine service (tms) reviews and makes recommendations to clinicians for all inappropriate cryoprecipitate orders. we aimed at analyzing the effectiveness of this intervention in reaching target fibrinogen levels in under-estimated and over-estimated orders. study design/method: we conducted a -month retrospective study (january-july ) of adult cryoprecipitate order quality assurance forms. the reference range for fibrinogen was - mg/dl with critical value of mg/dl. cryoprecipitate orders for massive transfusion protocol, from operating rooms and for extracorporeal membrane oxygenation were not reviewed by the tms. during the study period, tms evaluated orders for appropriateness of dosing and agreement with estimated required doses. post-transfusion fibrinogen levels due to intervention were compared with hypothesized no intervention levels. statistical analysis was performed using chi-square and t-tests. results/finding: there were adult (> years) orders reviewed by tms out of which were approved. of the approved orders, ( . %) were in agreement with tms's estimated dose. of ( . %) orders that were not in agreement with the tms's estimate, ( %) were underestimated and ( %) were overestimated. seventeen of orders had no post-transfusion fibrinogen levels. without intervention, there would have been a median deficit of . mg/dl (range . to mg/dl) and a median excess of . mg/dl (range . to mg/dl) of fibrinogen from the target. median difference between target and actual post-transfusion fibrinogen level was mg/dl above target, which is significantly higher with intervention than without (which could have been mg/dl below the target; p< . ). median differences between target and post-transfusion fibrinogen levels for the group with agreement between approved and requested units was not significantly different from possible differences without intervention ( vs. . mg/dl, p . ). median differences between target and post-transfusion fibrinogen levels for the group with non-agreement between approved and requested units was significantly different from possible difference without intervention ( vs. - . mg/dl, p< . ). seven of ( ) ( ) orders were for critically low fibrinogen (< mg/dl) and of these were under-estimated requests and reached target fibrinogen with tms's estimate and approval of required units to be transfused. overall most frequent orders were and units ( . % and %) i.e. and pools and the most frequent orders in the disagreement group were , , and units ( %, %, % and %). there is a significant difference between agreement and disagreement groups based on clinical service ordering the units (table) . conclusion: intervention by tms to review and approve cryoprecipitate orders was associated with increased accuracy of orders and achievement of desired target fibrinogen levels. further studies are needed to develop multidisciplinary strategies for accurate cryoprecipitate dosage. patient characteristics, medical records, vitamin k administration, and adverse events, were collected (table) . results/finding: the average pre-transfusion inr was . and posttransfusion was . . only % of patients had their inr corrected to . , while % had no change, or had increased inr. (table) . the majority ( %) of patients received units of plasma. the mean plasma dose was ml/kg. there were transfusion reactions reported, non-hemolytic and transfusion associated circulatory overload reactions in which required admission to the icu. two patients experienced bleeding during ir procedures (tips) and developed a hematoma (tunneled central line). the median of inr correction in this study was . with no relationship to the number of units of plasma transfused and/or if vitamin k was administered. this study suggests it may not be beneficial and may be harmful to transfuse plasma for correction when inr is . . randomized trials are needed to assess whether the inr is a rational tool to measure bleeding risk, and whether prophylactic treatment with plasma yields any benefit. of the patients experienced bleeding complications indicating that inr of . may be considered safe in some lower risk procedures. current practices may provide little or no benefit, with substantial risk of life threatening complications. background/case studies: group ab plasma, which lacks anti-a and anti-b antibodies, is considered to be the universal plasma donor and is used in the emergency setting before the patient's blood group is available. approximately % of the population is group ab, which limits the available inventory of group ab plasma. of group ab population, only plasma from male donors are considered suitable for transfusion since females, especially multiparous female donors, have a greater propensity to develop antibodies that can cause transfusion related acute lung injury (trali). this makes type ab plasma a limited resource. our hospital is a level one trauma center, where a significant amount of plasma transfusion is required for severely bleeding patients before their blood type is known. group o individuals make up % of the population and have no a or b antigens on their cells. group a is the second most prevalent blood group in the us population ( %) and has no b antigen on their cells. so, group a plasma is compatible with both group o and a patients, approximately % of the patient population. before patient's blood type is known, type o red cell units are transfused with a plasma, which decreases the chance of hemolysis. to conserve ab plasma, we instituted a policy effective july , as follows: units of group a plasma and units of group ab plasma is provided for the massive transfusion protocol (mtp) along with units of o negative rbc until patient blood type is known. study design/method: this prospective study is designed to monitor the use of group a plasma in mtps at our institution and to evaluate the risk and severity of hemolysis in patients transfused with incompatible plasma. direct antiglobulin test (dat) is performed if patient received incompatible plasma. if dat is positive, lactate dehydrogenase (ldh), haptoglobin and bilirubin levels are obtained to detect possible hemolytic transfusion reaction. results/finding: we reviewed mtps at our institution between july and march . twenty patients ( . %) were transfused with incompatible group a plasma ( group ab and group b patients). five patients died due to severe injury, and follow-up testing of these patients could not be performed. the remaining patients had negative dat, indicating the lack of significant amount of antibody coating their red cells, which could lead to hemolysis. none of these patients developed acute hemolytic reaction, or any other adverse effects of incompatible plasma transfusion. conclusion: our study adds more evidence of the safety of group a plasma transfusion in trauma patients requiring emergent massive transfusion before the patient blood type is known. based on this and other recently published studies, starting in april , our institute will provide only group a plasma for emergency release and mtp cases before the patient blood type is known. average ( background/case studies: in , bonfils immunohematology reference lab (irl) sent out approximately special platelets for patients with hla antibodies. by , hla platelet orders increased dramatically and the irl sent out over special platelet products. the purpose of this abstract is to illuminate the methods used to fulfill increased client need that occurred in a short period of time. study design/methods: bonfils blood center has over , donors in the database with historical hla typing. however, only approximately of those donors actively donate. in the denver area, one of the most common hla types is a a b b . only of the , donors have this type ( . %). therefore, to fill an hla platelet order request for a common hla type, only donors in the system would be a perfect hla match. with that low number of donors, it is not likely that there would be a platelet on the shelf ready to fill the order. after a donor is recruited and donates, it takes at least two days to fill an order. for a less uncommon hla type like a a b b , there is only out of , donors ( . %) that match perfectly. in those cases, there are no donors to recruit to fill such an order. in some complicated cases, the irl was provided with an hla antibody list or panel reactive antibody test (pra). in order to find product for these patients, lists of platelets in inventory with corresponding hla types were printed. if a patient had an antibody to a for example, all of the a positive platelets were crossed off the list. this cross-out process would continue manually until the only platelets on the list were the ones positive for hla antigens to which the patient did not have antibodies. these platelets are pra matched to the patient. in order to automate this process a report linked to the donor database was created to find both pra platelets in inventory and donors for recruitment. the blood center medical director began suggesting that hospital clients order a pra for each patient with platelet refractoriness. the pra test is fast and it is a definitive method to discern hla antibody mediated refractoriness from platelet refractoriness due to other causes. results/findings: in all but the most complicated cases with rare hla patient phenotypes, it was much easier to find a pra patient matched platelet on the shelf than an hla match donor. in , approximately % of these special order platelets were pra matched and the remaining % were hla matched by donor recruitment. by , approximately % of special platelets sent are pra matched. this change resulted in a . fold increase of finding product in inventory to fill orders quickly. conclusion: developing a system to provide pra matched platelets is a faster alternative to finding hla matched platelets thus contributing to better patient care. background/case studies: in urgent cases where large amounts of blood products are needed quickly, maintaining a standard massive transfusion protocol (mtp) is critical to the timely delivery of these products. each mtp pack at ucm contains packed red blood cells (prbcs), fresh frozen plasma (ffp) units, and plateletpheresis pack; a unit of prepooled cryoprecipitate is also given if the patient is in labor and delivery (l&d) or if one is requested. at ucm, blood products are generally transported through the pneumatic tube system (pts). we undertook a review of our mtp issuing practices and efficiency patterns over the last three and half years. study design/method: the electronic archives of the blood bank laboratory information system and electronic medical record at our institution were queried for patients who had mtp activations. the archives were correlated to paper copies of these activations to collect data pertaining to the relevant information such as where the order originated from, how quickly the first product was sent out, how many products were transfused, and so on. results/finding: between august to march mtps were activated at ucm, of which orders could be traced to the origin: on inpatient floor (including icus), in the operating rooms, in the emergency department, in labor and delivery, and in other procedure rooms. of the prbcs that were issued, were transfused ( % utilized); of the units of ffp that were issued, were transfused ( % utilized); of the platelet packs that were issued, were transfused ( % utilized); of the units of cryoprecipitate that were issued, were transfused ( % utilized). since march , the time of first product issue after the initiation of an mtp has also been tracked. of the events that fall within this time period, ( %), had the first product issued in minutes or less. another ( %) were issued between - minutes, resulting in over % of patients being issued their first blood product within the first minutes. only of ( %) events had an initial time greater than minutes and none were greater than minutes. conclusion: the majority of our activations currently come from inpatient floors (primarily icus). as our institution anticipates the introduction of an adult level trauma center, we anticipate this balance will shift. in addition, the data shows that (with the exception of cryoprecipitate) the utilization rate is nearly identical among the blood products sent during mtp activations ($ - %). again, we anticipate utilization rate of issued mtp products to increase with the introduction of a new adult trauma center. we have recently begun tracking time to last product issued during an mtp, but cannot report on that variable at this time. overall, our data show that our transfusion service is generally performing adequately to issue the first product within minutes of mtp protocol activation. this data only reflects time to issue in the pts; patient care areas can experience additional minutes delay in pts delivery and arrival of product at bedside. we must continue to collaborate with our clinical colleagues to collect accurate data to provide the best and most efficient mtp care. mehreen yasin* , shailesh macwan , arline stein , jane fischman , nancy nikolis , matthew bank , lennart logdberg , alexander indrikovs , sherry shariatmadar and vishesh chhibber . north shore university hospital, northwell health background/case studies: massive bleeding is generally defined as any patient who requires blood volume replacement within hours and/or receives transfusion of greater than or equal to units in one hour with a transfusion vol. supplement s ongoing bleeding. our mtp was officially implemented in in preparation for an initial verification as a level trauma center by acs. our mtp has the following packages: st pack has a ratio of : : (rbcs, plasma & platelets) and subsequent packs a ratio of : : . our mtp also includes prothrombin time (pt), activated partial thromboplastin time (aptt) and fibrinogen testing after each pack is transfused. this data is used to assess the patient and allows the transfusion service and clinical team to identify coagulopathies. however, attempts to supplement mtp packs with cryoprecipitate (cryo) and prothrombin complex concentrate (pcc) were challenging to accomplish in a timely manner. study design/methods: due to challenges in timely supplementation of mtp packages with cryo and pcc, the protocol was modified in march to add cryo and pcc at a defined point in the mtp (cryo is included in the rd pack and pcc in the th pack). in order to validate this modification of adding these products at defined intervals regardless of laboratory data, we decided to review all patients that received > rbc at our institution as these massively hemorrhaging patients would receive pcc based on our current protocol. we reviewed the blood products received by these patients and their available laboratory data. results/findings: we had patients who received > rbc in and . mtp had been activated for all patients and all patients received between . to unit of plasma for each rbc unit transfused. despite receiving these ratios of blood products, all patients had elevations of their pt > seconds and many had elevations of the aptt and fibrinogen levels less than our institution's target of mg/dl (table ) . as anticipated, improvement in the coagulation parameters was noted with cryo and pcc supplementation. conclusion: our data on massively hemorrhaging patients supports a role for supplementation of our mtp with cryo and pcc in patients who require transfusion of > rbc. our current protocol with the addition of cryo and pcc at defined intervals has streamlined the process and improved timely provision of these products in bleeding coagulopathic patients. background/case studies: red blood cell hemolysis is a key finding for a diagnosis of transplant-associated passenger lymphocyte syndrome (ta-pls). however, whether a hematopoietic stem cell or organ transplant recipient experiences hemolysis when a transplant contains unintended antibody-forming passenger lymphocytes depends, by chance, on the recipient's blood group phenotype. a living donor liver segment transplant resulted in a case of ta-pls with donor-derived anti-d that had the potential for causing a clinically significant hemolytic event. the donor's plasma contained anti-d. anti-d was absent in the recipient's pre-transplant plasma, but present in the recipient's -day and -day post-transplant plasma. although these findings established a diagnosis of ta-pls, hemolysis did not occur because the recipient's blood group phenotype was d-. the conventional focus on hemolysis, rather than on the transfer of antibody-forming lymphocytes, is a diversion from the primary pathophysiology of pls and limits capturing the true scope of the syndrome. study design/method: to determine the standard of practice for detecting and diagnosing ta-pls, a retrospective -year pubmed search for peerreviewed english-language journal articles was conducted using key words "passenger lymphocyte syndrome." cases were categorized according to the presence or absence of hemolysis and whether there was a routine antibody screen to detect donor-derived, passenger lymphocyte-formed blood group antibodies. results/finding: of published cases ( reports) of ta-pls, ( reports) were stem cell and ( reports) were organ transplants. all ( %) stem cell transplants and ( %) organ transplants were associated with hemolysis, reflecting an overwhelming bias for identifying ta-pls associated with hemolysis. of the reports of stem cell ta-pls, actively screened for antibodies in the immediate post-transplant period, and of the reports of organ ta-pls, actively screened for antibodies. these screens detected cases of stem cell ta-pls before hemolysis became apparent and cases of organ ta-pls with antibodies without hemolysis. it can be inferred that ta-pls is currently under-diagnosed, because hemolysis is not consistently present and/or antibody screens are not performed routinely. conclusion: a new category of "non-hemolytic ta-pls" is recommended to capture otherwise undiagnosed cases where ta-passenger lymphocytes form blood group antibodies in the recipient, but hemolysis does not occur, as in our aforementioned case. to ensure including the full scope of ta-pls, an antibody screen should be performed routinely one week after transplant and repeated as clinically indicated. occult hemolytic anemia due to anti-mur in a patient receiving blood from a region with a prominent asian donor population jean oak* , rosario mallari , marc de asis , elaine shu , jonathan hughes and tho pham , . stanford university, stanford health care, stanford blood center, bloodsource background/case studies: mur antigen is present in - % of individuals in southeast asia, taiwan, and parts of southeastern china, but is rare elsewhere. antibodies against mur antigens are clinically significant, hence many countries in asia routinely screen for it while other countries, including the us, does not include mur in the standard screen. we describe a case of an occult anti-mur antibody causing anemia and donor ethnicity distribution in a regional blood center with a large asian donor population. year old hispanic male with chronic myelomonocytic leukemia and plasma cell dyscrasia developed anemia. initial antibody screen and dat were negative, and the patient received - rbc units every - weeks to maintain a hemoglobin (hb) level of g/dl. the patient remained stable for months when his hb level acutely dropped to . g/dl. the antibody screen remained negative for an additional months when it became positive for anti-jka and anti-mur. donor ethnicity data was available for of the rbc units he received. units were from an asian donor, and a unit transfused days prior to the hb drop was from a caucasian/chinese donor. study design/method: we reviewed the ethnicity data of , donors at a hospital-associated blood center located in a region where asians comprise approximately % of the population. results/finding: . % of donors identified as chinese, vietnamese, filipino, or other southeastern asian. these donors account for of ( . %) rbc collections. conclusion: identification of anti-mur in this patient was triggered by the presence of a concurrent anti-jka alloantibody. since over % of the rbc supply in the local blood center was collected from chinese or southeast asian donors, chronically transfused patients are at risk of developing anti-mur-mediated hemolysis that could be missed on a standard screen. this finding raises a possible need for blood banks located in regions with a prominent asian population to implement screening for anti-mur. brian adkins* , princess maynie , carol chandler , shelia garret and pampee young . vanderbilt, vanderbilt university medical center, department of pathology, microbiology and immunology background/case studies: antibody titration is a testing modality vital to both obstetric and transplant services. manual direct tube testing is associated with variability in results (poor reproducibility/precision) and is also time and resource intensive. in fact, studies have shown a three-to eightfold inter-institutional difference between the antibody titers from the same samples using manual tube method. the orthovision automated analyzers offers automated titering of patient plasma using gel technology. although there is intense interest in adopting automated testing technology for titering, it is well-appreciated that titers obtained in manual gel testing are much higher than those obtained by manual/direct tube testing. the higher titer results lack clinical fetal anemia and outcome correlations, which is a barrier to their implementation. moreover, despite the increased sensitivity of gel testing, prior studies have found variable results with regard to reproducibility and precision. [ ] [ ] [ ] there is minimal information on the comparisons of tube titers to orthovision automated titers or assessment of the reproducibility of this automated method. study design/method: rh and non rh minor rbc antibody titrations were performed by manual direct tube method on clinical samples and the same samples were analyzed on three different ortho vision analyzers to assess precision and inter-instrument reproducibility. results/finding: a total of samples have been analyzed (table) , rh and non-rh antigens. titers via automated testing on orthovision resulted in a mean titration being . (range - ) times higher. the average fold change for rhd/c/e antibody titers were . , whereas the average fold change for non rh titers was . (range - ). the range for anti d titers was particularly variable, - , whereas for c/e, it was - . the overall reproducibility/precision of the automated analyzer was $ %. to correlate the a transfusion vol. supplement s increased titers observed with some classes of antibodies, particularly anti d, we will be performing parallel testing of obstetric samples and correlating with pregnancy outcome/fetal testing the obtained values. conclusion: automated titration of antibodies using the orthovision analyzers resulted in highly reproducible results between different instruments using the same sample. however, the automated analyzers consistently yielded higher values, particularly with rh d, with results $ times higher than in manual tube testing. interestingly, the difference in titers of non rh antibodies between manual tube and automated testing was not statistically significant, although our n thus far is small. in order to leverage the efficiency and reproducibility benefits of automated titering we will need to establish "critical titer ranges" which require active monitoring of the fetus. platelet additive solution reduces the isoagglutinin titer in apheresis platelet units maxim tynuv*, elizabeth j furlong and willy a flegel. dtm/cc/nih background/case studies: isoagglutinins in the plasma of apheresis platelets are a concern during transfusion, as high titer anti-a and/or anti-b may cause a hemolytic transfusion reaction (htr) in a recipient with cognate antigen. apheresis platelet collections are usually reconstituted with donor plasma, however most facilities do not test for high titer of isoagglutinins, exposing recipients to the risk of htr due to plasma incompatibility if given based on short outdate and not abo type. at our facility testing is performed on all apheresis platelets with a cutoff titer of . units above the cutoff are marked as "high titer" and only given to abo plasma-compatible recipients or washed with saline to reduce plasma. however, washing platelets is a time consuming process that results in a loss of up to % of the platelets. platelet additive solution (pas) is used as an alternative collection and storage solution, replacing approximately % of donor plasma in the final product. the goal of this study was to determine what affect pas has on isoagglutinin titers and whether using pas could lead to a revision of one facility's procedure for management out of group platelet transfusions. study design/method: isoagglutinin titers of whole blood edta samples were compared to the final apheresis platelet unit collected in pas (intersol, fresenius kabi, lake zurich, il). using two-fold dilution steps, plasma was tested with pooled red cells (equal mix . % suspension of a and b cells, ortho, raritan, nj) in a gel matrix test (mts buffered card, ortho, raritan, nj) with min incubation (room temperature) prior to centrifugation (mts ortho workstation). fifty two donors were group o, group a, and group b. results/finding: of the whole blood edta samples tested, ( group o and group b) exceeded a high titer threshold of . when the pas samples of these donors were tested, only one (group o) exceeded the same threshold. pas specimens showed a consistent two-fold decrease in titer compared with whole blood specimens. nearly half of the group o donors exceeded a titer of when whole blood specimens were tested. conclusion: only one sample from apheresis platelets collected in pas exceeded our clinically applied titer threshold of , a % decrease from the number of whole blood specimens exceeding the threshold. testing the platelet bag collected in pas instead of plasma from whole blood specimens would lower the number of units exceeding the high titer threshold, and reduce products needing to be washed. furthermore, facilities not collecting platelets on site or without access to whole blood specimens from donors could implement the process described here and screen platelet apheresis collections for potentially clinically adverse isoagglutinin titers, whether collected using pas or not. other components. the majority of blood components in israel are collected and distributed by magen david adom (mda), from main locations. several hospitals in israel also collect platelets in-house. as part of an effort to understand plt utilization, a nationwide survey of plt transfusion and expiration was conducted. study design/methods: data on the disposition of all plt units, acquired from mda and collected in-house, during the calendar year was requested from all hospitals in israel. the number of plt distributed to hospitals by mda was also collected. plt wastage was defined as the sum of plt that were returned and not reissued from the hospital blood banks and plt that expired on blood bank shelves. results/findings: sixteen of the ( %) hospitals in israel, along with mda, participated in the survey, listed as a to p. the results are presented in the table along with each hospital's distance from the mda facilities. for some hospitals, the sum of transfused and wasted plt was slightly less than the number of plt supplied by mda; this is likely due to the small number of plt that had not either been transfused or expired by the time the data collection period ended. three of the largest hospitals (c, b and a) collected plt in-house in addition to acquiring units from mda. these hospitals had a lower overall rate of wastage including their own donations than the other hospitals that did not collect in-house plt. the other hospitals had wastage rates ranging between - %. no correlation was apparent between the hospital's distance from the mda facility or its number of beds and the plt wastage rate. conclusion: there is considerable platelet wastage in israel. large hospitals in israel with in-house donations had the lowest overall wastage rates in comparison to the other hospitals. factors known to affect plt utilization and wastage such as patient diagnosis mix, policies about how plt are issued and accepted back into hospital inventory, plt inventory size and the time of pooling of whole blood platelets relative to the time they are issued and returned to the blood bank need to be investigated and optimized in order to reduce wastage rates. possible immune-mediated hemolysis due to platelet transfusion masked by underlying hemolysis in a patient with blast crisis sirisha kundrapu* , , christopher j gresens , anne capetillo , hollie m reeves , and katharine a downes , . case western reserve university school of medicine, university hospitals cleveland medical center, bloodsource background/case studies: transfusion-related hemolysis with abomismatched platelets is rare with a reported incidence of < . %. most commonly in such cases group o platelets having high titer anti-a result in clinically significant hemolysis when transfused to a group a or ab recipient. we present a patient with a possible hemolytic reaction following transfusion of abo mismatched platelets presenting in the setting of underlying disease associated hemolysis. study design/method: a -year-old male with chronic myelogenous leukemia in blast crisis was evaluated for possible transfusion reaction to a single donor platelet (sdp). two hours post transfusion he developed chills, rigors, and increased blood pressure ( / mm hg to / mm hg) followed by hematuria ( ml). chills and rigors resolved; blood pressure stabilized after min with diphenhydramine, solumedrol, and acetaminophen. negative. patient abo group, rh (d) type and antibody screen on pre-and post-transfusion specimens showed no discrepancies. laboratory indicators of hemolysis are summarized in table. notably, while total/ indirect bilirubin increased and hemoglobin decreased after transfusion other tests were indeterminate for hemolytic transfusion reaction with abnormal pretransfusion levels. despite underlying disease associated hemolysis, the blood supplier of the unit was contacted to investigate into the possibility of high titer donor anti-a. this revealed donor anti-a titer results of (igm) and , (igg); donor was deferred from future platelet donations. conclusion: while the post-transfusion sample had no visible hemolysis and a negative dat, increased total/ indirect bilirubin after transfusion and high titer donor anti-a are supportive of immune mediated hemolytic transfusion reaction. the key unique aspect in this case is baseline underlying hemolysis, which may mask needs for further investigation of donor for high titer anti-a. ana paula hitomi yokoyama* , leila patricia de sousa fontenele , isabel nagle reis , carolina bonet bub , araci sakashita , raffael zamper , cristiane nakazawa , tatiane almeida omura paula , patricia silva batista , marcio dias almeida , fernanda loureiro de andrade orsi and jose mauro kutner . hospital israelita albert einstein, hemocentro unicamp-universidade estadual de campinas background/case studies: orthotopic liver transplantation (olt) is a high complex procedure, fundamental to therapeutic approach for end-stage liver disease. despite improvements in hemostatic , surgical, and anaesthetic techniques, liver transplantation is still associated with massive blood loss and high rates of transfusion requirements. peri and intraoperative transfusion of red blood cells (rbc) have been previously reported as major predictors of post -operative mortality . identifying predictive factors for transfusion requirements may help optimise patient blood management strategies in olt. we conducted a single center retrospective analysis of cases of olt performed between and in brazil in order to identify predictive factors for red blood cell transfusion study design/method: a retrospective analysis in a single institution was performed, and charts of consecutive patients submitted to liver transplantation between and were reviewed. the following variables were collected for each patient: gender, race, primary diagnosis, presence of hepatocellular carcinoma, age, body mass index, corrected model for end-stage liver disease (meld), duration of warm and cold ischemia. categorical variables were analysed using pearson chi-square test. continuous variables were analysed using t-student test. a forward logistic regression model was used to analyse data in a multivariate fashion, to identify independent contribution of variables previously found to be significant. results/finding: in univariate analysis, female patients, absence of hepatocellular carcinoma (hcc), primary diagnosis, corrected meld and warm ischemia time were significantly associated with consumption of rbc use in the intraoperative period. multivariate logistic regression of these factors showed that female patients (or , - % ci: , - , , p: , ), absence of hcc (or , - % ci: , - , , p: , ), cirrhosis of any cause (or , - % ci , - , -p: , ), miscellaneous diagnosis (auto-immune, metabolic diseases, familial amyloid polyneuropathy, vascular complications) (or , %ic , - , ) and retransplantation due to primary non function of the graft (or , %ci , - , , , p: , ) were independently associated with rbc transfusion requirements. conclusion: in this study, female patients, absence of hcc, specific primary diagnosis and retransplantation due to primary non function of the graft were significantly associated with rbc consumption in intraoperative period. determination of rbc transfusion predictors before surgery might provide important information regarding management of blood components and help optimise utilisation of resources for blood conservation strategies. prevalence of high-titer anti-a /b in group o platelet products. charles k. childers* , mark destree , ashley rose and theresa nester , . madigan army medical center, bloodworks northwest, bloodworks nw, dept of laboratory medicine, university of washington background/case studies: with platelet substitution policies, minor aboincompatible platelets (where donor's plasma may contain antibodies to recipient's red blood cells) are often issued in an effort to best utilize the community supply. however, rare reports of acute intravascular hemolysis have been reported from such transfusions, and can be attributed to high anti-a or anti-b titers, typically in a group o donor. one method to reduce the risk of hemolysis is to identify high titer platelet units prior to transfusion with a subsequent intervention. the percentage of high titer anti-a /b in group o platelet products is presented from a large regional blood center collected over - months. data from both pre-storage pooled platelet units (pspp) and apheresis derived platelet units (aplt) is shown. study design/method: platelet component samples were collected in ml edta sample tubes. a single : dilution of plasma was prepared using a hamilton microlab series dilutor using . ml saline diluent and . ml platelet component sample. using a standard transfer pipette, two drops of diluted sample were transferred to each reaction tube along with one drop of a or b red blood cell reagent. reaction tubes were centrifuged immediately in a serological centrifuge at rpm for seconds. reactions were read using a lighted agglutination reader. the presence of macroscopic agglutination (weak or greater) with either the a cells or b cells was recorded as a positive reaction, indicative of a high titer anti-a or anti-b. retesting of samples was performed to confirm high titers. results/finding: the above results indicate that, when a titer cut-off of is used, approximately % of group o apheresis platelets will have a high titer, most commonly with anti-a . less than half of a percent of pspp units will have a high titer. testing units for the titer can help to change abo out-of-group platelet substitution policies. in our example, the bloodworks transfusion service was able to change from a policy of volume reducing any group o apheresis platelets being issued to a group a or ab patient, to giving high titer products to only group o patients. the subsequent decrease in episodes of volume reduction helped to improve overall availability of apheresis platelets, by maintaining their day outdate. after months of testing pspp units and verifying that the products rarely had a high titer ( . %), the blood center stopped performing this testing for pspp units. rh ) ] started complaining of worsening back pain two and half hours after receiving one unit of rbc for a drop in hematocrit to % (from % on the previous day). his hematocrit did not increase ( %), and over the ensuing hours, he became anuric and jaundiced. clerical checks confirmed that his forward type was a positive, which was also the type of the rbc unit transfused, but revealed anti-a at a titer of in his plasma. furthermore, the direct antiglobulin tests (dat) were positive for c in the pre-and post-transfusion blood samples. anti-a was not detected in his plasma collected three days earlier, however. although his plasma color was amber, he had signs of intravascular hemolysis: undetectable haptoglobin, increased lactate dehydrogenase (ldh) and total and indirect bilirubin results/finding: the positive dat in the pre-transfusion sample pointed to ongoing hemolysis prior to the transfusion of the a rbc unit. in the setting of recent abo-mismatched transplant, his picture was consistent with hemolysis from newly formed anti-a by proliferation of donor lymphocytes, or pls. we performed an emergent rbc exchange using o rbcs with a goal hematocrit of % while reducing the number of a rbcs in his circulation by approximately %. his pain improved rapidly thereafter, and he had complete recovery of renal function. conclusion: pls should be in the differential diagnosis when suspecting/ investigating clinically significant hemolysis in abo-mismatched hpc transplant recipients, especially when the hpc source is from peripheral blood. as in our patient, it usually takes - days for antibodies to develop and they are short-lived ( - weeks). due to the severity of his manifestations, we performed an emergent rbc exchange successfully. furthermore, this patient's event exposed a vulnerability in our system of issuing the proper blood type for abo-mismatched transplant recipients, which has since been remediated electronically background/case studies: group o rhd negative (oneg) red blood cells (rbcs) are a precious resource. to conserve the oneg inventory while minimizing the risk of rhd alloimmunization in oneg females of childbearing age, transfusion services may automatically provide group o rhd positive (opos) rbcs to rhd negative males and/or rhd negative postmenopausal females during bleeding emergencies. despite these conservation strategies, shortages of oneg rbcs occur. the goal of this study was to determine how the utilization of oneg rbcs can be optimized using agebased opos switching for routine transfusions in oneg patients. study design/methods: recipient age and abo/rhd group were obtained for all allogeneic rbc transfusions during the calendar year from hospitals. an additional hospital* provided data for august-december . rbc transfusions in patients < year of age, and in patients whose age and/ or abo group were unknown, were excluded from analysis. the abo/rhd group of each rbc unit was compared to that of the recipient to determine the number of oneg rbcs transfused to all patients, the number of rbcs transfused to oneg patients and the number of oneg rbcs transfused to oneg patients. the number of oneg rbcs transfused specifically to oneg patients >/ years was also determined. results/findings: see table . the fraction of all transfused rbcs that were oneg ranged from - % (row f). the percentage of oneg rbcs transfused to oneg patients ranged from - % (row g); thus, non-oneg patients received - % of the oneg units transfused (row h). hospitals differed widely in the practice of issuing oneg rbcs to oneg patients ( %- %; row i). overall use of oneg rbcs could have been reduced by %- % if opos units had been given to all oneg patients >/ years old (row j). conclusion: during times of oneg shortage, age based opos switching rules may be applied for routine transfusions. this would help to ensure the availability of oneg rbc units for oneg females of childbearing age. rasha eldeeb mohammed* , nehad mohammed , marwa aly and nashwa fahmy . national blood transfusion services, nbts background/case studies: sensitization to the transfused red cell may complicate further transfusion& make it increasingly difficult to find compatible blood components for those patients. splenectomy has been shown to increase human leucocyte antigen immunization. the aim of the study is to evaluated the effect of splenectomy on the occurrence of red cell alloimmunization in humans. study design/method: this study was conducted on multitransfused patients who received blood transfusion chronically at our central blood center. they were thalassemia patients ( bthalassemia patients, one patients with a thalassemia), sickle cell anemia patients and immune hemolytic anemia patients ( auto immune hemolytic anemia patients, one paroxysmal nocturnal hemoglobinemia patient, one immune thrombocytopenic purpra patients). oncology patients, chronic diseases patients. history and demographic data were documented. all the patients who received blood are examined for the presence of the spleen.our patients were subjected to direct & reverse blood grouping (abo& rh) tests, alloantibody screening and detection. results/finding: statistical study is done to determine what is the effect of splenectomy in increasing the rate of red cell sensitization in chronically transfused hemolytic patients. the study revealed that: out of ( %) alloimmunized patients and out of ( %) non alloimunized patients(p< . ) . statistical analysis show that there is high statistical significant difference between patients who performed splectomy& who did not perform splenectomy as regard form conclusion: patients who had splenectomy had a higher alloimmunization rate removal of the spleen is not recommended in those patients who are periodically in need of blood and blood components. restrictive transfusion triggers rather than specific evidence. therefore, two systematic reviews of a) rbc transfusion guidelines and review articles to determine if single or multiple unit transfusion strategies are recommended and b) to identify studies comparing strategies were performed. study design/method: methods medline, embase, cinahl, web of science, national guideline clearinghouse, and the trip database were searched from inception to june . screening and data abstraction were done independently by two assessors. for review a, the proportion of articles with recommendations and articles recommending single unit strategies were assessed; stratified by guidelines, systematic reviews, and other review articles. for review b, the primary outcome was rbc utilization. secondary outcomes included proportion of units transfused using a single unit strategy, length of stay, and mortality. meta-analysis was done using the mantel haenszel random effects model. results/finding: review a identified articles for data abstraction, where articles were transfusion guidelines. there were guidelines ( %) that made a recommendation, for a single unit and for multiple unit transfusion strategy (table ) . review b identified retrospective cohort studies that were eligible and data abstraction was performed. all utilized a policy encouraging single unit transfusion strategies and compared a pre-implementation period to a post-implementation period. meta-analysis could only be performed on the secondary outcome of the proportion of units transfused using a single unit strategy, which was higher after the policy intervention (or . , % ci . - . ), although heterogeneity was high (i %). conclusion: our systematic reviews demonstrated a lack of recommendations amongst guidelines pertaining to transfusing single units of rbcs and only a few retrospective cohort studies to support benefits of the use of single unit transfusion strategies. additional high quality studies are needed to identify the benefits of a single unit transfusion strategy and when it should be used. guidelines groups should review research in this area to determine if a recommendation can be made. background/case studies: platelets made with platelet additive solution c (pas c) and treated for pathogen reduction (pr) have been shown to have decreased post transfusion platelet counts from platelets stored in all plasma. with the advent of multiple types of platelets, we are evaluating whether a mixed platelet inventory has had an effect on component use. the literature from europe has shown that platelet and red cell use does not increase when pr and pas products are used. evaluation of rbc use at our institution has shown no change in the number of products transfused per patient per month. we are evaluating whether the mixed inventory has led to more platelet transfusions. study design/method: we looked at occasions when patients received all of their platelet transfusions on a single day. by doing this we were able to exclude refractory patients from the analysis. the information obtained from routine quality management audits of transfusions between december and february was used for this analysis. the information included the ordering service, product release time, product code, pre and post counts. statistical analysis was performed using minitab. results/finding: during the months, units of platelets were transfused to recipients. over the months, a median of units was given to each patient with a range of to . the overall distribution of products used was % plasma, % pr, % pas f and % pas c. thirty percent of patients (n ) received all of their products on a single day. single units were given to patients while , and received , , and units respectively. the distribution by product type was % plasma, % pr, % pas c and % pas f. this same percentage was present for single and multiple products and was not statistically significantly different from the overall distribution of the products given during the month period (p . ). the distribution by service was different for the groups receiving multiple units. for single units the distribution was % hematologic malignancy, % infusion clinic (nos), % solid tumor medicine, % surgery, and % pediatrics. for those receiving multiple units the distribution was % surgery and % each for solid tumor, hematology and infusion (nos). the chi-square test for associations showed the increase in multiple units to surgical patients to be significant with a p value of . . conclusion: the distribution of the type of platelets given during a single event of transfusion was not significantly different from the overall distribution of platelets given during the month period. the patient's clinical service was a better predictor of the use of multiple products than the type of product given. this suggests that surgical losses or the need to have a higher platelet count during a procedure was the leading factor in the use of multiple products in this transfusion scenario. the effect of red blood cell transfusion on iron metabolism in critically ill patients margit boshuizen* , , yvemarie b.o. somsen , maike e. van hezel , marleen straat , robin van bruggen and nicole p juffermans . sanquin research and landsteiner laboratory, academic medical center background/case studies: anemia of inflammation (ai) has a high prevalence in critically ill patients. in ai, iron metabolism is altered, as high levels of inflammation-induced hepcidin reduces the amount of iron that is available for erythropoiesis. ai is treated by red blood cell (rbc) transfusions. it is known that rbc transfusions increase iron level in neonates and thalassemia patients, but the effect of rbc transfusion on iron metabolism during inflammatory processes is unknown. since one unit of rbcs contains mg of iron and % of the rbcs are cleared by macrophages within hour following transfusion, rbc transfusion could increase iron levels and iron availability for erythropoiesis. we investigated the effect of rbc transfusion on iron metabolism in icu patients, and additionally compared the effect in septic patients to non-septic patients. study design/method: in a prospective cohort study in icu patients who received one rbc transfusion, different iron parameters were measured before and hours after transfusion, to determine the effect of a rbc transfusion over a period of time. next, the impact of a rbc transfusion on plasma iron parameters in septic patients compared to that in non-septic patients was analyzed. plasma iron concentration, transferrin (saturation), ferritin, haptoglobin, hepcidin and il- levels were determined. results/finding: in this cohort, serum iron levels were low and did not change following transfusion ( . vs. . mmol/l, p . ). also, the transfusion had no effect on transferrin saturation ( vs. %, p . ), ferritin ( . vs. . mg/l, p . ) and il- levels ( . vs. . pg/ml, p . ). hepcidin levels increased in these icu patients after rbc transfusion ( vs ng/ml, p . ). in septic patients, rbc transfusion induced a decrease in haptoglobin levels compared to baseline, which did not occur in non-septic patients (- . vs. . % change, p . ). other iron parameters did not differ between septic and non-septic patients. conclusion: transfusion of one unit of rbcs does not increase iron levels in icu patients. the increase of hepcidin suggests rbc transfusion induced upregulation of hepcidin, despite the absence of a significant increase in il- or plasma iron levels. this increase in hepcidin levels after transfusion can potentially further hamper iron availability for erythropoiesis. in sepsis, rbc transfusion decreases haptoglobin levels, suggestive of hemolysis. in conclusion, rbc transfusion might have a negative effect on erythropoiesis, due to the increase in hepcidin levels that are observed after transfusion. the effects of pas and pr on platelet use barbara mendez, judith delmonte, elizabeth mccabe and joanne becker*. roswell park cancer institute background/case studies: with the anticipated release of the fda guidance: bacterial risk control strategies for blood collection establishments and transfusion services to enhance the safety and availability of platelets for transfusion, the use of pathogen reduced platelets (pr) which are often produced from products made with platelet additive solution (pas) may become more common. our institution has been transfusing platelets made with additive solutions since and pathogen reduced platelets have been available since . in our data validating pas and pr, the post counts from transfusion of pas-c and pr products have been statistically lower than platelets in all plasma (pp) or pas f products. our study looks at whether this difference has led to a corresponding increase in the number of units of platelets transfused. study design/method: the data was obtained from the routine quality reports produced for the blood utilization committee at our facility between and . during this time pas c, pas f and pr went from % to % of all platelet products given. all recipients had an oncology diagnosis. the data collected included the service, unit number and product code. the number of unique recipients was determined monthly. the data was converted to plt/month/recipient for analysis. statistical analysis was performed using the two sample t-test results/finding: the data was normalized to plt/recipient/month. in patients received an average of . units/recipient/month and in the average was . units/recipient/month. the intervening data points for , , and were . , . , and . respectively. the year average was . . the slope of the graph for all points was y - . . . the two sample t-test showed that the plt/recipient/month from to was not statistically different with a p value of . . conclusion: the implementation of pas and pr platelets in the oncology environment has not increased in the number of platelet transfusions given. in additional analysis, the red cell use has decreased (data not shown). this can be interpreted as indicating that patients have not had increased episodes of bleeding. although the post platelet count from pas/pr platelets may be lower, we do not have evidence from our platelet transfusion data that this is leading to clinical outcomes necessitating additional products to be given. background/case studies: it is reported that the incidence of alloimmunization in aml patients is unrelated to the number of transfusions the patient receives and most patients who have hla antibodies do not exhibit platelet refractoriness. many cases are also found not to have any anti-platelet antibodies detectable by standard laboratory tests. recent data in leukemia and hematopoietic stem cell (hsct) recipients transfused exclusively with leukoreduced products show that % to % develop alloimmune platelet refractoriness. objective: to determine an improvement in platelet count with the match grade and/or the abo blood group of the hla matched platelets in highly alloimmunized patients with concomitant non-immune causes for platelet destruction. study design/method: clinically documented platelet refractory patients, who received hla matched irradiated sda platelets with their hla typings for hla-a/-b and hla antibody identification were reviewed. there were two strategies utilized, the hla strategy (matching recipient and donor hla-a/ -b types) and the antibody specificity prediction (patient provided with platelets from donors lacking only those hlas to which the patient had antibodies) strategy. statistical analysis: a one sample t-test using minitab statistical software was performed comparing the mean against a platelet increment of a hypothetical difference of at least k/ul. the analysis revealed that the mean of . k/ul (n ) had a percent lower bound confidence interval platelet increment of k/ul (p< . ) results/findings: (median range [ - ]) hla matched leucoreduced irradiated sda platelets were transfused to ( m/ f) patients, median age years (range - ). / ( %) patients showing broad alloimmunization to hla class i/class ii antigens. / ( %) patients had anti-hpa antibodies (gp iib/iiia and gp iib/iiia and gp ia/iia). the majority / ( %) had a diagnosis of hematologic malignancy (aml/mds/mpn/ cmml/mm); / ( %) female patients had prior exposure via pregnancy and / ( %) had a history of hsct. ( %) platelets were abo identical-platelet increment median k/ul (range - to ), ( %) were abo compatible -platelet increment median of k/ul (range - to ) and ( %) were abo incompatible with platelet increments median k/ul ( range - to ). platelet counts were performed within hours in ( %) transfusions. the hla match grade of the transfused platelets were as follows: the use of massive transfusion protocol (mtp) in a community hospital rohini patel* , renee leblanc , dongfu xie , alice cabe and yanyun wu . overlake hospital, bloodworks northwest the use of massive transfusion protocol (mtp) in a community hospital background/case studies: the establishment and use of massive transfusion protocol (mtp) have become common practice, especially in trauma centers and tertiary hospitals due to significant number of patients with massive bleeding. however, it is not well established if the use of mtp also has value in small hospitals and community hospitals, and how mtp is used in fig. these settings, such as indication for mtp, blood products used, and the outcomes of these patients. study design/method: retrospective review of transfusion data from a community hospital with a bed size of about for years (from to ) was performed. patients with mtp requested are included in this study. results/finding: please see the table below for the summary of data. notably, patients with gi bleed and ob bleed are the two most common indications for mtp, and % of patients survived with the support of mtp. in one case, no blood product was used. the establishment and readiness of mtp can be very important in supporting patients who experience massive bleed in small hospitals and community hospitals. in these settings, mtp is most commonly used for patients with massive gi bleed and ob bleed. if the patient develops antibodies to a high incidence antigen, finding compatible units may become impossible. included in the mns system, and residing on glycophorin b (gpb), the u antigen is absent in less than . % of the black population. those with altered forms of gpb, known as u variants, can produce a diverse group of antibodies capable of causing mild to severe hemolytic transfusion reactions and hemolytic disease of the fetus and newborn (hdfn). this case illustrates the balance between the need to transfuse and avoiding complications thereof. a -year-old ghanaian woman with scd and history of chronic transfusion presented with diffuse pain and a hemoglobin value of . g/dl (baseline - g/dl). she is known to be e, c, k, fya, jkb, s, s negative, u variant, and has anti-e, c and u antibodies. there were no eligible family donors and a nationwide search for compatible blood yielded four crossmatch compatible u variant units. the decision to transfuse was made. the patient had no change in symptoms or vital signs during transfusion but post-transfusion hemoglobin was . g/dl. a transfusion reaction work-up was ordered. post-transfusion serum sample was negative for hemolysis and no new antibodies were identified. the post-transfusion dat was weakly positive only with complement and laboratory data revealed a decrease in total bilirubin ( . to . mg/dl). two additional u variant, crossmatch compatible units were transfused over the next two days restoring her hemoglobin to . g/dl. the patient was discharged to home in stable condition and follow-up hemoglobin levels continued to rise back to baseline. study design/methods: molecular genotyping was used in donor unit selection prior to compatibility testing by transfusion services. conventional methods were used to monitor the patient's condition pre and posttransfusion. results/findings: each donor unit came from a different donor but all were the same gpb genotype as the patient. the patient did not experience an acute or delayed hemolytic transfusion reaction and genotype matching successfully facilitated donor unit selection in this case. conclusion: transfusion of u variant red cells to a u variant patient should be undertaken with great caution due to epitope and antibody heterogeneity. this case highlights the importance of genotype compatibility in selecting donor units for a chronically transfused, scd patient with anti-u. sound transfusion management of such patients requires planning and good communication on the part of clinicians and the laboratory staff. background/case studies: patients with decompensated waiha may require transfusion with red blood cell (rbc) products that are cross-match incompatible due free autoantibodies. the feasibility of blood transfusions in waiha patients is controversial because of difficulty in cross-matching and increased risk of transfusion reactions, since transfused rbcs may be destroyed more rapidly in patients with active hemolysis. to study the actual vs. theoretical risk of increased hemolysis in waiha patients, we investigated the post-transfusion (post-tfn) hematocrit (hct) change in waiha patients who were transfused compatible rbcs compared to those who received li blood. we further hypothesized that a post-tfn hct would be inversely related to the degree of ahg-phase incompatibility. study design/method: we reviewed all transfusions to patients in our quaternary-care hospital with a history of waiha from october to march . patient hcts were ordered by prescribing physicians for clinical purposes. a transfusion episode was defined as all units released in the interval before a post-tfn cbc. ahg-phase crossmatch was tube tested in saline per clinical procedure. transfusion medicine physicians determined the release of least-incompatible units. statistical tests were performed with statcalc (epiinfo, cdc) and www.socscistatistics.com. results/finding: there were rbc products transfused to waiha patients. twenty-three ( . %) patients received at least incompatible unit. the mean age was . years (range - yrs) with % women. ethnic composition was % african-american, % caucasian, and % patients of mixed/other ethnicity. one hundred fourteen ( %) of these products were released as li products and ( %) were compatible. ninetythree ( . %) of the li product transfusions had a post-tfn hct change of < % whereas only ( %) of the compatible product transfusions resulted in a post-tfn hct change of < % (p . , v ( ), exact methods). the mean hct increase in the compatible group was . % per unit vs. a slightly lesser per-unit increase of . % in the li group (p . , t-test, -tailed) within the li group, there was no difference in the per-unit hct change according to strength of incompatibility (table) . strength of ahg incompatibility was not available for units. units that were incompatible had a lower mean post-tfn hct rise compared to all other li units ( . % vs. . %); however, this difference was not statistically significant (p . ). conclusion: the post-tfn hct change for transfusions of li units to patients with waiha was less than the expected % per unit more frequently than it was for waiha patients who received compatible products ( . % vs. %). however, likely due to our small sample size, the mean differences were not statistically significant. interestingly, there was no difference in the per-unit post-tfn hct according to differing strengths of incompatibility in our sample, although the mean increase for the li products was less than all other li products combined. the increase was unexpectedly low for weaklyincompatible units, which we are further studying. future work includes consideration of inpatient vs. outpatient clinical status, effect of co-incident alloantibodies, comorbidities, and medications. transfusion management was summarised by individual hospital, type and total cases. in-hospital mortality (adjusted for age, sex, comorbidity, bleeding context and number of rbcs in the first -hours from mt onset) was calculated with % and . % control limits to indicate potential outliers. data were analyzed using statistical software (stata). results/finding: there were mt cases from hospitals ( tertiarylevel, smaller/medium sized acute-care and specialist women's). number of mt cases per hospital ranged from to . patient median age was years (iqr , ), % were male and % required admission to intensive care. the most common clinical groups were cardiac surgery ( % cases), trauma ( %) and gastrointestinal hemorrhage ( %); however there was marked variation between hospitals. ratios of transfused products, analyzed according to bleeding context, varied between hospital types. the pooled average adjusted in-hospital mortality for the tertiary-level hospitals was % (range % to %) and / ( %) were within the % control limit. cb that required ! rbcs within -hours of mt onset occurred in % of cases. comparison of transfusion management for this subset of mt cases showed that patients treated in smaller/medium sized acute-care were less likely to receive cryoprecipitate than patients treated in tertiary-level hospitals ( % versus %; p . ). conclusion: patient characteristics and transfusion practice varied between hospitals and hospital types, however in-hospital mortality outcomes were comparable. results are made available to participating hospitals in the anz-mtr to initiate discussion, practice review, and examination of compliance with national standards, patient blood management guidelines and to highlight areas for further investigation. data are also available for review by governance and policy bodies at state and national level to support practice improvement activities and highlight priority areas for future research. background/case studies: in hospitals and medical centers, in case of big traumas often an intraosseous entrance via a bone needle is combined with a fast flow fluid warmer. with this, infusion fluids, including blood products, are administered under pressure. this is done because veins of trauma patients are often not suitable for infusion of fluids. suppliers of pump and needles describe the possible transfusion of blood products, but this is mainly limited to plasma and erythrocytes. there is no information available concerning transfusion of platelets under pressure via a bone needle. the aim of the study was to investigate the effects of warming and administration of a platelet concentrate (pc) under pressure via a bone needle on the in vitro quality of platelets. study design/method: pools of bcs and ml of platelet additive solution iii (pasiii) were used to produce pcs (n ). pcs were stored on a flatbed agitator ( cycles/min) in a temperature-controlled cabinet at c for - days. to mimic hospital conditions, pcs were warmed using a blood warmer and transfused via a bone needle to a transfer bag. on the pcs a pressure of mm hg was applied. using clamps, a flow velocity of - ml/minute was realized. platelet quality before and after pressurized simulated transfusion was determined by means of various in vitro parameters. results/finding: due to priming of the transfusion disposable with saline, the pcs were diluted - %, resulting in a significantly increased pc volume and decreased platelet concentration after simulated transfusion. because of loss of platelets in the disposable set, also the total number of platelets was decreased after simulated transfusion. after simulated transfusion, the pcs still fulfilled the requirements for platelet concentration ( . - . x /l) and number (> x /unit). simulated transfusion had no effect on the percentages of cd p and annexin v positive cells, indicating no activation or induction of apoptosis. ph was not influenced by simulated transfusion. due to the dilution effect, glucose and lactate concentrations were slightly lower after simulated transfusion. conclusion: warming and simulated transfusion of pcs under high pressure via a bone needle has no negative effect on the in vitro quality parameters of platelets. transfusion of warmed pcs via an intraosseous entrance via a bone needle is not expected to have a negative effect on the in vivo functionality of platelets. it is recommended to study the in vivo effects in a limited clinical study. alesia kaplan* , , joan sevcik and joseph e. kiss , . university of pittsburgh, blood systems inc. background/case studies: low titer a plasma has been safely used as a substitute for ab plasma in trauma patients. low inventories of ab plasma can cause a delay in life saving therapeutic plasma exchange (tpe) procedures in ab patients needing plasma replacement. here, ab non-bleeding patients are presented who safely received ab and low titer a plasma for tpe. one ab patient who received ab plasma only was used as control to compare hemolysis laboratory data over tpe course. study design/method: a retrospective review of tpe procedures for patients was conducted from medical records. number of procedures, volume replaced, total number of plasma units, number of a plasma units, quantity of a plasma and hemolysis laboratory data were recorded. average quantity (ml) for a plasma and % of a plasma out of total volume of plasma used were calculated. all a plasma units were low anti-b titer units. in the laboratory, plasma dilution : is prepared and tested with reagent b cells. if agglutination is not observed, the unit is labeled as "low titer anti-b". hemolysis laboratory data was traced with linear graphs and trends were compared between patient and and (control). results/finding: all patients were ab blood type. patient , a year old female with recurrent adamts deficient ttp, received courses of tpe (total tpe procedures) for relapse and exacerbation. ten out of procedures were performed with ab and a plasma (average ml of a plasma or % of total plasma volume for tpe procedures). patient , a year old female with thrombocytopenia, schistocytes and presumed ttp, received a total of tpe procedures. four out of procedures were performed with ab and a plasma (average . ml of a plasma or % of total plasma volume for tpe procedures). patient , a year old female with adamts deficient ttp who served as a control, received a total of procedures with ab plasma only. haptoglobin, ldh, hemoglobin and total bilirubin were graphed and compared between patients. the trends of hemolysis laboratory data for patient and were comparable with patient . all patients had negative dat. only patient received rbc transfusions. all patients had a favorable clinical outcome with tpe treatments and adequate platelet recovery. conclusion: in this study, tpe was effectively performed without evidence of increased hemolysis using up to % of low titer a plasma. this approach can reduce strains on limited supplies of ab plasma while providing a vital treatment alternative for ab patients undergoing tpe who require plasma replacement. when cd negative platelet unit is not available for a patient with anti-cd antibodies sameer khatri* , charles harmon , brian r curtis and chisa yamada . background/case studies: refractoriness to platelet (plt) transfusion can be caused by antibodies (abs) against human leukocyte antigen (hla) class i antigens (ags) or less frequently against plt specific ags (psas). glycoprotein iv (cd ) is one of the identified plt surface ags and deficiency is rare, but found in asians ( - %), sub-saharan africans ( - %) and also in some people from mediterranean descent. two types of cd deficiency have been described. type deficiency is the complete lack of cd on both plts and monocyte-macrophages whereas type deficiency lacks cd on plts with variable expression ( - %) on monocytemacrophages. transfusing plts in a patient with cd deficiency is challenging given the rarity of cd negative phenotype and risk of further immunization when giving ag non-matched platelets. study design/method: a patient with cd negative phenotype who received multiple plt units was reviewed in the electronic medical record. results/finding: a year old man developed aplastic anemia following liver injury possibly due to a supplement for body building and required multiple plt and rbc transfusions. he received more than units of apheresis plt units over a week period without any significant increase in plt count. cross-match compatible plt unit found in of units and hla matched units were tried without success. at that point, a cd ab was identified in the serum and the patient's type cd deficiency was confirmed by flow cytometry. his hla class i panel reactive ab (pra) was % due to multiple plt transfusions, although all abs were low levels. the patient initially received high-dose prednisone and thymocyte immune globulin infusions without significant improvement in plt increase. following three doses of ivig, he received a cd- negative (but blood type different and hla a transfusion vol. supplement s unmatched) plt unit from his relative with only a slight increase in plt count. however, he started to respond to cd non-tested apheresis plts after receiving a fourth ivig and two rituximab infusions. since then, he has received ivig every weeks. other medications include filgrastim, eltrombopag, and cyclosporine for treatment of aplastic anemia. the mean corrected count increments (cci) when post-transfusion plt count was available are shown in table. with desensitization therapy, his cd antibody positive reactivity in serial dilutions has reduced from : to : dilutions and his hla class i pra has decreased to %. he is currently receiving apheresis plt units twice a week and rbc units periodically. his bone marrow (bm) has been slowly recovering evidenced by increased wbc count from zero to up to . k/ml and slow increase of reticulocyte counts. current plan is rbc/plt transfusion support until bm recovers or a haplo-identical transplant if bm recovery fails. conclusion: we report a case with anti-cd abs that received multiple plt transfusions. this case demonstrates that decreasing ab level with immunomodulation can be an alternative option for successful plt transfusion when compatible plts are not available for patients with rare or multiple abs to plts. table: mean available cci for plt transfusions a blood center's experience screening donations for babesia microti using enzyme-linked immunoassay methodology nancy van buren*, jed gorlin, vanessa reynolds and deborah anderson. background/case studies: our blood center, located in an area considered to be moderately endemic for babesia microti, implemented universal screening of red cell collections from minnesota and wisconsin under an investigation new drug (ind) study in oct utilizing the immunetics investigational enzyme-linked immunoassay (elisa) performed by creative testing solutions (cts). this test was selected as the most cost-effective approach for universal screening of blood donors, as opposed to the investigational ifa/pcr test combination. study design/methods: we performed a retrospective analysis of our screening test results and deferral rates for to evaluate for seasonality, donor abo bias, deferral rates, and outcomes of lookback investigations. since an opt-out of this research test was originally offered, we report donor opt-out rates. results/findings: from jan through dec , , blood donations were screened for b microti by immunetics elisa. of those, ( . %) were positive. the percent of positive donations was evaluated monthly revealing a variable reaction rate between . % and . %. no patient babesia transmission has been reported since implementing this test, but we only had documented babesia ttd cases from - . donors who previously tested negative demonstrated an increased seroconversion rate during the summer months, consistent with historical seasonal variation corresponding with tick season in minnesota and wisconsin. test performance characteristics were analyzed by abo group with no demonstrable differences in positive rates. the opt-out rate of donors who chose not to be tested significantly decreased over time, reflecting an increased acceptance of this test. of positive test results, lookback investigations were initiated representing % of positive donations. lookbacks were only performed when there was a donation within months of the new positive screening test, according to ind protocol. no confirmatory testing was performed per ind protocol or for donor counseling, so the true positive rate is unknown. in the prior ind trial, up to % were unlikely to transmit infection in our region, i.e. were pcr and blood smear negative. although a small number of antibody positive, pcr negative donors may be actively infected, no transfusion-transmitted babesia infections were identified by lookback investigations. notification of blood donors with positive screening results was also performed and information provided for healthcare provider followup. overall, donor deferral represented . % loss of eligible donors during this follow-up period. deferred donors were invited to participate in other research collections not requiring volunteer donor eligibility. conclusion: testing for b microti may help improve blood safety, particularly in endemic regions. although only . % of donors have a positive reaction, this represents a significant loss of eligible donors over time, most of whom are unlikely to transmit infection. a direct test capable of detecting babesia in individuals with very low levels of organisms without the need for concurrent antibody testing would be ideal. a reinstatement protocol for donors who test positive should also be considered. nonetheless, the current method of screening is inexpensive compared to pcr-based methods. background/case studies: human anelloviruses are the smallest in particle size, smallest in genome size, and least complex in genetic organization of all human pathogens. they establish a chronic persistent infection in infancy or early childhood and produce a constantly detectable load in plasma thereafter. some studies suggest they are ubiquitous, present in > % of the human population, and that immune surveillance is required to control the level of the virus load. study design/methods: we have developed a quantitative dna pcr assay for the most conserved region of the anellovirus genome that detects all known genotypes of the virus. we used this assay to examine viral loads in the plasma of us blood donors and transplant recipients pre-transplant and three months post-transplant. results/findings: for blood donors, were positive with an average load of . x copies/ml of plasma, a median value of . copies/ml of plasma, ranging from to . x copies/ml. pre-transplant viral loads were similar. for transplant candidates, were positive with an average of . x copies/ml of plasma, a median value of copies/ml of plasma, ranging from to . x copies/ml. post-transplant viral loads were remarkably different. for transplant recipients, all were positive with an average of . x copies/ml of plasma, a median value of . x copies/ml of plasma, ranging from to . x copies/ml. conclusion: these results validate the pcr assay that was developed and confirm that detectable viral loads of around - copies were present in > % of the blood donors surveyed. in addition, the effect of post-transplant immunosuppressive therapy has caused an increase in the viral load of at least orders of magnitude above that of non-immunosuppressed individuals. background/case studies: the screening of blood donors and travelers returning from endemic/epidemic areas has highlighted the importance of multiplex diagnostic approaches for the simultaneous analysis of various pathogens. furthermore, in the context of similar clinical signs, the differential diagnosis of arboviruses during acute infection is essential to discriminate the causative agent for patient management and epidemiological surveillance. the development of a flexible diagnostic approach is a key challenge to face the continuing emergence of arboviruses, belonging to flavivirus and alphavirus, such as dengue virus (denv), west nile virus (wnv), zika virus (zikv), yellow fever virus (yfv), usutu virus (usuv) and chikungunya virus (chikv). study design/method: an innovative diagnostic approach combining generic rt-pcr amplification and identification on low cost microarrays has been developed. we have patented original polythiolated probes grafted on maleimide-activated microplates for the robust, sensitive and specific mean cci pre-ivig: all plt ( ) . post-ivig: all plt ( ) . post-ivig: cd -negative plt from relative ( ) . post-ivig: single donor apheresis ( ) . post-ivig: cross-match compatible ( ) . post-ivig: flow cross-match compatible plt ( ) . a transfusion vol. supplement s detection of the viral genomes. analytical performances of the test were evaluated on viral standards and on clinical samples: denv ( / / / ), wnv, zikv and chikv. forty human plasmas from blood donors with no history of contact with arboviruses were used as negative controls. we have designed two sets of degenerated primers for the generic rt-pcr amplification of all flaviviruses and for chikv. biotinylated amplicons were captured on complementary grafted polythiolated probes on microplate. after addition of streptavidin-europium label, the molecular hybridization events were detected by time-resolved fluorescence using a microplate reader. results/finding: one original generic probe for denv and specific probes designed for each of the four denv serotype, wnv, the two zikv lineages and for chikv, were validated. the use of our methodology combining the amplification of the viral genomes and their identification using polythiolated probes shows % of specificity, with no false positive results on the control samples, and no cross reactions. using viral reference standards, we have observed sensitivities of tcid /ml for denv- , denv- and chikv and of tcid /ml for denv- , denv- and zikv. finally, the first results obtained on denv( ), zikv( ) and chikv( ) clinical samples show %, % and % correlation respectively between our approach and commercial or in house real time rt-pcr methods. conclusion: this innovative strategy allows the development of flexible, highly sensitive and easy to handle platforms dedicated to the multiplex screening and identification of emerging viruses. this methodology is adapted for the easy inclusion of additional molecular targets to improve the surveillance and the prevention of arboviral infections. babesia microti serological testing with pooled samples: a feasibility study laura tonnetti*, aaron thorp, letitia dixon and susan l stramer. background/case studies: blood donation screening for babesia microti, a tick-borne intraerythrocytic parasite endemic in the northeast and upper midwest us, is performed under an investigational study using nucleic acid and immunofluorescence assays (ifa). however, ifa is a time consuming and labor intensive procedure. with the possibility of an fda licensed screening assay(s) in the near future, we investigated if b. microti testing by ifa in pools of plasma or serum could be a feasible screening approach. study design/method: to test if the increased amount of plasma or serum interferes with background fluorescence, pools of , , and were prepared from plasma or serum samples determined to b. microti-negative by individual ifa screening. the pools were tested by ifa with or without a blocking step using bovine serum albumin (bsa) and goat serum to minimize background fluorescence. potential interference from multiple pooled plasma or serum samples on the endpoint titer of positive samples was investigated by including positive samples with endpoint titers from : to : ( -fold dilutions) in the pools. results/finding: non-specific fluorescence was visible in pools of or higher and was not eliminated by the addition of a blocking step. pools of or samples did not show significant increased background. there was no difference between testing of pooled serum or plasma samples. when one single positive sample was included in the pools of or samples, the pool tested positive and the final titer was the same as the positive sample tested individually. when two or more positive samples were included in the pools, the final titer of the pools was equal to the sample with the highest titer. conclusion: this study represents a proof of concept that serological testing for b. microti by ifa in pools of up to plasma or serum samples does not increase false positivity while maintaining the sensitivity of the test. background/case studies: the rapid detection of bacterial contamination in platelets is key to reducing the risk of infection in transfusion of blood components. the bact/alert virtuo* is an advanced, next generation system with improved automation, connectivity and with data management systems. the virtuo's new algorithm significantly reduces the time to detection (ttd) of microorganisms during quality control testing of platelet preparations using bact/alert (bta) bpa (aerobic) and bpn (anaerobic) bottles. as plasma is known to be bactericidal, a study was completed to evaluate plasma susceptibility/resistance for organisms considered for virtuo studies. study design/method: human plasma (thawed and pooled) and saline controls were seeded with $ cfu/ml of organisms associated with platelet contamination and incubated at room temperature for - hours. colony counts were performed initially and after incubation. plasma resistance was determined if the colony count of the seeded plasma was equivalent or higher ( log) than the colony count of the seeded saline after incubation. results/finding: the serially diluted strains and all bioball tm strains except p. aeruginosa, nctc , were determined to be plasma resistant. the bioball tm p. aeruginosa was susceptible to the antimicrobial effects of human plasma, but when spiked into ml of leukocyte reduced apheresis platelets (lrap) and inoculated into bta bpa bottles and loaded into the bta d and virtuo the organism was recovered % . conclusion: results confirm that previously tested organisms and additional strains are plasma resistant with the exception of p. aeruginosa, nctc . however, the bpa bottles still recover p. aeruginosa in the presence of lrap. bpa/bpn bottles inoculated with select organisms from this panel in the presence of ml lrap demonstrated % recovery when loaded onto the virtuo and d ( table ) . further studies may be required to determine if higher test volumes of lrap could affect the recovery of plasma sensitive strains. * virtuo is not fda cleared for platelet testing a. notoscriptus, identified as a major urban vector of rrv, is also capable of transmitting dengue virus - , and has recently been found in los angeles, illustrating an expansion in range. with the growing geographical distribution of aedes species mosquitoes, the potential for rrv to enter local transmission cycles outside of australia is significant. in , a probable transfusiontransmission (tt) was confirmed as the cause for an rrv infection in australia, validating the reality that rrv tt can occur. rrv morbidity leads to clinical manifestations that are similar to chikv infection, with varying degrees of arthralgia, which can become debilitating. various asymptomatic to symptomatic infection ratios have been reported, but this further increases the risk of additional tt in endemic areas and could mask the spread of the disease globally. study design/method: platelet concentrates (pc) prepared in pas were inoculated with rrv, amotosalen was added to final concentration of mm and the units were treated with uva light. pre-and post-treatment illumination samples were collected for titration. as- rbc units were contaminated with rrv, mixed with processing solution/glutathione (gsh) and treated with amustaline at a final mm concentration. pre-and post-treatment samples were removed prior to amustaline treatment and hrs after amustaline addition, respectively, for titration by plaque assay on vero cells. log reduction was calculated as the difference between the mean infectious titer in pre-vs. post-treatment samples. results/finding: inactivation of rrv was achieved to the limit of detection in pc and rbc. in pc, > . log or log /ml of rrv was achieved, with > . log or > . log /ml of rrv inactivated in rbc. conclusion: these studies illustrate that amotosalen/uva and amustaline/ gsh treatments are effective at inactivating rrv in pc and rbc, respectively. these data corroborate previous results achieved with other alphaviruses, including chikv and mayaro virus which are inactivated at high titers in pc and rbc, demonstrating the ability for these systems to mitigate tt potential and maintain safe blood component availability in endemic areas. (data have not been submitted for fda review and intercept for red blood cell is not approved for commercial use). background/case studies: the interceptv r blood system for platelets is designed to inactivate pathogens and contaminating leukocytes. this photochemical treatment process utilizes amotosalen and low energy ultraviolet a (uva) light. the current available sets include small volume (sv; - ml), large volume (lv; - ml) and dual storage containers (ds; - ml) designed to treat platelet doses between . and . x . the new triple storage (ts) set was designed to expand the dose range to . x and the maximum volume to ml, generating either or doses of pathogen reduced platelet components (pc). the objective of this study was to evaluate the effectiveness of the system by performing log reduction assays using representative gram positive and negative bacteria and enveloped and non-enveloped viruses in platelets suspended in pas, or % plasma using ts set. study design/methods: for each experiment, a platelet pool was prepared either in % plasma/ % pas or % plasma with a final volume of $ ml and a dose of - platelets. these conditions represent inactivation using the lowest amotosalen concentration ( mm) and highest concentration of platelets. platelet units were inoculated with high titers of viruses, or bacteria and treated. control (pre-uva) and test (post-uva) samples were serially diluted and cultured. plates with suitable media were used for bacteria, whereas viral titers were determined using plaque assays. log reduction was calculated as the difference between the log titers in control (pre-uva) and test (post-uva) samples. conclusion dromedary camels were identified to be the reservoir of mers cov, transmission to humans occurs through direct and indirect contact. mers cov has been detected with high genomic titers of - logs in respiratory secretions of mers patients, and with lower genomic titers of - logs in blood. the presence viral particles in the blood of acute patients gives rise to concerns, especially in endemic areas. the high mortality rate, especially for critically ill patients, which often require blood transfusion, raises the need for a method to safely exclude mers cov contamination of blood products. pathogen reduction with amotosalen/uva technology is a widely established technology with a broad range of data supporting clinical efficacy and safety of amotosalen/uva treated blood products. the aim of the study is the assessment of the mers cov inactivation efficacy in human plasma with amotosalen/uva pathogen inactivation technology to safely exclude the presence of infectious virus in human plasma units. pre-uva titer post-uva titer log reduction/ml (log /ml) %plasma/ % pas e .coli . <- . > . e. cloacae . <- . > . k. pneumoniae . < . > . s. aureus . <- . > . blue tongue virus . <- . > . bovine viral diarrhea virus . <- . > . adenovirus- . <- . > . %plasma k. pneumoniae . - . > . s. aureus . <- . > . adenovirus- . <- . > . n a transfusion vol. supplement s abstract study design/method: four therapeutic human plasma units were spiked with a fully characterized mers cov clinical isolate followed by pathogen inactivation with amotosalen/uva (intercept blood system, cerus corporation) at four different days. pathogen reduced samples were taken preand post-pathogen reduction after various processing steps to assess the infectious titer by plaque assay titration and the genomic titer by real-time-pcr. samples post pathogen reduction have been passaged times up to days, assessing the infectious titer and genomic titer every rd day to exclude the presence of low-titer infectious particles. results/finding: all viral particles in the plasma units were completely inactivated with an average efficacy of ! . log infectious titer. no viral replication was observed after days of passaging post inactivation. the genomic titer was only slightly affected by pathogen inactivation, which is designed to target the infectious titer, but not the physical titer. conclusion: amotosalen alone had a slight effect on the infectious titer while amotosalen/uva effectively inactivated all infectious mers cov viral particles in the plasma units with an inactivation efficacy above logs infectious titer, giving evidence for improved blood safety of amotosalen/uva treated plasma in mers cov endemic regions. estimating the prevalence and incidence in a national blood service in taiwan for hcv eradication program yun-yuan chen* , jen-wei chen , chi-ling chen , sheng-nan lu and pei-jer chen . department of research, head office, taiwan blood services foundation, graduate institute of clinical medicine, college of medicine, national taiwan university, division of hepatogastroenterology, department of internal medicine, kaohsiung chang gung memorial hospital background/case studies: world health organization (who) has set a goal to eliminate hcv by , and the epidemiological indicators generated from a national blood service is useful to monitor the effectiveness. this study aimed to evaluate the prevalence and incidence of hcv infection in taiwan. study design/method: in taiwan, anti-hcv (since ) and -sample mini-pools triplex nucleic acid test of hcv, hbv and hiv (since ) have been used in the routine blood screening. prevalence of anti-hcv and hcv rna were estimated in the first-time donors during - and - , respectively. age-standardized prevalence and its % confidence interval ( % ci) were calculated with adjustment of who world standard population - . for the incidence study, donors who have donated blood two or more times during - and who were without a history of anti-hcv positive before the follow-up period were included. the incidence and its % confidence interval was estimated from the number of new hcv rna positive cases divided by the person-years of follow-up. results/finding: the crude prevalence of anti-hcv in the first-time donors was dramatically decreased from . per , donors ( % ci: . - . ) to . per , ( % ci: . - . ) during - , and the agestandardized prevalence was also decreased from . per , donors ( % ci: . - . ) to . per , ( % ci: . - . ). the agestandardized prevalence of anti-hcv was generally higher in female donors before , but it was significantly higher in male donors at (p-value . ). a total of , hcv rna positive cases, . % of them were anti-hcv negative, identified from , first-time donors during - , and the crude and age-standardized prevalence of hcv rna was . per , ( % ci: . - . ) and . per , ( % ci: . - . ), respectively. crude prevalence of hcv rna was significantly higher in female donors (p value < . ), but no significant difference was found after age standardization (p value . ). both the prevalence of anti-hcv and hcv rna were increased with age (p for trend< . ). in the incidence study, a total of new hcv rna positive cases, . % of them were anti-hcv negative, found from , , donors followed for , , person-years. the incidence of hcv rna was . per , person-years ( % ci: . - . ), and no significant difference was observed between both genders (p-value . ) and between age groups (p for trend . ). conclusion: the prevalence of hcv infection has been dramatically decreased by . % during - . it becomes significantly higher in male donors and that needs to monitor in the future. incidence of hcv rna is low in repeat blood donors and it needs to identify more incident cases to observe the epidemiological characteristics. dalia ashour* , sahar muhmmad and dalia el dewi . national blood transfusion services, azhar university background/case studies: blood safety is a challenge in egypt because of the high prevalence of hcv and hbv. nucleic acid amplification test (nat) technologies have the potential to detect viremia earlier than current screening methods, which are based on seroconversion. the primary benefit of nat is the ability to reduce residual risk of infectious wp donations. the estimated reduction of the wp utilizing nat for hcv is - days, hiv from to days, and hbv from - days. study design/method: this cross sectional study was conducted in national blood transfusion center (giza, egypt) from to , the total number of donor samples to be screened is , the age of the donors ranged from to years, and they were of both sexes (m: f : ).screening by nat ulterio assay (grifols diagnostics; formerly novartis diagnostics) was done in parallel with eia testing for hbsag, hcv-ab and hiv ag/ ab. using individual donation nat (id-nat). multiplex nat yield samples are further tested using the discriminatory assay in order to ascertain which viral nucleic acid is present in the donor sample. statistical analysis chi-square (v ) test was used to measure the association between two qualitative variables. results/finding: nat screening detected a total of nat yield donations among ( . %) seronegative donors. among these nat yields cases, ( . %) were reactive for hbv, ( . %) were reactive for hcv and ( . %) were reactive for hiv- . we stratified the age of the donors into groups; group a ( - years), group b ( - years) and group c ( - years). the prevalence of nat yield to the three viruses was significantly higher in either group b or c, compared to group a (p . ; with % confidence interval (ci) . - . & p . ; with % ci . - . respectively). prevalence of nat-hbv; was significantly higher in age group b, as compared with group a (p . ; with % ci . - . ). on the other hand, there was no statistically significant difference between groups c and a and between groups b and c. comparing groups b and c combined with group a found a significantly higher prevalence of hbv in the former (p . ; with % ci . - . ). nat-hcv; did not differ significantly between the three groups (p . ; with % ci - . to . between groups a and b & p . ; with % ci - . to . between groups a and c & p . ; with % ci - . to . between groups b and c). nat-hiv; did not also differ significantly between the three groups (p . ; with % ci - . to . between groups a and b & p . ; with % ci - . to . between groups b and c). in either group a and c, no nat-hiv detected. nat yield to the three viruses was significantly higher in males than in females (p . ; with % ci . to . ). nat hbv was significantly higher in males (p . ; with % ci . - . ), but the prevalence of either hcv or hiv did not differ significantly between males and females (p . ; with % ci - . - . & p . ; with % ci - . - . ; respectively). conclusion: in this study the nat yield of in assumes more significance when one considers the fact that single donation is used for generating components that can be used by recipients. hence, in effect the nat yield becomes times that is, in . saving recipients from tti out of ( . %) is indeed very significant. results/finding: of the , donors who were tested by our donor center, , ( . %) were repeat reactive. a seasonal pattern in the prevalence was observed with the highest number of donors being positive in summer, and then progressively declining during the fall and winter months and increasing again in spring. there was a single case of transfusion transmitted babesiosis reported from our center during this period. a patient who was transfused with two units of packed red blood cells (rbcs) from two donors in the beginning of july presented in august for further transfusion and was found to have parasitemia in the peripheral blood smear and was subsequently diagnosed with babesiosis. the donors were called back, however one of them could not be tracked. samples were sent to the state for further testing: an immunofluoresence assay was performed (combination of igg, igm and iga). the test was positive at : titer. the screening elia s/co of this donor was . . both donors were indefinitely deferred as blood donors. conclusion: our data confirm a decreased risk in transfusion transmission with the use of a screening assay. prior to implementation of the screening there were - transfusion transmitted babesia cases per year from - (table ). in the months after implementation of pre-transfusion babesia screening, one break through case of transfusion transmitted babesia was observed ( in , donors tested). thus the babesia eia screening test effectively prevents ttb. however, there was a substantial loss of donors due to being screen positive. four years of experience with id-nat at a tertiary care centre in north india: implications for transfusion transmission and donor screening. jasmeet singh*, amarjit kaur, gurpreet kaur, rajesh kumar and sonia gupta. dayanand medical college and hospital background/case studies: transfusion transmitted diseases are a challenge for transfusion medicine specialists and patient care providers around the globe. blood safety is a formidable task especially in a high population country like india. newer technologies like id-nat equip us to screen and prevent transfusion transmitted viral infections and prevent their transmission by improving over the sensitivity and specificity of conventional methods. this study aims at examining the effect of id-nat as an additional test on the safety of blood supply. study design/method: a retrospective observational study was conducted to analyze the data of years of additional nat testing at blood bank, dmch, ludhiana from september to december . results/finding: results . % ( of ) units were initially nat reactive. these units were further tested, of which . % were discriminated ( hiv, hcv, hbv and co-infections). the remaining . % ( ) were repeat non-reactive and . % ( ) could not be discriminated. overall, nat yield rate was one in , whereas virus-specific nat yield rates were one in , for hiv, one in for hcv, one in for hbv and one in , for hbv/hcv coinfections, respectively. conclusion: id-nat screening of all blood donations at our institution over past years has increased the screening sensitivities to check viral load and prevented transmission of probable transfusion transmitted viral infections. assuming % component preparation it saved transfusion recipients from harm. implementation of nat along with routine serological tests for screening of the blood donations definitely improves the transfusion safety and should be mandated across all transfusion centers. min xu , , wei mao , tao he , yashan yang , , zhan gao , , chunhong zhang , hongmei liao , jingxing wang , and miao he* , . institute of blood transfusion, chinese academy of medical sciences & peking union medical college, sichuan blood safety and blood substitute international science and technology cooperation base, chongqing blood center background/case studies: many emerging infectious pathogens are known to be existed in heathy blood donations, and could be transmitted via transfusion with potential hazardous consequences against recipients. with more convenient application of high through put sequencing, it becomes much easier to investigate uncultured microbiome in qualified blood donations. therefore, metagenomics analyses were used to reveal emerging and re-emerging infectious diseases in healthy donations which might potentially threat the blood safety. study design/method: pooled plasma sample were collected from , voluntary blood donors from chongqing, china. total dna and rna were extracted and amplified with random primers pcr respectively in order to construct a pe library to peform deep sequencing by illumina miseq. all reads were trimmed to remove low quality bases and adapter sequences. the fully overlapping paired-end reads passing the quality filter were concatenated using pear. we classified the final reads using kraken and a kraken database made from complete refseq bacterial, archaeal and viral genomes, along with the grch human genome. the unclassified reads by kraken were aligned to ncbi nt database using blastn with cut-off evalue as e - . the best alignment hits were used to classify the reads. krona was used to generate all taxonomic distribution plots. finally, the potential emerging and re-emerging infectious pathogens were identified out of the classified microbiome by experience. abstract results/finding: . gb raw data with , , reads were generated in the dna library. meanwhile, . gb raw data with , , reads were generated in the rna library. after cleaning the human background, reads from bacteria, reads from viruses, and reads from parasites were identified (table ) . no hazardous viruses were identified as potential threats to blood safety. except for viruses and bacterias which would do limited hazards to blood safety, plenty of parasites were identified in which some were already considered as threats to blood safety in some developed countries were also discovered such as plasmodium sp. and leishmania infantum (table ) . conclusion: the investigation has revealed the metagenomics of the qualified blood donations in chongqing, china. the results showed a thoughprovoking discovery of genomic fragments of some microbes which might threat the blood safety. the displayed serious results let us have to think about regulating some reasonable screening methods as well as donor recruitment strategy in certain epidemic areas or seasons to ensure the blood safety. however, on the contrary, the results should be considered more cautiously because the existing of genomic fragments could not represent the existing of infectious pathogens. the validity of the metagenomics hints were suggested to go through epidemiological investigations and specifically tested under laboratory ways such as bacteria or virus culturing to ensure the vitality of those pathogens. background/case studies: the caribbean has become an endemic region for several emerging viruses in the last decade. after a chikungunya outbreak in most recently zika was shown to be endemic on the caribbean island of curacao. to effectively provide safe blood products in an endemic region the conventional international recommendations of donor exclusion and testing do not seem a viable option and could severely affect the local blood supply. pathogen reduction (pr) is considered an important new approach with potential benefits. the introduction and experience of use of pr platelets in the dutch caribbean over a period of one year is presented. study design/method: pathogen reduction of thrombocyte concentrates by use of riboflavin and ultraviolet treatment (mirasol prt, terumo, belgium) was introduced. all thrombocyte concentrates provided to the general hospitals on the dutch caribbean islands of curaçao, bonaire and sint maarten were pr and data collected over the period of february to february . thrombocyte concentrates are prepared out of single donation units by the buffycoat method. results/finding: over the period platelet concentrates were provided to adult and pediatric patients. these included patients on the intensive care and neonatal intensive care departments. no adverse events were reported and the cci for each transfusion was within the expected outcome. introduction of pr had minimal impact on the logistics of thrombocyte concentrate preparation and availability. furthermore no transfusion related bacterial contaminations were reported. conclusion: pr of platelet concentrates seems viable and safe for use in a small scale caribbean setting with endemicity for emerging viruses like chikungunya and zika. it offers a realistic alternative for conventional recommendations of donor exclusion and testing, thereby helping to maintain sufficient labile blood product availability. michael phillips* , germ an leparc , phillip c williamson , lani palmer , ben reynolds , maria noedel and lindsey houghton . creative testing solutions, oneblood background/case studies: due to the risk of travel and sexually transmitted zika infections, the food and drug administration issued a guidance document on february , recommending that blood centers in puerto rico cease distribution of locally collected blood products unless donors are tested or products are pathogen reduced by march , . with the high incidence of zika virus (zikv) in puerto rico and uncertainty of the impact to the continental u.s. blood supply, there was intense pressure to implement a donor screening test for zikv. the project was initiated on february , and included clinical trial requirements, client onboarding and laboratory operations. stakeholders consisted of clients, the manufacturer, institutional review boards (irb), informational technology (client and lab based), the food and drug administration (fda), the centers for disease control cdc, and the florida department of health. clinical trial requirements included development of instrument and assay validations, sop creation, result reporting, assay and clinical trial training, deviation management, donor notification, and follow up sample handling. client onboarding began with confidentiality agreements between the client and the sponsor. a zika based webinar was created to provide an overview of the sponsor protocol, lab test system and client responsibilities. the complexity of the project increased when mosquito borne zika transmission was identified in two counties in florida. this required zikv testing to be performed on collections in both florida and puerto rico. the zikv-nat is performed in singlet, unlike the mpx and wnv assays which are run in minipools. this had a significant impact on instrument capacity. despite these obstacles and the changing regulatory requirements, the zikv screening test was implemented within six weeks. study design/method: one metric used to measure client service levels is our ability to meet established upload time goals for individual clients. the percentage of samples released on time is evaluated daily with a running monthly total. our upload time goals were negatively impacted from july through september due to the unexpected increase in zikv testing, the requirement to perform testing in singlets and the resulting instrument capacity issues. additional instruments were sourced in october and operations stabilized. conclusion: on february , , the project to implement a zikv ind test was initiated. six weeks later, testing was performed on the first batch of samples. despite the changing regulatory requirements over time, the implementation was extremely successful. initiating a new ind testing within weeks is unprecedented and required exceptional collaboration between all participants and stakeholders. background/case studies: plasmodium falciparum (pf), an intraerythrocytic protozoan parasite, is accountable for nearly all malaria mortality in africa. in , who reported $ million new cases worldwide, resulting in > , deaths. malaria prevalence is highest in sub-saharan africa, home to % of all infections accounting for % of mortalities. both the incidence and prevalence of malaria in africa significantly increase the potential for transfusion-transmission (tt), with little to no screening of products in developing countries. the objective of this study was to evaluate the inactivation of pf in whole blood (wb) using a system specifically developed for the realities of the developing world and in support of the swiss red cross humanitarian foundation for whole blood pathogen inactivation for africa. the inability to consistently supply blood components leads to routine wb transfusion, and as transfusion-transmitted diseases are prevalent in the developing world, the establishment of a robust wb pathogen inactivation system is desirable. the approach uses the small molecule amustaline to form covalent adducts and crosslinks within nucleic acids of leukocytes and contaminating pathogens to prevent replication. the process includes addition of . mm amustaline and mm glutathione (gsh) and a h at room temperature (rt) incubation after which the treated wb unit is suitable for storage up to days at rt. study design/method: for each experiment, a wb unit was spiked with ring-stage pf-infected red blood cells (irbc). a pre-treatment sample was removed prior to addition of amustaline and a post-treatment sample was removed h after amustaline addition to determine the pre-and posttreatment titers to calculate the level of inactivation. these samples were serially diluted in flasks containing medium with % fresh rbcs. the diluted samples were used to inoculate flasks in quadruplicate and monitored for parasitemia by counting irbc in blood smears and by flow cytometry. pretreatment cultures were terminated after reaching > % parasitemia, while no residual pf was detected in post-treatment cultures. log reduction was calculated as the difference between the mean titer in pre-and posttreatment samples. results/finding: robust inactivation of pf in wb was achieved to the limit of detection, at > . log or > . log /ml. conclusion: pf was inactivated to the limit of detection in wb after treatment with amustaline/gsh, illustrating that the system has potential to mitigate the risk for pf transfusion transmission in endemic regions that lack testing capacity and operate under the constraint of a very limited blood component supply and rely on wb transfusion. (this system for wb is not approved for commercial use). increased patient safety and improved inventory management with day apheresis platelets nancy m. dunbar* and zbigniew m. szczepiorkowski. background/case studies: a pathway currently exists for apheresis platelet (ap) outdate extension from to days using an fda cleared rapid test (rt). in february , our hospital based transfusion service implemented the use of rt on day , and to routinely extend ap shelf life to days. prior to this, we tested aps by rt on day and transfused day or day units with physician approval when deemed medically necessary. this report describes changes observed in transfusion practice and platelet inventory management one year following routine use of day platelets. study design/methods: data were obtained for two -month study periods: october -september (pre-implementation) and february -january (post-implementation). the interval transition period was intentionally excluded. for each study period, we determined the total number of aps transfused, rt status on the day of transfusion, total number of rts performed, expired ap units, and aps obtained from suppliers using ad-hoc ordering. we also obtained hospital data including inpatient admissions, surgical volumes, average length of stay and case mix index. results/findings: data are shown in table . the number of ap transfusions increased by % post-implementation, comparable to a % increase in inpatient admissions and an % increase in surgical volumes. the hospital length of stay and case mix index were similar for both periods. the average number of platelet transfusions per patient was not statistically different ( . pre; . post, p . ). the number of rts performed increased by %. the percentage of transfused units tested at least once by rt prior to transfusion increased by % (p< . ). the outdate rate decreased from % to % (p< . ). ad-hoc ordering decreased from % to % (p< . ). conclusion: use of an approved rt for routine ap outdate extension to day was associated with increased patient safety as more transfused units underwent secondary testing prior to transfusion. increased cost of rt was offset by reduced ap waste and less frequent need for ad-hoc ordering. sheila o'brien* , vito scalia , carla osiowy , michael carpenter , anton andonov and margaret fearon . canadian blood services, public health agency of canada background/case studies: the rates of hepatitis b (hbv) and hepatitis c virus (hcv) positive donations are low ( . and . per , donations, respectively) and most are among first time donors. we aimed to determine the frequency of various genotypes of hbv and hcv in canadian blood donors confirmed positive for hbv and hcv. study design/methods: in the roche multiplex assay (hcv/hiv/ hbv) was implemented in minipools of units. hcv nat was in place since (using minipools of ) but this is the first time donors have been screened by hbv nat. hbsag, anti-hbc and anti-hcv were tested using the abbott prism assay. confirmatory testing for hbsag was by the prism neutralization assay. anti-hcv repeat reactivity was confirmed by the inno-lia hcv score line immunoassay. since march all samples testing hbv nat positive, or confirmed positive for hbsag and all hcv nat positive or anti-hcv confirmed positive samples were considered positive and samples were sent to phac for sequencing. a sample from each positive donation was aliquoted and frozen at - o c. genotyping was carried out by sequence and phylogenetic analysis of the hbv surface antigen coding region. hcv viral rna was extracted and subjected to reverse transcription and pcr amplification in the ' ntr-e and ns b regions. sanger sequencing of these regions represents approximately % of the genome. results/findings: all confirmed positive donations were whole blood donations. there were hbv positive donations. of these, had tested hbv nat positive. genotypes were type a, b, c, d and e. there were samples hbv nat negative but hbsag positive ( were anti-hbc reactive). of these, could not be sequenced and one was genotype a (also anti-hbc reactive). there were samples considered hcv positive. of these, samples were hcv nat positive. genotypes were type a, b, c, b and a. there were also samples hcv nat negative but anti-hcv positive. none of these could be sequenced. conclusion: the first months of molecular surveillance show a range of genotypes for hbv and hcv for samples identified as nat positive. to date no samples that were nat negative anti-hcv reactive could be sequenced, however one nat negative sample that was positive for hbsag and anti-hbc reactive was hbv genotype a. surveillance over a longer period is background/case studies: the bact/alert d microbial detection system (bta d) is currently fda cleared for the quality control testing of leukocyte reduced apheresis platelets (lraps). the bact/alert virtuo microbial detection system (virtuo) (biom erieux, st. louis, mo) is a new generation of bact/alert instrumentation. the underlying colorimetric technology used in previous generations of bact/alert is used in the vir-tuo and incorporates new instrument architecture to improve temperature stability, workflow improvement via automation of processes that are currently performed manually, an improved user interface and an enhanced algorithm to shorten time to detection. the objective of this study was to compare the performance of the virtuo and bact/alert d (bta d) instruments, using bact/alert bpa (aerobic) and bact/alert bpn (anaerobic) bottles, for the detection of a range of typical bacterial contaminants seeded into leukocyte reduced apheresis platelets (lraps).* study design/method: the study was performed at two institutions, one in the us and the other in the uk. aliquots of lraps were seeded with low levels ( - cfu/ml) of bacterial species commonly associated with platelet contamination, and replicates ( per instrument) of ml aliquots per bottle were inoculated into bpa and bpn bottles. one set of bottles was loaded into bta d and the other into virtuo and incubated until signaled positive by the instruments or for up to days. overall detection rates and time to detection of bacterial contaminants between instruments were compared. additionally bottles were tested in each instrument (lraps only, no organism) to evaluate differences in the overall negative agreement rates (detection of false positives) between instruments and to serve as sterility controls for the platelet preparations. background/case studies: the implementation of nucleic acid testing (nat) blood screening is still a challenge in resource-limited countries. at the same time, in these countries, higher to similar proportions of replacement to voluntary blood donors are recruited. a higher prevalence of infections is observed in relation to developed countries. as a consequence, more incident cases of infections can be expected. in our country, some hospital blood banks could not afford nat due to high costs, but belong to a net that centralizes nat in a reference blood center. the process to consolidate small blood banks in regional blood centers, which will be able to implement nat, is not yet complete. although efforts to reduce replacement /familiar blood donations are in progress, these goals have not been completely achieved. the aims were to compare the prevalence of hiv, hcv and hbv by nat screening in a blood center recruiting only voluntary blood donors with the prevalence in centers recruiting replacement and voluntary blood donors, and describe the nat yield rates for hiv, hcv and hbv in a period of three and a half year experience. study design/method: a regional blood donor center (rbdc) has centralized nat screening from centers in different regions of the country due to since august . this process required to achieve adequate laboratory conditions and staff qualification and a development of software to assure sample traceability and interface for transmission of results. when a window period was suspected, the nat screening was repeated from the plasma unit and a second sample of the blood donor was required to confirm nat results. this rbdc have also been developed a % voluntary donor program since and is the only center in the country that has achieved this goal. results/findings: a total of , blood donations were studied from august to december . in the rbdc, where only voluntary blood donations are recruited, the prevalence was per , donations for hiv (ic % - : , ); per , for hbv (ic % - : , ) and per , for hcv (ic % - : , ). in all other centers together, where voluntary and replacement blood donations are recruited, the prevalence was per , donations for hiv (ic % - : , ); per , for hbv (ic % - : , ) and per , for hcv (ic % - : , ). window period infections were detected only in centers recruiting voluntary and replacement blood donations, giving nat yield rates of : , for hbv; : , for hiv and : , for hcv. conclusion: the hiv, hbv and hcv prevalence was lower in a center where the tasks to sustain a voluntary blood donor program were developed. nat yield rates could be reduced in the region if this program could completely be applied in all centers. mechanisms leading to obi include various factors such as imperfect host's immune response and viral variation factors. this study was to determine the viral loads of obi under currently recruitment and screening among blood donors in five blood services of zhejiang province, china. study design/method: before donation, the donors were screened and precluded with hbsag preliminary test positive and alt level abnormal. following, the samples were detected for hbsag twice using different elisa reagents and hbv dna using tma or qt-pcr techniques. then, the samples with hbv dna positive and elisa negative were tested for the viral loads using taqman technique in cobas s system. hbv s region was also sequenced. results/finding: obi were found in the , donations. in the viral loads assay, samples were negative and samples' viral loads were lower iu/ml. the mean viral loads was . . (log ) iu/ml in other samples,while the mean viral loads with hbsag /hbv dna samples was . . (log ) iu/ml. samples of obis have analyzed the hbv genotype, which b was the most prevalent subtype ( . %) and the other was hbv c genotype( . %). compared the samples with hbsag /hbv dna ,we found two obi samples carrying with t>c mutation, which could cause an amino acid s f. conclusion: in this study, the viral loads of obi infection in donors was much low than hbsag /hbv dna , and some unique variation was identified in the obi individuals. a transfusion in general population. screening of blood donors for hbv in india is primarily based upon detection of hepatitis b surface antigen (hbsag) in donor's sera. the current study was undertaken to determine the prevalence of occult hbv infection (obi) in voluntary blood donors and to analyze the burden of hbv window period donations. study design/method: this is a prospective, observational, mono-centric study performed in a national accreditation board for hospitals (nabh) accredited apex blood bank, located in maharashtra state, india. monolisa hbsag ultra (bio-rad, france)sandwich type elisa using monoclonal and polyclonal antibodies was used for hbsag detection in donor's sera. all the elisa non-reactive samples were also tested by an additional real time multiplex polymerase chain reaction (mpx-pcr) by cobasv r taqscreen mpx test. the donors which were found to be positive for hbv dna were followed upat th days, month, months& months by monolisa hbsag ultra (bio-rad, france) to analyze interval of window period and to delineate the window period donations (wpd) & true obi. background/case studies: occult hepatitis b infection (obi) is characterized by hepatitis b virus (hbv) dna-positive, but hbv surface antigen (hbsag) -negative. since may , we have been testing apheresis donors for hbv nucleic acids and improvements in laboratory testing have reduced the risk of transfusion-transmitted infection. the number of apheresis collections increased significantly year by year, however, data on hepatitis b virus marker rates among these donors continue to be lacking. the aim of this study is to evaluate the epidemic characteristics, incidence and estimate the risk factors of obi among apheresis donors in a region of central china. study design/method: apheresis donors' data from may to dec was retrospectively analyzed. all samples were tested for hbsag, hbv dna, and other markers. nucleic acids testing (nat) was performed on the roche cobas s platform using pools of serologically negative samples and any pools positive would undergo nat again individually. hbsag negative, but hbv dna positive were further tested for hbv dna quantitative pcr, antibody to hepatitis b surface antigen (hbsab), antibody to hepatitis b core antigen (hbcab), hepatitis b e antigen (hbeag) and antibody to hepatitis b e (hbeab). results/finding: in the evaluation, seronegative donations were screened by nat and a total of hbv dna-reactive/hbsag-negative donors were detected. no hiv rna -reactive or hcv rna -reactive sample was detected. complete serologic screening of the index donations indicated that the majority of these donors had an occult hbv infection and the majority of which were married men and the fixed donors with many whole blood or apheresis donations. age distribution of the age group - years old showed a large proportion, who accounted for % of reported infections. most of the hbv dna cases (about . %) reached senior high school education. the average hbsag dna positive rate was . % ( / ). incidence among apheresis donors in this period for hbsag dna were . / . these estimates were comparable to those among repeat whole blood donors. we developed pathogen reduced (pr) cryo derived from ffp and pf with day stability at c. study design/method: six replicates of type-matched pools of whole blood derived (wbd) and apheresis (aph) plasma were split to produce conventional control ( ml) and test components ( ml ml). test components were pr with amotosalen and uva light. aph and wbd ffp were produced by freezing plasma within hr and wbd pf within hr. cryo was manufactured according to site sops and frozen at - c (test ml, control ml ). test and control cryos were thawed at c, and characterized immediately post thaw (t ), and after d storage at c and tested for fb and fviii function, thromboelastography (rotem) and thrombin generation (cat). results/finding: pr cryo retained sufficient fb and fviii activity post thaw and over d at c (table) for hemostatic capacity. rotem (extem) showed retention of fibrin formation (a angle) and clot quality (mcf) (table) . thrombin generation was robust as demonstrated by multiple parameters (lag time, peak thrombin, endogenous total thrombin potential (etp), and time to peak (tt) despite lower fviii levels. these parameters were maintained through d storage at c. conclusion: pr cryo can be processed from plasma sources, including pf , and stored at rt for days. pr plasma provides adequate levels of fb with hemostatic capacity equivalent to control as demonstrated by rotem and cat. use of pf with stability over days can increase the availability of cryo with a reduced risk of transfusion-transmitted infection. cryo produced with psoralen-treated (pr) plasma is not approved for use in the us. performance of a new automated alinity s assay for antibodies to t. cruzi darwin smith* , ed bakker , anton van weert , jane bryant , mark paradowski , lynne fleischmann , mirjana sarac , george chen , george schlauder and gregg williams . abbott diagnostics, sanquin diagnostics, abbott gmbh & co. kg background/case studies: the parasite, trypanosoma cruzi (t. cruzi), is the cause of chagas disease which is endemic to the americas and infects - million people. in order to prevent transfusion mediated transmission of this parasite in endemic countries, blood collection centers require high throughput anti-t. cruzi assays with good specificity and sensitivity. in nonendemic countries, selective testing of at risk donors is a strategy to avoid temporary donor deferrals. in addition, continued pressures on laboratory operations demand that assays perform on platforms capable of increased walk away time and enhanced automation in areas of reagent management, retest options, and commodity/waste management. study design/method: the performance of the new automated chemiluminescence immunoassay for the detection of antibodies to t. cruzi was evaluated on the alinity s automated platform and compared to another onmarket chemiluminescent immunoassay. precision was assessed over days using a panel of positive and negative samples. sensitivity was evaluated on presumed antibody positive specimens and specificity was evaluated on random blood donor samples. results/finding: precision was % cv or less for positive samples over days. the overall specificity in a blood donor population was . % ( / ). sensitivity was . % for presumed antibody positive samples. conclusion: these results indicated that the new automated alinity s chagas assay provided very good performance in sensitivity and specificity, comparable to the current on-market anti-t. cruzi assay, and is equally suitable for use of universal screening in endemic and selective donor screening in non-endemic countries. performance of a new automated alinity s assay for hepatitis b surface antigen and hepatitis b surface antigen confirmatory randal makela* , anton vanweert , ed bakker , jane bryant , mark paradowski , lynn martin , daniela kaleve , george chen , gregg williams and george schlauder . abbott laboratories, sanguin diagnostics, abbott gmbh & co. kg, abbott diagnostics background/case studies: despite the development of sensitive nat methods, blood transfusion in many parts of the world relies on serologic screening for hepatitis b surface antigen (hbsag) to prevent transfusion transmitted hbv infection. sensitive hbsag assays must be capable of coping with a wide range of mutants while exhibiting an uncompromised specificity. in addition, continued pressures on laboratory operations demand that assays perform on platforms capable of increased walk away time and enhanced automation in areas of reagent management, retest options, and commodity/waste management. study design/method: the performance of a new automated chemiluminescence immunoassay for the detection and confirmation of hbsag was evaluated on a next generation automated platform, abbott alinity s. precision was assessed over days. sensitivity was evaluated using known positive samples, commercially available seroconversion panels, the who standard, hbsag mutants, and hbsag genotyped specimens (a through h). specificity was evaluated on random blood and plasmapheresis donors. results/finding: precision was less than % cv for positive samples over days. the blood donor specificity was . % ( / ). sensitivity was % for presumed positive samples. sensitivity was % for all genotypes. % of the mutants were detected vs % for the comparator assay. seroconversion detection was equivalent to the comparator assay with reactive samples detected with the alinity s assay and reactive samples detected by the comparator assay. analytical sensitivity ranged from . to . iu/ml. the alinity s hbsag confirmatory assay confirmed all known positive hbsag specimens, including hbsag mutant samples that were not confirmed by the comparator hbsag confirmatory assay. conclusion: the new automated alinity s hbsag assay provided precision, specificity, and seroconversion sensitivity comparable to the current onmarket comparator assay. however, the alinity s hbsag assay demonstrated a gain in sensitivity over the comparator assay through the detection and confirmation of a wider range of mutants. performance of a new automated alinity s immunoassay assay for hiv darwin smith* , ed bakker , anton van weert , jane bryant , mark paradowski , kevin callear , susan sullivan , george chen , george schlauder and gregg williams . abbott diagnostics, sanquin diagnostics background/case studies: blood donations are commonly screened to detect the presence of antibodies (or antibody and antigen) to human immunodeficiency virus types and (anti-hiv- / ). blood centers require very high throughput anti-hiv- / assays with high specificity and sensitivity to prevent unnecessary donor deferrals while maintaining a safe blood supply. in addition, continued pressures on laboratory operations demand that assays perform on platforms capable of increased walk away time and enhanced automation in areas of reagent management, retest options, and commodity/waste management. in the response for the need for such screening assays, we have evaluated an improved automated assay for the detection of anti-hiv- / antibodies and hiv- p antigen. study design/method: the performance of the new chemiluminescence combination immunoassay for the detection of anti-hiv- / antibodies and hiv- p antigen was evaluated on the abbott alinity s system. precision was assessed over days evaluating positive samples. specificity was evaluated on samples obtained from random blood donors and plasmapheresis donors. sensitivity was evaluated using presumed positive samples for hiv- , hiv- and hiv group o antibodies and hiv- p antigen. seroconversion sensitivity was evaluated with commercial seroconversion panels. results/finding: precision was less than % cv for positive samples over days. the blood donor specificity was . % ( / ). sensitivity was % for presumed antibody positive samples comprised of hiv- , hiv- and hiv- groups o, n, p, crf and urf samples. also, sensitivity was % for antigen positive viral isolate samples comprised of hiv- , hiv- and hiv- groups o, n, p, crf and urf samples. seroconversion detection was equivalent to the comparator assay with reactive samples detected with the alinity s assay and reactive samples detected by the comparator assay. conclusion: these results indicate that the new automated alinity s hiv ag/ab combo assay provided acceptable performance in specificity, sensitivity and precision, while providing similar seroconversion sensitivity as the comparator assay. performance of a new automated alinity s immunoassay for the detection of anti-hbc antibodies randal makela* , anton vanweert , ed bakker , jane bryant , mark paradowski , joyce siregar , angela vockel , george chen , gregg williams and george schlauder . abbott laboratories, sanguin diagnostics, abbott gmbh & co. kg, abbott diagnostics background/case studies: in countries with a low prevalence of hepatitis b, blood donations are commonly screened to detect the presence of antibodies to hepatitis b core antigen (anti-hbc) alongside hbsag and hbv nat to detect donors with occult hepatitis b infections (obi). blood centers require anti-hbc assays with high specificity and sensitivity to prevent unnecessary donor deferrals while maintaining a safe blood supply. in addition, continued pressures on laboratory operations demand that assays perform on platforms capable of increased walk away time and enhanced automation in areas of reagent management, retest options, and commodity/waste management. in the response for the need for such screening assays, we have developed an improved automated assay for the detection of anti-hbc on the alinity s system. study design/method: the performance of a new chemiluminescence anti-hbc assay for the detection of anti-hbc antibodies was evaluated on the next generation automated abbott alinity s system. precision was assessed over days evaluating positive samples. specificity was evaluated on samples obtained from random blood donors. sensitivity was evaluated using specimens characterized as anti-hbc positive by means of serologic methods. analytical sensitivity was assessed using the who st international standard. seroconversion sensitivity was evaluated using commercial seroconversion panels. results/finding: precision was less than % cv for positive samples over days. the blood donor specificity was . % ( / ). sensitivity was % for samples presumed to be anti-hbc positive. analytical sensitivity results on the alinity s anti-hbc assay ranged from . to . iu/ml. seroconversion detection was equivalent to the comparator assay with reactive samples detected with the alinity s assay and reactive samples detected by the comparator assay. conclusion: these results indicate that the new automated alinity s anti-hbc assay provided good performance in specificity, sensitivity and precision versus the comparator assay. performance of a new automated alinity s immunoassay for the detection of htlv i and htlv ii antibodies melanie anderson* , anton vanweert , ed bakker , mark paradowski , jane bryant , tuan bui , joyce siregar , george chen , george schlauder and gregg williams . abbott laboratories, sanguin diagnostics, abbott diagnostics background/case studies: in endemic countries, universal blood screening is necessary to prevent transfusion transmitted htlv infections (anti-htlv i/htlv ii). in non-endemic countries, selective testing may avoid unnecessary temporal deferrals for donors at high risk, such as returning travelers from or donors born in countries with a high htlv prevalence. blood centers require high throughput anti-htlv i/htlv ii assays with high specificity and sensitivity to prevent unnecessary donor deferrals while maintaining a safe blood supply. in addition, continued pressures on laboratory operations demand that assays perform on platforms capable of increased walk away time and enhanced automation in areas of reagent management, retest options, and commodity/waste management. in response for the need of an assay with high specificity on a high throughput instrument we have developed a new assay for the detection of antibodies against htlv-i/ii antibodies for the alinity s system. study design/method: precision was assessed over days using htlv i and htlv ii positive samples. specificity was evaluated using , blood donor specimens from europe and diagnostic samples obtained from the united states. sensitivity was evaluated using preselected htlv i and htlv ii positive samples. sensitivity and specificity samples were split across reagent lots during testing. confirmation of repeatedly reactive samples was done using the mp diagnostic htlv blot . . results/finding: imprecision was less than . % for positive samples over days. clinical sensitivity was . % ( / ) on preselected htlv i and htlv ii positive samples. the specificity was . % ( , / , ) on a blood donor population and . % ( / ) on diagnostic samples. conclusion: these results indicate that the new alinity s automated htlv i/ ii assay provided very good performance in specificity, sensitivity, and precision. sensitivity and specificity were comparable to the comparator assay. claudia ramirez , michel garcia* , fernando palomino and guillermo orjuela-falla . national blood bank colombian red cross, universidad del rosario, fuats background/case studies: current hepatitis c virus (hcv) supplemental testing algorithm for blood donations in colombia, requires that an immunoblot assay be performed on every hcv enzyme immunoassay (eia) repeatreactive sample. a higher proportion of indeterminate (ind) results by immunoblot assays has been documented for non-us donor samples, affecting donor counseling and eventually increasing costs and opportunity for the notification of infected donors. this work aimed to establish the distribution of immunoblot results in colombian repeat-reactive samples, as well as the frequency of band detection in both positive and indeterminate blots. study design/method: in total, anti-hcv-reactive donor samples (signal-to-cutoff (s/co) ratio greater than . ; abbott architect i sr) underwent supplemental testing by immunoblot (either chiron riba hcv . sia or hcv blot . test, mp diagnostics). negative (neg), indeterminate (ind) and positive (pos) blot results were grouped by s/co ranges as follows: - . , - . , > . band detection and intensity were independently analyzed for indeterminate and positive results. results/finding: immunoblot results were negative in . % ( / ) of samples, indeterminate in . % ( / ) and were positive in . % ( / ). a direct relationship was observed between positive immunoblot and increased s/co. the proportion of ind results were higher in the s/co group - . ( . %) compared with the - . ( . %). in samples with indeterminate results, ns _ was the most frequent band detected ( , %). in contrast, the most frequent band in the group of positive results was core ( , %). only one sample from the indeterminate group ( . %) had a strong band intensity ( ), compared with samples from the positive group ( . %). conclusion: the proportion of indeterminate immunoblot results in this sample of colombian donors is one of the highest ever reported, being twice as much as the proportion found in larger samples of us donors. the high proportion of ind results found in the s/co group ( - . ) suggests that the optimal s/co ratio for predicting a confirmed anti-hcv result in this population should be higher than the one recommended by the cdc for us population (> ). overall, these results suggest that the supplemental testing algorithm for blood donations in colombia could be improved not only by using high s/co ratios as an alternative to immunoblot, but also by introducing hcv genomic assays instead of immunoblots, at least for samples with intermediate s/co ratios. ns _ and ns _ cross-reactivity in colombian population warrants further investigation. performance of the alinity s immunoassay for the detection of syphilis antibodies melanie anderson* , ivanka mihaljevic , manuela miletic , miljana stojic vidovic , irena jukic , jane bryant , mark paradowski , angela vockel , george chen , gregg williams and george schlauder . abbott laboratories, croatian institute of transfusion medicine, abbott gmbh & co. kg, abbott diagnostics background/case studies: blood donations are commonly screened for syphilis in order to detect the presence of antibodies to the bacterium treponema pallidum. in addition, continued pressures on laboratory operations demand that the full panel of ttid assays perform on a single platform capable of increased walk away time and enhanced automation in areas of reagent management, retest options, and commodity/waste management. in response to those needs, we have evaluated a new automated immunoassay for the detection of antibodies to t. pallidum. study design/method: performance of the new automated chemiluminescence immunoassay for the detection of antibodies to treponema pallidum was evaluated on the alinity s system. precision was assessed over days using positive samples. specificity was evaluated on samples obtained from , blood and plasmapheresis donors from the united states and europe and diagnostic samples obtained from the united states. sensitivity was evaluated using preselected positive samples. sensitivity and specificity samples were split across reagent lots during testing. confirmation of repeatedly reactive samples was done using a testing algorithm with confirmatory assays, inno-lia tm syphilis score, and mikrogen recomline treponema igg and igm blots. results/finding: imprecision was less than . % cv for positive samples over days. clinical sensitivity was . % ( / ) on preselected syphilis positive samples. the specificity was . % ( , / , ) for blood donor specimens and . % ( / ) on diagnostic samples. conclusion: these results indicate that the new automated alinity s syphilis assay provided good performance in precision, specificity and sensitivity in line with data found for the comparator assay. performance of the new automated alinity s assay for anti-hcv melanie anderson* , ed bakker , anton vanweert , jane bryant , mark paradowski , tuan bui , lynn martin , george chen , gregg williams and george schlauder . abbott laboratories, sanguin diagnostics, background/case studies: serological screening for antibodies to hepatitis c virus (hcv) often in conjunction with nucleic acid testing (nat) is used worldwide to prevent transfusion transmitted hcv infections. while nat provides improved sensitivity and detection of hcv in the pre-seroconversion window, serological testing provides continued detection of hcv in infected individuals and individuals with resolved infections with no detectable hcv rna. blood and plasma centers require very high throughput anti-hcv assays with high specificity and sensitivity to prevent unnecessary donor deferrals while maintaining the safety of the blood and plasma supply. in addition, continued pressures on laboratory operations demand that assays perform on platforms capable of increased walk away time and enhanced automation in areas of reagent management, retest options, and commodity/waste management. study design/method: the performance of a new automated chemiluminescence immunoassay for the detection of antibodies to hcv was evaluated on the alinity s system. precision was assessed over days evaluating positive samples. sensitivity was evaluated using preselected positive samples and seroconversion panels. specificity was evaluated on samples obtained from , blood and plasmapheresis donors from the united states and europe and diagnostic samples obtained from the united states. sensitivity and specificity samples were split across reagent lots during testing. confirmation of repeatedly reactive samples was done using a testing algorithm consisting of the inno-lia tm hcv score and nat/hcv discriminatory nat assays. results/finding: imprecision was less than . % cv for positive samples over days. overall clinical sensitivity was % on preselected anti-hcv positive samples. seroconversion sensitivity was better than the comparator as evidenced by the new anti-hcv assay identifying more bleeds than the comparator assay. the specificity was . % ( , / , ) for blood donor specimens and . % ( / ) background/case studies: zika virus (zikv), which has been outbroken in south america and the united states since middle of , was declared as the public health emergency of international concern by who in feb . in addition to mosquito, zikv can be transmitted via maternalneonatal relationship, sexual intercourse or blood transfusion. the potential for transfusion-transmitted zika virus was shown in french polynesia where . % of asymptomatic blood donors tested were positive for zika virus rna using nucleic acid test (nat). several case reports have confirmed that zikv can be transmitted by transfusion. it has been shown that among blood donors, . % of the zikv infections were asymptomatic and the ratio of symptomatic to asymptomatic patients observed in micronesia was approximately : to : . thus zikv has raised a great challenge to transfusion safety. measures should be taken to prevent transfusion-transmitted zikv, including temporary deferral of blood donors in epidemic locations, donor self-reporting of zikv symptoms after donation with or without quarantine of blood components, supply by blood collected from non-endemic areas to epidemic regions, nat of blood donations, and pathogen inactivation of blood products. in this study, we evaluated zikv inactivation in plasma by using methylene blue photochemical treatment (mbpt). study design/methods: plasma units from randomly selected healthy donors were collected and spiked with zikv. samples were added by mb at a final concentration of lm and assayed after illumination with visible light from both sides for , , and min. viral infectivity and zikv rna loads (reverse transcription pcr) were measured in spiked plasma before and after mbpt and confirmed using repetitive passages in cell culture. control was zikv spiked plasma without photochemical treatment. results/findings: zikv titer of control sample was . log % tissue culture infectious dose (tcid )/ml. no viral infectivity was detected after mb photochemical inactivation treatment for min, min or min and the losses of the infectivity were further demonstrated by repetitive passages of cell culture. meanwhile, zikv rna loads decreased significantly during the initial min of treatment whereby ct-value jumped from . (control) to . (mbpt for min) (table ) . conclusion: it showed that mb photochemical treatment could effectively inactivate zikv in plasma. rna lesions were induced during mbpt process so that nucleic acid reverse transcription and amplification were inhibited. mbpt is proved to be an efficient method to prevent plasma transfusiontransmitted zikv infections. gilles delage* , margaret fearon , susan l stramer , megan l nguyen , france bernier , sheila o'brien , vito scalia , sakina smith , yves gr egoire and boris hogema . h ema-qu ebec, canadian blood services, american red cross, sanquin background/case studies: hepatitis e virus (hev) is known to be transfusion-transmissible. as part of the risk assessment for this infection, a study was carried out in , canadian blood donors in . in a subset of , donor samples the seroprevalence was . %. however, no donor samples were positive for hev by an in-house nucleic acid test (hev-nat). since that study suggested exposure to hev in canada but used an hev-nat with a limit of detection of iu/ml, a larger study was performed using a more sensitive hev-nat assay. study design/method: donors were informed about the study in the predonation reading materials. linked samples from approximately , canadian whole blood donors including , from canadian blood services (cbs) and , from h ema-qu ebec (hq) were collected. clinics were selected to ensure representative sampling of the donor population. all a transfusion vol. supplement s donations with available plasma samples were tested by individual donation nat at the american red cross laboratory in gaithersburg, md, using the cobas v r hev test ( % lod . iu/ml, % ci . - . ) for use on the cobas v r / system. this test is not currently approved in canada or the usa, but is available as a ce marked test. all nat-reactive donors are questioned concerning risk factors for recent hev infection (travel, animal contact, food and water exposure), undergo confirmatory testing (alternate nat, viral load, genotyping and igm/igg serology), are notified by letter, and deferred from donating for months; in-date products collected from the donor, and any frozen red blood cells or plasma from the previous months are destroyed. recipients will be traced in the event of any products transfused in the previous months. results/finding: as of april , , of , ( , cbs, , hq) tested samples with valid results have been found hev-nat reactive: donors have been confirmed by further testing to date. confirmation is pending in donor. of the donors, were from quebec, and one each from nova scotia and alberta ( male, female). ages ranged from to years. only two donors reported non-specific symptoms (fatigue). in terms of risk factors: ate pork (including who ate pork liver), ate shellfish, ate venison, and drank well water. one donor had no identifiable risk factor. viral loads ranged from to iu/ml, of which were < , were - , and were > iu/ml; were anti-hev igm positive and anti-hev igg positive at index (wantai assay). conclusion: the prevalence rate of acute hev infection in this donor population appears to be around / . the data from this study will contribute to the ongoing risk assessment of transfusion-transmitted hev infection in canada. prevalence of malaria parasite in donated blood at nakasero blood bank, uganda gerald nsubuga* and musiisi ezra. uganda blood transfusion service background/case studies: introduction infectivity of donated blood with malaria is a significant health problem facing humanity. in uganda, screening for malaria parasite is neither routinely done in blood banks, nor stipulated in the current uganda national blood transfusion service (ubts) guidelines by the ministry of health. as a result, the proportion of donated blood that is infected with malaria is largely unknown. malaria infection places more than half of the world's population at risk and in majority of the tropical and sub-tropical regions of the world and about to million cases and to million death occur per year. however the study aimed at determining the prevalence of malaria parasites in donated blood at nakasero blood bank, kampala, uganda study design/method: a cross sectional study was carried out in nakasero blood bank, kampala, uganda in four hundred and seventy randomly selected donor samples at the blood bank between june and august . both thin and thick glass stained blood smears of blood samples with giemsa was examined using microscope. results/finding: of the donated blood samples, ( . %) tested positive for malaria parasite (p. falciparum), although there was no significant difference in occurrence of plasmodium in relation to sex, age and blood group (p> . ), majority of the blood donors that tested positive belonged to blood group o ( . %). the prevalence of malaria parasite in the study was . %. regardless of the prevalence, the presence of malaria parasite (plasmodium falciparum) in donated blood from donors that were presumed to be healthy raises a serious concern on the safety of donated blood in uganda. the ministry of health should review the existing guidelines for screening malaria and mandatory universal blood donor screening policy for malaria, for exclusion of blood donors with plasmodia parasitaemia. using methods like pathogen inactivation compared to tedious microscopic procedure to screen donated blood to be introduced to further enhance blood safety in our communities. components. the bact/alert virtuo* (virtuo) is an advanced, next generation system with improved automation, connectivity, and with data management systems. most importantly, the virtuo's new algorithm significantly reduces the time to detection (ttd) of microorganisms during quality control testing of platelet preparations using bact/alert bpa (aerobic) and bpn (anaerobic) bottles. bpa and bpn bottles were tested on virtuo and bact/ alert d (bta d) to evaluate repeatability to detect growth in seeded leukocyte reduced apheresis platelets (lrap) without platelet additive solution (pas), throughout platelet shelf life ( , and days after collection). study design/method: pooled lrap were seeded with low levels of organisms commonly associated with platelet contamination at , and days post collection. the seeded lrap were inoculated into bpa and bpn bottles on different days (not consecutive) alternating between teams of people each. seeded bottles were loaded into a virtuo and a bta d and incubated until declared positive or negative (up to days). additionally, bpa and bpn bottles inoculated with ml of unseeded lrap were tested on the virtuo and the bta d ( and bottles respectively), to serve as negative controls, sterility controls, and to evaluate the risk of false positives caused by lrap results/finding: the repeatability of the virtuo to detect organisms in lrap was demonstrated by a recovery rate of seeded bottles of . % for the virtuo and . % for the bta d. the virtuo demonstrated an average improved ttd of . hours, when compared to the bta d in the presence of ml lrap platelets. the lrap did not cause false positives. additionally, the age of the lrap units (within day expiry),did not impact the ttd when seeded with organism background/case studies: zika virus (zikv) is an emerging flavivirus that is transmitted by the aedes aegypti mosquito and sometimes a. albopictus mosquito. most infections are asymptomatic. zikv nucleic acid testing (nat) became a required test for blood donors per the fda guidance entitled, "revised recommendations for reducing the risk of zika virus transmission by blood and blood components". based on our geographical location, implementation of this testing began weeks after this guidance was issued. we performed zikv nat for donors of whole blood and blood components under an investigational new drug (ind) study (sponsored by hologic, inc.). we performed a retrospective analysis on all nat results as there is a potential to defer donation due to false positive screening results. study design/method: donors that consented to donate blood and be tested for the zikv were obtained from three blood banks in colorado and nebraska. nat was performed using the procleix virus assay which is a qualitative in vitro nucleic acid assay system that detects zikv rna in plasma specimens. the assay was performed on the automated procleix panther system. all testing was performed according to the manufacturer package insert. results/findings: in the event of a reactive result, donors would be retested by nat in addition to other testing (igm antibody testing, neutralization test). donors are deferred for days barring continued zikv testing and nonreactive results. a total of , donors were screened for zikv. all donors screened for zikv were nonreactive by nat. no invalid test results were obtained. in addition the number of failed test runs due to instrument or assay issues were experienced were quite low ( . %). this data indicates that both the assay and instrument are robust. there was a low frequency for additional testing which allows the laboratory to publish timely infectious disease results for our blood bank customers. conclusion: the reactive rate data presented here demonstrate that there is a low/zero incident rate in our region for whole blood and blood component discard due to reactive results. this screening is important to continue to ensure blood safety in the united states. robust inactivation of the yellow fever virus d strain can be achieved using amotosalen and uva light for pathogen reduction treatment (prt) of platelet components andrew laughhunn , felicia santa maria , yvette girard , peter bringmann , marion lanteri* and adonis stassinopoulos . microbiology department, cerus corporation, scientific affairs department, cerus corporation background/case studies: yellow fever virus (yfv) is known to cause explosive outbreaks, such as the one in angola in . the rapidly increasing number of infections in brazil, with hundreds of fatalities since december , is of concern. yfv is a flavivirus transmitted by aedes mosquitoes and could spread, like zika virus, to other parts of the americas where the vector is endemic. with no effective antivirals and only supportive therapy available, the best mitigation strategy is through vaccination with live attenuated vaccine strains, like the d-yfv strain. yfv vaccine is considered an effective and safe vaccine; however major adverse events have been reported including neurologic and visceral adverse effects. in addition, transfusion transmission (tt) of live attenuated yfv has been reported with severe clinical outcomes, especially in immunosuppressed patients. in order to prevent tt by yfv vaccine strain, the aabb recommends a weekperiod deferral after yfv vaccination. yfv outbreaks and vaccination campaigns may therefore reduce blood availability. this pilot study evaluated the ability to inactivate d-yfv using amotosalen (s- ) and uva light prt of platelet components (pc). study design/method: pc in %pas (n ) or % plasma (n ) were spiked with high titers of d-yfv and treated with s- /uva prt. samples were taken pre-and post-uva illumination and infectious titers were determined, by plaque assay using vero cells. the extent of inactivation was quantified by comparing titers before and after inactivation. results/finding: pre-prt infectious titers were . . log pfu/ml for pc in % plasma and . log pfu/ml for pc in % plasma while titers in post-prt samples were <- . . log pfu/ml for pc in % plasma and <- . log pfu/ml for pc in % plasma. inactivation to the limit of detection of > . . log or inactivation of > . . log pfu/ml was achieved for pc in % plasma. inactivation to the limit of background/case studies: the use of biotin as a supplement has increased in recent years and many health care professionals may not be aware of the high dosage intake by their patients. this high dosage has resulted in an increased prevalence of individuals being exposed to biotin levels much greater than the recommended daily dose and as a consequence, has led to inaccurate lab results for assays that utilize the free capture biotin-streptavidin methodology. although abbott's alinity s assays do not utilize this free capture biotin-streptavidin methodology, eight assays developed for blood screening on the alinity s system were evaluated for biotin interference to ensure there are no unknown consequences of high biotin levels. study design/methods: the purpose of this study was to determine if the eight developed abbott alinity s assays would be susceptible to biotin interference by evaluating their performance in the presence of a high concentration of biotin. for each of the alinity s assays evaluated (hiv ag/ab combo, htlv i/ii, anti-hcv, chagas, hbsag, anti-hbc, syphilis, and cmv igg), samples spiked with a concentration of biotin at approximately ng/ml were tested against a control (unspiked) sample preparation to determine if there was a difference between the control and biotin containing samples. two samples, one negative and one positive, were tested with all assays, except the hiv and htlv assays, which each tested two positive samples ( hiv- antibody and hiv- p antigen, and htlv-i antibody and htlv-ii antibody, respectively). results/findings: for the negative samples, the sample to cutoff (s/co) differences between the biotin spiked and control were . for hcv, hbc, syphilis, cmv igg, and chagas, . for hiv ag/ab and htlv i/ii, and . for hbsag. for the positive samples, the mean s/co % differences between the biotin spiked and control were . % (antibody sample) and . % (antigen sample) for hiv ag/ab combo; . % (htlv i antibody sample) and . % (htlv ii antibody sample); - . % for anti-hcv, - . % for chagas, - . % for hbsag, - . % for anti-hbc, - . % for syphilis, and - . % for cmv igg. conclusion: eight abbott alinity s assays were evaluated to determine if they were susceptible to biotin interference. these results indicate that the eight alinity s assays do not show susceptibility to biotin interference at an approximate concentration of ng/ml. robustness of the abbott prism methods to biotin interference c fischer , r schneider , w leonard , m cobb , g schlauder , g williams , m zuske m janulis* . transfusion medicine, abbott diagnostics, wiesbaden, germany, add diagnostics, transfusion medicine, abbott laboratories, chicago, united states background/case studies: the use of biotin as a dietary supplement has increased significantly in recent years and many health care professionals do not realize their patients are taking high doses. the increase has resulted in an increased prevalence of people being exposed to biotin levels much higher than the recommended daily dose and as a consequence, potentially inaccurate lab results for assays that utilize the free capture biotin-streptavidin methodology. the purpose of this study was to identify any abbott prism assays that may be susceptible to biotin interference based on assay design and then evaluate the performance of those assays with high concentrations of biotin. after a comprehensive review of abbott's current on market prism assays, no assays were identified that utilize biotin-streptavidin capture; however, assays were identified for subsequent testing as they contain biotin in their assay design. background/case studies: bacterial contamination of platelets is the highest residual infectious risk in transfusion despite the current preventive strategies. while bacterial contamination may affect any blood component, the ambient storage temperature conditions for platelets make them most likely to facilitate bacterial growth. based on all the precautionary measures, the final platelet concentrates include in the worst cases a very limited viable bacteria number estimated from to colony forming units (cfus)/bag (i.e. . to . cfu/ml). one major difference between viruses and bacteria is that bacteria have the ability to grow up to a concentration of - cfu/ml over the days product shelf-life. moreover, a large diversity of strains is found in contaminated platelets representing a key challenge for the development of a generic bacterial test. the aim of this study was to develop an economic and easy diagnostic approach for the early, rapid, sensitive and generic detection of bacteria in platelet concentrates. the adaptability of the process with the blood transfusion services requirements was of major concern. hence, attention was focused on an easy to automate technique able to deliver results on day after collection. study design/method: a large panel of bacteria involved in transfusion reactions including clinical isolates and reference strains was established and used for mouse immunizations, antibody screening and platelet spiking steps. an original approach was used to produce and select monoclonal antibodies directed against bacteria to develop our generic immunoassay. as recommended, hours (day ) after collection a sampling volume of spiked platelets ( . - cfu/ml) was tested after a short generic culture, lysis and capture of bacteria on magnetic microparticles in a microplate format. an immunoassay was performed for the detection of the captured bacteria. results/finding: this approach was tested on a panel of bacterial strains involved in transfusion reactions. the pre-analytical steps and the capture of bacteria on microparticles were improved to avoid false negative results and to enhance the sensitivity of detection. the full test developed in this study combining a pre-analytical culture step followed by an there are many stakeholders are involved in hcv eradication program, including government authority such as centers for disease control and prevention, national health insurance and health promotion administration, and private property like hospitals, medical societies, pharmaceutical and vaccine industries, npos and academia. results/finding: tbsf is a private nationwide single blood services program in taiwan, and performs anti-hcv screening test and nat confirmatory test on every collected blood, which is a large-scale population screening of hcv in taiwan because of its high blood donation rate ( . %). tbsf confirmed positive test result of repeated blood donors, and can identify hcv rna seroconversion cases as recently-infected hepatitis patients. those infected patients would be referred to physician for further medical care and deferred permanently by tbsf to secure blood safety. by interviewing the newly-infected cases, the risk factors of hcv patients can be studied and then help identifying and eliminating sources of hcv infection. tbsf also contribute to health education by teaching our donors being aware of potential risks of hcv infection and keep monitoring every parameters of hcv epidemiology to evaluate the efficacy of hcv eradication program. conclusion: in hcv eradication program, tbsf can not only secure blood safety but also participate in health education, disease screening, etiology finding and prevention, surveillance and evaluation. thus, among all stakeholders, tbsf is particularly important and can play a pivotal role in eradicating hcv by in taiwan. the theraflex uv-platelets technology efficiently inactivates transfusion-relevant bacteria species in contaminated platelet concentrates ute gravemann , frank tolksdorf , wiebke handke , thomas h. m€ uller and axel seltsam* . german red cross blood service nstob, maco pharma international, gmbh background/case studies: the theraflex uv-platelets system (macopharma) is a uvc-based pathogen inactivation system for platelet concentrates (pcs). inactivation efficiency has been shown for a broad range of viruses, bacteria, and protozoans. previous studies with the first set of bacteria species of the who international repository of platelet transfusion relevant bacterial reference strains revealed a high inactivation capacity for clinically relevant bacteria. aim of the current study was to investigate the bacteria inactivation efficacy of the theraflex uv-platelets system for enterobacter cloacae, pseudomonas fluorescens, staphylococcus aureus and streptococcus bovis which have recently been added to the who international repository. study design/method: pcs were produced from buffy coats using the additive solution ssp (macopharma) with a residual plasma content of %. for inactivation kinetics, pcs (n ) were spiked with bacteria to a final concentration of approx. colony forming units (cfu)/ml and irradiated with increasing doses until the full uvc dose was achieved. samples were taken for the bacterial titer determination after each irradiation step. for sterilization studies, two pcs were pooled and inoculated with bacteria to a final concentration of approximately . cfu/ml. bacteria were allowed to grow for h in the pcs at c under agitation. after splitting, one pc remained untreated (growth control) while the other one was uvc-treated. after storage for seven days, samples were taken from both bags for sterility testing by bactalert (biomerieux) and for determination of the bacterial titer in the untreated control units. results/finding: bacteria in pcs were inactivated in a dose-dependent manner by treatment using the theraflex uv-platelets system. mean log reduction factors ranged from to for enterobacter cloacae ( . . , pei-b-p- ), pseudomonas fluorescens ( . . , pei-b-p- ), staphylococcus aureus ( . . , pei-b-p- ), and streptococcus bovis ( . . , pei-b-p- ). pcs (n for each species) spiked with these different bacteria species were efficiently sterilized ( out of ). treated pcs remained sterile during storage for days, while bacteria in non-treated pcs grew to high titers of - cfu/ml. the theraflex uv-platelets system efficiently inactivates a broad range of different bacteria species, including the who reference strains. sterility is maintained over a storage period of days. these results suggest that the uvc-based pathogen inactivation technology will significantly improve the bacterial safety of platelet transfusions. transfusion transmissible infections among blood donors and strategy on direct laboratory testing cost of blood screening at national blood bank center, addis ababa, ethiopia abraham zewoldie*. national blood bank service background/case studies: blood and its components are life saving; however, they are also associated with life threatening hazards such as transfusion transmitted infections (ttis). hepatitis b virus (hbv), hepatitis c virus (hcv), human immunodeficiency virus (hiv) and syphilis are the most serious infections transmitted during blood transfusion. serious of blood shortages especially in developing countries and reliance on unsafe family replacement or paid donors also contribute to an increased risk of ttis. knowing the current prevalence of ttis among blood donors will be crucial in donor program strategy development and cost effective alternative strategies of blood screening are highly required especially in resource limited setup. study design/method: a retrospective analysis of blood donors' record covering the period from july , to july , was conducted. the data was collected from the nation al blood bank (nbb) center donor data base. in addition, direct laboratory costs of parallel versus sequential strategy of blood screening were compared using the current price of the laboratory costs. data was first exported to spss version software for analysis. data analysis was performed using scores and odds ratio using same software to look for an association between dependent and independent variables. p values less than . were considered significant. results/finding: a total of , consecutive blood donors were screened between and . the overall seroprevalence rate of hbv, hiv, hcv and syphilis of blood donors was . %, . %, . % and . % respectively. the hiv-hbv co-infection was higher among blood donors ( . %) followed by hbv-hcv co-infection whish accounts about ( . %). significantly increased sero-prevalence of ttis was observed in among family replacement donors, factory workers, daily labors and the age group of - . in this study the difference in cost between the current in use strategy (parallel) versus the newly proposed designed sequential testing algorism was , . ethiopian birr. conclusion: a significant percentage of the blood donors harbor ttis. the nbb center should work on voluntary blood donor mobilization and develop culture of voluntarism. the direct laboratory cost analysis using current in use strategy (parallel) was higher than the newly designed sequential testing algorithm. thus, the new strategy can be implemented to make screening of ttis cost effective in nbb center. transfusion transmitted malaria in a month old infant patricia davenport* , geeta paranjape and laurie sutor , . carter bloodcare, ut southwestern medical center background/case studies: in at a large pediatric hospital, a month old infant was supported for days by extracorporeal membrane oxygenation (ecmo). over this time blood products were transfused. about days after end of ecmo support, a routine blood smear examination revealed inclusions in some of the patient's red cells. the patient had also been having intermittent fever. malaria was confirmed by pcr as plasmodium ovale (p. ovale). because the patient had no other risks, the infection was suspected to be transfusion related and was reported to our blood center which had supplied all transfused products. study design/method: the investigation began by focusing on donors of red cell products, since the chance of an apheresis platelet product transmitting malaria is relatively small, and that of a frozen product is remote. we identified donors of red cell products. each donor was contacted and was asked four questions. additional questions were asked for clarification if needed. based on donor response, risk for active malaria infection was assessed. we also considered areas where p. ovale is, or is not found. donors identified as having possible risk were tested for antibodies and parasitic dna. results/finding: the five donors who had been ill all had common cold or bronchitis like symptoms. donors who traveled went only to non-risk areas. three donors were former residents of another country and may have risk because they lived in malaria endemic countries since birth and came to the u.s. as adults. it was discovered that one of these three did not meet all donor criteria. the donor had failed to disclose that he had not completed years stay in the u.s. after emigrating from cameroon, an area endemic for p. ovale. he had not travelled anywhere after coming to the united states in october and answered "no" to travel. antibody tests on this donor were positive for p. ovale and p. falciparum, but pcr tests were negative. another possible at-risk donor, a former resident of iran was tested and was pcr and antibody negative. the third donor has not yet been tested but the country of residence does not have p. ovale malaria. conclusion: while it could not be definitively proven that the donor with antibodies to p. ovale had active malaria at the time of donation, the donor was indefinitely deferred and referred to an outside physician for treatment. transfusion-transmitted babesiosis outside an endemic area: a case report german felix leparc*. oneblood background/case studies: an y.o. male patient was admitted to the emergency room for severe acute gastrointestinal bleeding, caused by an arterio-venous malformation later located in the proximal jejunum that was clipped endoscopically. during this admission, he received a total of units of red blood cells. approximately weeks later, he was re-admitted due to another episode of gi bleed manifested by melena. as part of his routine evaluation, a cbc was performed in which a blood smear revealed the presence of intraerythrocytic parasites consistent with babesia sp. study design/method: upon notification of a suspected case of transfusion-transmitted babesiosis, lookback of all donors involved in prior transfusion event was initiated. results/finding: to confirm the presumptive diagnosis of babesiosis, pcr was performed and babesia microti dna was detected. an evaluation of the patient's risk factors revealed that prior to the gi bleed episode for which he received transfusions, eight months earlier he was also transfused during open heart surgery. no travel history to the us midwest, and while he travelled to new england two years ago he did not spend time outdoors. he was splenectomized in his mid 's. donor lookback identified a donor who lived in new london county, connecticut but spent the winter season in central florida, where the blood donation (double rbc collected by apheresis) took place. he had never been diagnosed with babesiosis, but participated regularly in outdoor activities in connecticut that put him at risk for tick bites (although he never noticed being bitten or showing signs of it). upon testing, he was found to be negative for b. microti on pcr as well as igm antibodies, but had igg antibody titers of : . the recipient of the other rbc unit collected in the same donation was deceased within hours of transfusion, so no follow up could be performed. during phone interviews, none of the remaining donors had risk factors for babesiosis, and all but four were tested and found serologically negative. conclusion: while transmission of babesiosis through the zoonotic route is confined to regions were the appropriate hosts and vector coexist, people from areas where it is endemic may establish temporary residency and donate blood in non-endemic locations facilitating transmission through transfusion as illustrated in this case. once licensed assays for babesia microti become available, testing schemes will have to be formulated through policies that take this issue into consideration. transfusion-transmitted stenotrophomonas maltophilia from a red cell unit: a case report ashley c gamayo* , andrea j linscott and donny dumani . background/case studies: transfusion-transmitted bacterial infections (ttbi) are rare, but serious complications of blood product transfusions. from - , % of transfusion-associated fatalities reported to the fda were attributed to bacterial contamination. red cell units are rarely implicated in severe and fatal ttbi. when present, contaminants are often gram-negative rod (gnr) bacteria with psychrophilic properties. we present a case of a sickle cell patient who developed definitive sepsis after receiving a red cell unit contaminated with stenotrophomonas maltophilia (s. maltophilia). study design/methods: a -year-old female with sickle cell disease was admitted to the hospital for possible pain crises. pre-transfusion blood and urine cultures collected on day of hospitalization showed no growth after five days. on day , the patient required a blood transfusion for which she was issued a cmv-safe, irradiated, hbs-negative, crossmatched, o-negative red cell unit. the ml unit had been aliquoted via sterile connecting device days prior for a pediatric patient. all ml of the pediatric aliquot were transfused without adverse effects. the patient's pretransfusion temperature was . c. within minutes of starting the transfusion, the patient's temperature increased to . c and subsequently reached a maximum of . c. the transfusion was stopped and the blood bank notified immediately. gram stain of the remainder of the transfused component revealed gnr bacteria. blood was collected from the patient for culture and antibiotic treatment initiated. results/findings: initial transfusion reaction work-up revealed no evidence of clerical errors with negative post-transfusion antibody screen and direct antiglobulin test. blood cultures from both the patient post-transfusion and the implicated red cell unit grew gnr bacteria identified as s. maltophilia. further microbial testing revealed the cultured pathogen was able to proliferate at - c; a finding not characteristically observed in s. maltophilia. conclusion: this is the first definitive case of ttbi with s. maltophilia. this bacterium is a globally emerging gnr that is widely spread in the environment, causing both community-acquired and nosocomial infections in immunocompromised and debilitated patients. contamination was unlikely due to an asymptomatic donor. there was laboratory evidence of the pathogen in both the transfusion recipient and the transfused component. the patient was not infected with the pathogen prior to transfusion, and no other potential exposures could be identified. the patient recovered following appropriate antibiotic treatment, but endured prolonged hospitalization. the transfusion reaction was classified as definitive, severe tti of definite imputability. validation of commercial immunoassays for detecting hbsag and hiv antibodies in production pools karen leighton, izekial butler and scott jones*. qualtex laboratories background/case studies: plasma fractionators test plasma production pools for hbsag and hiv antibodies as a qualitative limit test for the control of impurities, to safeguard against errors in donation testing or pooling. the european medicines agency (ema) has published guidelines for the validation of immunoassays for the detection of hbsag and hiv antibodies in production pools. the aim was to validate commercial immunoassays for the testing of production pools for hbsag and hiv antibodies utilizing the ema guidelines. study design/method: a lower calculated cutoff value for the abbott prism hbsag and hiv o plus assays was determined by calculating the mean signal-to-cutoff ratio (s/co) plus standard deviations of four different types of plasma production pool samples. the calculated cutoff values were utilized for the rest of the validation. the detection limit was determined by testing in triplicate, serial dilutions of who hbsag and hiv antibody standards diluted in plasma. a normalized detection limit was calculated for the hbsag assay using production pools containing low, typical and high anti-hbsag titers. intra-assay variability was determined by testing a minimum of determinations of a low positive control in run. inter-assay variability was determined by testing at least representative negative production pool samples, at least low positive sample (about s/co) and a titration series of who standard spiked into plasma production samples. runs were performed on six separate days using two different instruments and two different lots of assay reagents. results/finding: the lower calculated cutoff values for the hbsag and anti-hiv assays were both below the manufacturer cutoffs of . and were . and . respectively. the hbsag assay detection limit was . iu/ ml for source plasma and . iu/ml for recovered plasma samples. the normalized detection limit study demonstrated that one and a half hours was the maximum amount of time the pool samples could sit at - c where all samples were still reactive for hbsag. the anti-hiv lowest positive dilution for all replicates varied between : , to : , , depending on subtype and group. the % cv of the s/co values of the replicates of the intra-assay variability validation were less than % for both assays. the %cv of the s/co values of the panel of samples of the inter-assay variability validation were less than %. conclusion: a lower calculated cutoff value could be determined for commercially available immunoassays for hbsag and anti-hiv. these immunoassays could meet all of the recommendations in ema validation guidelines. the abbott prism hbsag and hiv o plus assays can be utilized to test production pool samples. was performed on donors ( - days after the index donation) - donors in the follow up study and tested by the doh. no donors tested by the doh participated in the follow up study. follow up testing was negative for all donors. denv antibodies were negative in donations and equivocal in . our initial reactive rate is higher than that reported to date for the procleix zikv tma of per , [p. williamson, et al transfusion, in press] . conclusion: universal testing under ind was successfully implemented and incorporated into blood center operations. we have noted an initial reactive other demographics that should be analyzed for their potential to be used to predict cmv seroconversion rate include gender, age, race, ethnicity or a combination of these. background/case studies : growing the geographic footprint has been a priority for the organization since . over a four year period, the organization doubled the number of blood centers, with continued growth expected. with the current challenges in the blood industry, the audit program needed to be flexible, maximizing efficiency and capacity utilization, and without increasing compliance risk. the internal audit function was centralized in late , for which the program consisted of types of audits, an operational compliance audit and a support systems compliance audit. each type was performed twice per year at each main center. this model was no longer serving the changing organization. study design/methods: lean six sigma concepts were applied to this project. survey results and brainstorming aided in capturing the strengths of the current program, opportunities for improvement, and ideas for a redesigned program. this information was the primary input to the swot analysis (strengths, weaknesses, opportunities, and threats) for the purpose of understanding performance of the current program, as well as elements that could impact the future design. potential solutions were placed into a pugh matrix, which was used to facilitate a disciplined, team-based process for concept generation and selection. each potential solution was compared to criteria for evaluation and selection of the best solution. results/findings: the program was re-designed to perform internal audits annually as a single, team-based comprehensive audit. remote auditing was incorporated to require less on-site time, less disruption, improved auditor work/life balance, and cost savings. a formula was created to determine on-site audit time that included adjustable risk factors. the audit reporting process was also automated for simplification, efficiency, and to meet stakeholder needs. the team-based approach leverages auditor strengths, fosters a learning environment, and increases detectability of organization-wide concerns. conclusion: the comprehensive team-based approach, and other program improvements, has been effective in responding to organizational growth without sacrificing quality or increasing compliance risk. external inspection performance has achieved record performance levels the past year. diversity of auditor skills led to a stronger skill presence, which was consistently applied across system. auditing is more efficient and effective. stronger collaboration among audit team members provided stronger objectivity, fairness, and consistency across the system. auditors and auditees have increased in knowledge, and the internal quality audit program has improved. background/case studies: in many places, blood banking is using semiautomated systems to perform fractioning in different blood components (red blood cells, platelets and plasma). banc de sang i teixits (bst), adopted the fully automated reveos system (terumo bct inc, lakewood, co) few years ago to manufacture blood components. in june , bst started a validation of new blood bags manufactured by terumo bct with different variables on platelet volume after processing and a kit to perform platelets pools with a new filter. study design/method: to perform this validation, blood donations were used under different conditions (see table below ). the current filter evaluated for the platelet pool (lrf-xl, haemonetics corporation) was compared to a new filter (terumo bct inc.). the new blood bags were manufactured using a new vinyl supplier. a portion of these processed blood components (red blood cells, platelets and plasma) was used for different quality control (qc) tests (routine qc performed at bst following european directorate for quality of medicines & healthcare; cytokine analysis, such as p-selectin and platelets recovery through the filter). results/finding: the results are very similar between both bags, current and new one, as well as filters. all the analysis done to evaluate the quality of the blood components were similar in all conditions. also, it was shown a better performance on platelets pools, when they came from bags centrifuged with ml of plasma, vs. ml of plasma and additive solution. conclusion: these new bags and filter have shown a similar behavior when using them for manufacturing blood donations with reveos system in our blood bank. regarding the new platelets pooling kits, a better manipulation by the operator was observed; although the tubing is shorter and it meant being more difficult when manipulating the pools. no issues should be found if they are implemented in routine use. it's planned to start this implementation during this year, ; so then there will be larger results in order to have a proper procedure qualification. conclusion: patients requiring rare blood products are rare, and those lacking high prevalence antigens are the most challenging for whom to obtain antigen negative blood. it is clear that some requests for exquisitely rare types are not able to be filled with current donors. molecular testing of large numbers of donors has likely helped to identify more rare donors in recent years. it is recognized that commercial platforms do not include many of these making these rare types even more challenging to find. consideration should be given to testing more donors of all ethnicities to identify more rare donors. recommendation # : updating donor educational material to provide more comprehensive information on risks of iron deficiency and recommendations on iron supplementation. updating our educational materials will likely have a minor impact. recommendation # : implementing strategies such as iron supplementation, ferritin testing or increasing interdonation intervals for all donors or those groups most at risk for iron deficiency. initial implementation would likely be either iron supplementation or ferritin testing for at risk groups only and implementation of either one of these strategies would potentially affect over , donors. the recommendation to limit the number of donations would have a substantial impact. for this analysis, the focus was on - year olds and premenopausal women (ages - ) donors. on average, - year olds donate . times a year and premenopausal women donate . times a year. if both of these groups were limited to donating once a year, a total of , donations from - year olds and , donations from premenopausal donors would not be collected. conclusion: after analyzing the impact of the aabb association bulletin # - , the bulletin will have a significant impact on both donors and our local blood supply. more than half of donors would receive either ferritin testing or iron supplementation. if the only measure employed is limiting the number of times a donor could donate for - year olds and premenopausal women, this recommendation would have a substantial impact on our ability to provide blood products to local hospitals. background/case studies: transfusion medicine knowledge deficits are apparent among medical students, residents and practicing physicians. these deficiencies may be due to the frequency and type of education. the majority of medical students in the united states receive four or fewer hours of transfusion medicine education. the transfusion medicine academic award group published educational content guidelines for medical school, residency and fellowships. however, the frequency and educational methods remain poorly evaluated and with little guidance. we investigated the effects of different educational techniques on transfusion medicine knowledge acquisition in novice learners. study design/method: three educational pathways were developed to teach principles of transfusion medicine while allowing learners to recognize problems and develop solutions for transfusion medicine complications. the simulation group received all educational activities within a . hour inperson, high-fidelity live session. the hybrid group received some educational component online and also attended an in-person high-fidelity simulation session. the online only group received all educational materials online, including a pre-recorded-video simulation session. the learners were second year medical students enrolled at one institution. the same faculty members taught all live sessions and developed all online materials ensuring the content was the same. a pre-and post-test was created to address blood groups, blood donation, blood testing, blood component indications and transfusion complications. the educational session was evaluated by the likert scale survey which ranges from zero (poor/unsatisfactory) to five (outstanding). results/finding: % ( / ) of the simulation group students improved their post-test scores and had an average likert scale rating of . (very good). % ( / ) of hybrid group students improved their post-test scores and had an average likert scale rating of . (very good). % ( / ) of online only students improved their post-test scores and had an average likert scale rating of . (good). the average changes in scores were statistically significant within all training groups (p value < . ). additionally, the simulation group had a larger increase in average post-test scores when compared to the online only group (p< . ) and the hybrid group (p< . ). conclusion: our study demonstrated that a faculty taught high-fidelity transfusion medicine simulation curriculum consisting of an in-person didactic session and simulation session for second year medical students produces greater knowledge acquisition compared to an online only or hybrid curriculum. the high-fidelity simulation curriculum is also preferred over the online only education as indicated by the likert survey results. aaron j wyble*, yeon mi kim and barbara j bryant. university of texas medical branch background/case studies: diagnostic management teams (dmts) are an innovative way to bridge the communication gap between the laboratory and clinical services thereby facilitating the delivery of improved patient care. dmts employ a multidisciplinary approach which integrates clinical and laboratory data into succinct interpretations and recommendations. the interpretations must be of moderate to high complexity in order to be clinically valuable. recommendations are made regarding future testing, timing of testing prior to blood component needs, and other pertinent concerns to allow for improved coordination of patient care. the timeliness of the dmt reporting is vital to patient management. the inherent design of a dmt also provides an educational opportunity for trainees at academic centers. study design/method: the transfusion medicine service at a large university-based academic medical center implemented a dmt in . all cases involving complex antibody identification workups, transfusion reactions, deviations from standard operating procedures, consultations for blood component utilization, and massive transfusion protocols from july through january were evaluated by transfusion medicine residents. the electronic medical record (emr) of each patient was also reviewed to determine relevant clinical history. all significant findings were presented at the transfusion medicine dmt conferences. the dmt was comprised of physicians from transfusion medicine, hematology/oncology, anesthesiology, transfusion service technical staff as well as visiting clinical staff from surgery, obstetrics and gynecology, transplant services, and pediatrics. the dmt integrated the clinical and laboratory data to formulate relevant interpretations and recommendations. the final dmt reports were placed into the emr for access by health care providers. financial benefits of a transfusion medicine dmt were also evaluated. results/finding: in a -month period, cases of complex antibody identification workups ( %), transfusion reactions ( %), consultations for blood component utilization ( %), and deviations from standard operating procedures and massive transfusion protocols ( %) were presented at the transfusion medicine dmt conferences. the placement of dmt narratives in the emr as progress notes and laboratory reports provided informative and timely communications. residents participating in dmts demonstrated improved clinical and laboratory correlation skills. as a result, resident competency in transfusion medicine was enhanced. over $ , of revenue was generated utilizing the standard professional component cpt codes. conclusion: dmts encompassing multiple aspects of transfusion medicine improved patient care through enhanced communication between laboratory and clinical services. additional benefits of a dmt program include resident, clinician, and technical staff education and the generation of revenue for the institution. streamlining a blood center and hospital transfusion service supply-chain with an informatics vendor-managed inventory solution hamilton c. tsang* , david lancaster , dianne geary , robert scott , anh thu nguyen , adam garcia , raina shankar , leslie buchanan and tho pham . stanford health care, stanford blood center background/case studies: inventory management is both a major challenge and an integral part of hospital transfusion service (hts) and blood centers (bc) operations. the current process at our institution involves twice-per-day shipments from the bc to the hts, with each shipment predicated upon current stock levels at hts. manually obtaining inventory levels for each product is time-consuming. the manual determination is also errorprone. we aim to enhance inventory management operations by developing an informatics solution to ( ) streamline the ordering process to accurately reflect inventory status and transfusion practices and ( ) re-allocate valuable hts tech time. study design/method: at our hts, the general inventory accounts for over product categories broken down by component, blood type, irradiated status, and cmv-serology status. we therefore sought to establish an electronic method to reliably infer the general inventory level. since the raw electronic inventory report comprised both the general inventory and physically sequestered units (e.g. special antigen units, cross-matched units), over a -month calibration period we performed linear regression between electronic and the gold-standard manual count to impute from the electronic census the number of units of each product category in the general inventory. once we had a reliable electronic method to determine inventory levels, we implemented a -month pilot period. we analyzed various metrics pre and post pilot implementation to ensure non-inferiority of our electronic system: ( ) the ratio of units transfused per week to the number stocked (t:s), ( ) the number of products ordered as stat, and ( ) the number of expired products. we created in-house programs on visual basic for applications (microsoft, redmond, wa) for both the calibration and pilot periods. lines of code were written for both programs, including class modules and distinct subroutines. results/finding: during the pilot period, we investigated our system's noninferiority. the average weekly t:s ratio for cryoprecipitate, plasma, and rbc, respectively, were . , . , and . before the pilot period compared with . , . , and . during the pilot period. these differences did not reach statistical significance (p . ). we also monitored the number of stat ordered products before and during the pilot period, which were and stat units per week, respectively (p . ). lastly, we also monitored the number of monthly wasted products due to expiration as an indicator of inventory mismanagement before and during the pilot period, which were and units, respectively (p . ). an estimated hours per week of technologist time was reallocated to other tasks once the electronic census was adopted. this translates to . fte and $ , per year saved from labor costs per year if permanently adopted. conclusion: we created an in-house electronic ordering system to enhance information fidelity, re-allocate technologist time, and further standardize ordering. our system showed non-inferiority to the labor-intensive manual system, by not changing the number of stat orders, having the same t:s ratio, and not increasing the number of expired products. this is achieved while freeing up over hours of staff time per year. future directions include full automation with involvement from hts informatics department. transfusion practice improvement: gaining traction through the use of a provincial transfusion quality improvement plan denise evanovitch* , yulia lin , troy thompson , allison collins and sheena scheuermann . ontario regional blood coordinating network, sunnybrook health sciences centre background/case studies: a provincial regional blood coordinating network (prbcn) held a "quality focus day" (qfd) in to explore transfusion quality indicators to be included in a province wide quality improvement plan (qip). the plan's main goal is to reduce patient harm by improving transfusion practice in hospitals through the reduction of inappropriate use. the following recommendations were made: select a blood component that most hospitals could monitor display progress in a public forum so that hospitals could compare themselves to peers strike a province-wide transfusion qip committee to guide the development of the plan, supporting resources and ongoing improvement initiatives study design/method: a provincial transfusion quality improvement plan (ptqip) committee was formed and included broad representation: the provincial patient blood management coordinators, physicians, technologists, nurses, administrators, clinicians, quality/risk managers from all regions of the province and the provincial blood advisory committee, the blood supplier and a patient. there was further collaboration with other organizations such as the provincial health quality division, choosing wisely after the launch, an informal survey indicated that of the province's hospitals were interested or had already adopted portions of the ptqip. to further assist hospitals in advancing their qips, a technologist prospective screening educational module was developed in addition to an electronic tracking tool with which hospitals can enter their baseline data and subsequent audit data and track their success. both hospital and provincial reports can be generated from the tracking tool. a more formal survey conducted in indicated that % plan to implement or already have implemented the ptqip and % of the respondents already have put prospective order screening by technologists in place. conclusion: helping hospitals through the development of standardized templates, instructions, education and other tools for transfusion quality improvement increases the ability of hospitals to uptake quality improvement initiatives. taking a standardized approach across the province allows for both aggregate and hospital data comparison analyses. background/case studies: military and civilian trauma-based studies have demonstrated the advantages of transfusing blood products prior to a patient's hospital arrival, a process known as pre-hospital transfusion (pht). helicopter emergency medical services (hems) worldwide have implemented this protocol with great success, despite a current lack of guidance or advisory publications. there is a need for literature that addresses the regulatory requirements and logistical challenges associated with developing a pht program. herein, we report our experience as a large hospital system embarking on the development of a multi-state pht service. study design/method: in october a work group was formed to establish pht services for the hems providing care to over thirty regional hospitals. composed of flight care staff, emergency physicians and transfusion medicine specialists, the group identified the major tasks to be addressed: federal/state regulations; inventory structure/management; product storage/testing; tracking/traceability; emergency release protocol; and staff training. while there are no specific regulations governing pht, the regulations pertaining to blood product storage, validation, and monitoring apply. the fda, aabb, and state agencies were each consulted to ensure compliance with all directives. results/finding: the largest hospital within this system, already acting as a reference site, was designated to perform all confirmatory testing on products supplied to the multi-state hems. similarly, this hospital was tasked with remote monitoring of all blood refrigerators at the helipad sites. the system's fda licensed blood supplier was deemed responsible for product consignment and transport between the four hems sites. the blood inventory at each site was designed to contain: group o positive rbcs, group a low anti-b titer liquid plasma, and four-factor prothrombin complex concentrate. a military-tested in-flight medical record system will be used to transfer transfusion information to non-affiliated hospitals as needed. validated inflight coolers, protocol for product emergency release, inventory tracking system, and re-stocking schedule were also requisite to this plan. staff competencies regarding emergency release guidelines, transfusion reactions, and the handling/storage of products are maintained by the hems medical director with additional oversight provided by transfusion medicine physicians. conclusion: our work group successfully identified the challenges associated with a multi-state pht helicopter based service, which spans blood product management, adaptation of existing transfusion procedures and operating policies, licensing requirements, and personnel training. our pht service will go live in . publishing this experience may benefit future sites as they launch similar pht initiatives. blood transfusion during humanitarian emergencies yetmgeta e. abdella* , rana hajjeh and cees th. smit sibinga . world health organization regional office for the eastern mediterranean, international quality management (iqm) consulting background/case studies: more than million people are affected by humanitarian emergencies in the eastern mediterranean region of the world health organization (who), where some of the most affected countries in the world are located. in these countries, the health systems have been weakened or destroyed and health workers provide health services under difficult circumstances. humanitarian emergencies increase the demand for blood transfusion and make its delivery challenging and complex. despite these obvious needs, across the region, there is a lack of information on the emergency preparedness and response capacity of blood transfusion and on the challenges countries and health responder's face in meeting the needs of the patients during emergencies. study design/method: we searched pubmed and index medicus for the who eastern mediterranean region for data on availability and safety of blood transfusion in humanitarian emergencies. we conducted a structured survey of blood transfusion services (bts) in all countries in the region to identify the following: type of humanitarian emergencies between and ; current strategies to ensure availability and safety of blood transfusion during emergencies; coordination and collaboration between countries; and gaps and challenges. additional information was collected during a regional consultation (eastern mediterranean region) held in may in tunisia. results/finding: we found publications on disaster from five countries in the region and publications on disaster preparedness and blood transfusion in casualties and severe trauma outside the region. however, none dealt with the questions of availability and safety of blood transfusion during emergencies. twelve countries ( . %) responded to the survey. armed conflicts and terrorism are the commonest types of emergencies with estimated - % of the injured requiring blood transfusion. nine countries have emergency preparedness and response plans for bts. potential blood donors are mobilized through public calls, besides a direct appeal on regular and replacement donors. seven of the responding countries keep an emergency blood stock. collaboration between the different stakeholders exists in seven countries. lack of adequate and competent human resource, transport and cold chain deficits, shortages in supply of consumables and maintenance of equipment, lack of reliable power supply, and shortage in finances are the gaps identified. conclusion: there is a need to integrate bts in the overall national emergency preparedness and response, collect and disseminate updated information on factors affecting provision of blood transfusion in humanitarian emergencies, provide technical and financial assistance to affected countries, strengthen mechanisms for coordination and collaboration among different parties, and develop a regional emergency blood services system and management expertise. ( , , and for - ) . the number of collections per registered trt donor varied significantly, ranging from to therapeutic draws/donor per year. excluding those that didn't present for a therapeutic blood collection, the average number of trt collections/donor per year decreased from . to . between and . conclusion: our blood center has experienced an increasing number of therapeutic phlebotomies, as well as individuals on trt referred for therapeutic phlebotomy due to elevated hemoglobin values from through . it is not clear from information provided by the ordering physician whether this is intended as a temporary measure to decrease the hemoglobin while the patient is on trt, or whether the dose was being adjusted or discontinued due to the known risk factor of cardiovascular disease in patients with polycythemia; however, the average number of donations per trt donor decreased during this timeframe. the percentage of men on testosterone who present as regular blood donors at our blood center is not known, since this hormone is not reason for deferral. our findings raise the concern, however, that regular phlebotomy is necessary to reduce the risk of testosterone-associated polycythemia in this population. as it is our duty to provide a safe and adequate blood supply, our blood center also has concerns about perpetuating the misperception that repeat phlebotomy, particularly if required more frequently than days, is sufficient to mitigate the risks of testosterone therapy. hence, we have made the decision to discontinue offering phlebotomy services to this population of donors other than for those on testosterone that meet all donor eligibility requirements. approaches involving the use of a vein illumination device in a blood donor center sara matheson*, kimberly j duffy, audrey e traun, mary m benike, james r stubbs and justin d kreuter. mayo clinic background/case studies: venipuncture is a critical step in blood collection and locating a suitable vein for this procedure can be a challenge. unacceptable vein selection or incorrect needle placement can lead to incomplete collection or infiltration. in a blood donor center, the primary selection of a vein is done by palpation within the antecubital area. prior to needle insertion, the skin at the site must be prepared and contact avoided until after needle is placed. vein illuminator (vi) devices are available to aid in visual display of potentially suitable veins. such a device was made available to staff in march of . after an initial testing and instructional period, the vi has since not been used by staff. the objective of this study is to discover reasons why staff does not use the vi to identify potentially suitable veins. study design/method: a staff survey was developed and distributed to staff in march to inquire about usage of the vi and obtain feedback about the device. at the time that the survey was sent, the device had been available for several years. the survey included questions involving frequency of use, adequacy of training, comfort with using the device, knowledge of the device's storage location, willingness try the device, and general feedback. results/finding: the survey had a % response rate (n ). of these, . % have never or very rarely utilized the vi. self-reported reasons for low utilization focused on two dominant themes. first, that the device is not needed and second that it doesn't accurately show veins. . % of respondents are aware of where the vi is stored and a more accessible location to share the device was not identified. although . % of respondents have been provided training on using the vi, the group was mixed regarding their comfort level in using the device independently. only % of the group was willing to try vi. conclusion: infiltration and incomplete collection account for approximate % ( units/year) of qualified blood donors, yet vi does not appear to be a viable solution for our blood donor program. there seems to be both an opportunity and challenge with vi implementation. the opportunity is to create critical awareness of problems with vein cannulation. the challenge is to identify a device that is more effective at visualizing deeper veins necessary for blood donation. benefits of converting from mcs to alyx penny schroeder* and elizabeth parker. indiana blood center background/case studies: in , apheresis red cells (arc) represented . % of total red cell collections at our center. hae mcs ln was utilized to collect arc. due to the age of the instruments, challenges with collections on mobiles as well as the need to increase collection of right type products, the decision was made to change technologies. study design/method: fresenius kabi demonstrated the fenwal alyx technology as well as the business case to the primary stakeholders. all implicated departments were involved in the initial impact assessment. a multidepartment kick off meeting was held and project team formed. due to product demands, the decision was made to validate arc and plasma apheresis. the primary departments affected were blood collection and production. fresenius kabi provided sample validation plans, sops, training and training materials for use. four mobile-carts were purchased for easy transportation of alyx and quick-connect feet for installation on mobile buses. the lead trainer and the bc technical administrator traveled to an affiliate blood center to observe their alyx program and identify best practices. a team of blood collection trainers and preceptors were the initial group trained and validation performed. this team also served as the subject matter experts and field preceptors. fresenius kabi returned for advanced alyx operator training. the training plan targeted previous mcs operators first and then operators new to apheresis with a training goal of % of mobile staff. validation of the alyx began / / and took approximately days to complete. during this time, fresenius kabi conducted alyx education and apheresis recruitment training to all collection and recruitment staff. the mcs machines were removed from service / / . alyx go-live occurred / / . additional operator training continued through september . results/finding: due to ease of mobility and use of alyx, reduced procedure time compared to mcs and donor conversion training we increase components collected. alyx disposable kit includes pre-attached solution containers reducing ancillary items required to pack and carry to mobiles. this decreased kit cost by $ . each providing an estimated annual savings of $ , . conclusion: with the multiple alyx donation types we were able to increase our collection of right type procedures by approximately . % and decrease our kit costs by %. with alyx the collection plasma on mobile blood drives is now possible. due to ease of use, operators have embraced this technology and we have consistently met our monthly collection goals from october -march . background/case studies: high frequency of donation is a risk factor for iron deficiency. because females' iron stores are generally lower than males' before they start their donation career, females who donate frequently are particularly high risk. minimum hemoglobin (hb) has long been the same for males and females at g/l, but for males this falls below the normal limit. as a first step to mitigate iron deficiency, criteria for whole blood donors were modified for males (minimum hb increased to ! g/l) and for females (minimum interdonation interval increased from to days). the longer interdonation interval in females was gradually implemented, starting with donor messaging in october , changes in rebooking of donation appointments in december and culminating with eprogesa criteria changes on march , . both these changes are expected to initially result in donation loss, but may be partly counteracted by a decrease in hb deferral rates in female donors. we aimed to assess the impact of these changes on hb deferral rates. study design/method: percentages of hb deferrals were calculated as the number of donation attempts that resulted in hb deferral divided by the number of successful donations plus hb deferrals multiplied by . percentages were calculated for male and female donors before and after changes were made. results/finding: the percentage of hb deferrals increased in male donors from . % in the weeks pre-implementation to . % in the weeks post-implementation of the change in the hb criterion. hb deferral rates for female donors were . % in september, . % in october/november, and . % from december to march, . conclusion: hb deferral was more frequent in male donors after the minimum hb was increased to g/l. the gradual implementation of increased interdonation interval for females resulted in a reduction in deferrals, thus the initial donation loss associated with this change may be partly offset over time by decreased hb deferrals. a longer observation period is necessary to confirm these findings and assess impact on phenotyped blood and donor retention. in the past years, , blood products, derived from , procedures, were distributed to different investigators in over laboratories. whole blood was the most common product ( . %), followed by unmanipulated mononuclear cell collections ( . %), and elutriated monocytes or lymphocytes ( . %). less common requests included platelets ( . %), plasma ( . %) and granulocytes ( . %). adverse donor reactions were infrequent ( . % of procedures). conclusion: we report the feasibility of a program for collecting and distributing blood for investigators to obtain blood components for in vitro research use, utilizing the staff and resources of a hospital-based blood bank. research blood donation is essential to support laboratory research and to maintain positive relationships with donors who have been deferred from allogeneic transfusion. hospital-based blood donor center's experience with implementing platelet pathogen reduction system kimberly j duffy*, mary m benike, james r stubbs and justin d kreuter. background/case studies: the safety of platelet products has been continually improving due to testing despite the continued emergence of microbial threats. the recent fda approval of platelet pathogen reduction technology will protect transfusion recipients regardless of the new microbial dangers. in order for platelet products to use the pathogen reduction technology, the volume, platelet yield (dose), and concentration must be collected within tight specifications. the objective of this study was to determine the optimal collection settings to enable % collection of pathogen reduced platelets while limiting the loss of products. study design/methods: the collection instrument evaluated for this study has fda approval for platelets suspended in % plasma. the corresponding pathogen reduction system used for the study has kits with different collection specifications. all apheresis collections occurred at a fixed site and pre-platelet counts were performed on a hematology analyzer. the yield scale factor has been established for correlation between the hematology analyzer and apheresis collection device. in order to determine the optimal collection targets, the apheresis collection instrument had a variety of multiple yields and volumes established for collections. staff was instructed to collect the highest available yield per donor. after collection, volume, platelet yield, and concentration data was obtained. this data was used to determine if the product met the specifications for one of the available kits, and if the actual platelet yield was higher than . x , thus meeting the criteria for a double product. results/findings: a higher platelet concentration product is ideal to produce a double product, but targeting products with a platelet concentration greater than x /ml was more likely to be outside the specification of the pathogen reduction kit. the platelet concentration target of x / ml results in discarding products and was quickly removed from instrument settings. collections with a platelet yield as low as of . x and platelet concentration of x /ml were more likely to produce a product that was not within the specification of the pathogen reduction kit. abstract conclusion: the loss of both triple platelet products and lowered postprocessing platelet recovery requires the collection of platelets to be far more precise. the goal of platelet collection has shifted from simply maximizing each platelet collection to an approach that considers optimal collection within the limits of kit specifications. final collection instrument configurations are platelet yield of . x and . x at the volume of mls and platelet yield of . x , . x , and . x at the volume of mls. moving from subjective to objective donor eligibility screening platforms: a blood center's journey angela dirr* and steve cihura . bonfils blood center, bbc / bsi background/case studies: in , the device used by bonfils blood center to determine donor hemoglobin and donor eligibility was reaching its end of life, and bbc needed to define a path forward for a reliable replacement device. study design/method: bbc evaluated devices with the following criteria in mind: ) device disposable costs, ) reagents/controls/quality control, ) objective hgb/hct measurement, ) portability and durability for a mobile environment, ) ease of use, ) donor experience, ) battery life, ) validation requirements plans, ) blood center suitability, and ) ability to link to becs. multiple departments including donor care, equipment management and validation, quality, and regulatory affairs were involved in the evaluation and product selection. bbc tested donors per each device at both a fixed and a mobile site. bbc also considered donor feedback for the choice of replacement technology. the project started february with a targeted implementation date of july . after creating necessary sops and adopting existing sops, bbc successfully completed the validation of the devices, and chose the compolab technology from fresenius kabi as the new device for bbc blood bank. results/finding: the compolab was selected as it met project scope and selection criteria. it was important for bbc to reduce paperwork and daily tasks. the compolab eliminates daily qc reducing paperwork, time and improves error management. after converting to the new technology, bbc donor deferral rates increased by approximately %. as a consequence to this increase, bbc conducted reminder training with bbc staff to ensure proper sampling technique and higher sample quality. over time, bbc deferral rates stabilized to . % in and . % in . during this time period, bbc also successfully recruited new blood donors to bbc program, which may have contributed to an increase in deferral rates. in , the deferral rate increased again, probably due in part to the fda final rule "requirements for blood and blood components intended for transfusion or for further manufacturing use", which went into effect in may . conclusion: during the evaluation for new equipment, bbc learned that it is critical to understand the equipment's life cycle and the effect the equipment has on all aspects of the business. after comprehensive evaluation of multiple donor eligibility screening platforms, the compolab device was selected at bbc facility. it met the majority of all aspects of the project scope and qualifying criteria. bbc also learned that continuous refresher training of the staff ensured optimal device performance, and how external factors such as changes to the regulatory environment may impact deferral rates. flowmetry on platelet apheresis. tetsu yamamoto* , ayumi araki , hiromi sanyoshi , hiromi kanai , hiroya kikuchi , katsushi tsukada and kazuhide mure . hokkaido red cross blood center, japanese red cross hokkaido blood center background/case studies: vasovagal reaction (vvr) is known to be the most common adverse reaction to blood collection, but effective measures for preventing vvr have not yet been developed. effective timing of interventions during apheresis donations in particular should hold the key to predicting vvr, but no research has been done on the topic. study design/methods: this study investigated the potential to predict vvr from fluctuations in peripheral blood flow measured by laser doppler flowmetry in platelet apheresis donors, a population highly likely to experience vvr. data were collected from individuals who donated platelets during the -month period between february and august , and data from the donors who experienced vvr were analyzed. to calculate the level for issuing vvr alert, the percent decrease in blood flow (dbf) and the percent decrease in heart rate (dhr) were calculated, the time from alert to vvr was estimated for three dbf levels, and the detection performance of each alert level was calculated. results/findings: eight of the men ( . %) and of the women ( . %) experienced vvr. one donor did not experience vvr during blood collection, but had a delayed reaction while resting afterward. mean maximum dbf in the donors in the vvr group was . . %, which was significantly higher than the . . % in the non-vvr group. at a maximum dbf threshold of %, sensitivity for discriminating between vvr and non-vvr donors was . % and accuracy was . %. when % dbf was used as the alert level, alerts were issued for donors, including in the vvr group. therefore sensitivity for predicting vvr was . % and specificity was . %. mean time from alert to diagnosis in the vvr group was . . minutes, and accuracy of the alert was . %. some of the vvr could not be predicted even the value of maximum dbf exceeded %. the reason was supposed to be the difference of donor susceptibility on dbf. conclusion: we investigated whether vvr in platelet apheresis donors can be prevented by prediction and found that it is possible to predict vvr early enough before onset to intervene by monitoring dbf in real time during blood collection using laser doppler flowmetry. future research must also investigate whether the incidence of vvr can actually be reduced by interventions such as adjusting extracorporeal circulation. the risks of alloimmunization in sickle cell patients using c, e, k negative blood: experience of a hospital apheresis and transfusion service grace banez sese* , , salam abdus and shabrina shah . inova blood donor services, inova fairfax medical campus, inova fairfax medical campus transfusion services background/case studies: red blood cell (rbc) transfusion is often a lifesaving measure for patients with sickle cell disease (scd). it is critical in the management of scd complications such as splenic sequestration, stroke, priapism, iron overload and acute chest syndrome. a wellrecognized complication of chronic transfusion in scd patients is alloimmunization to rbc antigens. to prevent alloimmunization, transfusion with rbcs negative for c, e, and k antigens has been advocated. this has led to reports of reduction in the rate of alloimmunization and a decrease in hemolytic transfusion reactions. we report a summary of our three year experience with the prophylactic transfusion of rbc units negative for c, e, k antigens for scd patients during red blood cell exchange transfusions (rbcx). study design/method: retrospective review of scd patients with a history of stroke, refractory sickle pain crisis and priapism was done. rbcx was performed every to weeks from december to march . blood bank work-up used the mts gel method for antibody screen and identification. our hospital-based donor center proactively works with the hospital blood bank in preparing these units in a timely manner. results/finding: a total of patients, females and males, who underwent a total of rbcx from october to march , using an average number of rbc units per rbcx. rbc units negative for c, e, and k antigens were used during rbcx for patients. two patients positive for c antigen underwent rbcx, using e and k antigen negative rbc units. review of the antibody screen test results performed prior to each of the rce showed that no new clinically significant alloantibodies were formed after exposure to multiple rbc units. conclusion: although there is no consistent standard of care in transfusion practice related to the extent of antigen matching for scd patients, studies suggest that the standard of care for transfusion of all patients with scd is to provide rbc negative for c, e, and k antigens. this ability to find these rare units is also affected by the characteristics of one's institution and blood supplier. it is an advantage to have a hospital based donor center to work with, as we proactively collaborate with them to provide these rare units. the approach by our institution to transfuse rbc units negative for c, e, k or study design/method: venous blood specimens of healthy volunteers were collected before blood donation and after blood donation immediately, day, week, weeks, and weeks among men and weeks among women. immunoglobulin g (igg), immunoglobulin m ( igm) , immunoglobulin a ( iga)and complement component ( c ) , red blood cell (rbc), white blood cell count ( wbc) , hemoglobin (hb), hematocrit (hct), and serum iron (fe) , were measured to monitor he dynamic changes of these biomarkers and blood quality. results/finding: the level of igg slightly decreased after blood donated immediately, iga and c decreased significantly but still within their normal ranges, igm did not change after blood donation. the level of iga significantly decreased at weeks among men and weeks among women, while c significantly increased at the same time period. igg, rbc, hb, hct and fe started to recover week after blood donated and reached their levels before blood donated within weeks among men and weeks among women. conclusion: the biomarkers mutually changed over the course of weeks among men and weeks among women. donating ml blood will not significantly affect overall blood quality. utilizing amicus dxt relay data managment solution to increase platelet split rate and improve amicus productivity janelle wilhelm* and jennifer kaluza. memorial blood centers background/case studies: with the increase in platelet demand and the opportunity to export products we set an initiative to increase the platelet products collected form our existing donor base. we also faced the challenge of managing multiple collection sites in multiple states. the decision was made to implement amicus dxt relay data management solution to provide us insight into procedure details to make data driven decisions. day to day variability previously dipped as low % split forcing reactive planning. study design/method: incorporate dxt to strategically plan our day to day operations. dxt reports were monitored by management and with the fresenius kabi team for productivity by site, phlebotomist and device. reports measured target vs actual yield, donor parameters, and procedure events to perform a donation opportunity analysis. this allowed us to adjust configuration settings when appropriate to improve the accuracy of the yield prediction. reports by phlebotomist were utilized for training on how to optimize the donor's gift to donate an additional platelet or plasma product(s) and increase procedure success rate. results/finding: the monthly dxt report analysis resulted in device configuration improvements, phlebotomist and center manager accountability, effective training, and donation optimization we increased our overall platelet split rate percent and increased concurrent plasma collections by percent. with utilization of the dxt reports we are able to take a proactive approach allowing us to predict product availability, with day to day variability dropping no lower than percent split. phlebotomist qns rates were easily monitored regularly (daily, weekly monthly) resulting in a decrease in our overall qns rate to consistently below percent. conclusion: dxt was easy to implement, is very user friendly and will continue to help improve our platelet collection and process improvements between donor centers. dxt provides invaluable tools for the operational supervisors to monitor their staff and improve productivity at their multiple sites. next step is to develop the plan for implementation of paperless documentation with dxt and healthcare-id. the ability to immediately review data directly from amicus was key in the productivity improvements realized. evaluating the impact of a background/case studies: as blood and blood products are limited and expensive resources, they are prescribed, handled, stored and transfused according to hospital guidelines established to ensure that the best practice standards are maintained for patient safety. it is a prerequisite for all registered nurses (rns) involved in blood and blood product administration to possess fundamental knowledge of transfusion practice. aim: the aim of this study is to evaluate the impact of a hospital-based transfusion practice training program among registered nurses, through administration of a knowledge-based questionnaire before and after implementation of the program. the results gathered would identify gaps in assimilation of knowledge and suggest improvements to the design and implementation of specific content in the nurse-led transfusion training programme. study design/method: all rns from various units and departments were invited to participate in the blood transfusion knowledge questionnaire in october . after which, a formal transfusion practice training programme was introduced, consisting of an online learning platform and in-service training sessions. the same questionnaire was administered to the rns one year later in september for post-training programme evaluation. individual item scores and proportion of nurses with perfect scores was compared pre-and post-implementation. results/finding: in and , a total number , rns and rns completed the questionnaires, giving a response rate of . % and . % respectively. the overall mean score in was . points (range to ). the mean score in was . points (range to ). the percentage of rns having perfect scores of increased from . % in to . % in . table i below shows the results for each question item. the implementation of a hospital-based, nurse-led transfusion practice training programme has led to encouraging improvement in blood transfusion knowledge amongst rns. further training may be needed in the preparation of blood sets and management of fever. background/case studies: clinical use of blood has shown to be the least developed part in the vein-to-vein transfusion chain. this global survey was therefore carried out in order to investigate the level of awareness, accessibility and utilization of e-continuous learning and quality of blood use among blood prescribing clinicians and nurses. study design/methods: a descriptive 'ex-post facto' survey design was used; purposively selected blood prescribing clinicians and nurses from hospitals in countries of the human development index (hdi) groups (low, medium, high, and very high) participated. three research questions were answered, while seven null hypotheses were tested at . level of significance. descriptive statistical tools (frequency counts and percentage) were used to analyze the demographic backgrounds, while inferential statistics -pearson product-moment correlation coefficient (ppmc), analysis of variance (anova), were used to analyse the hypotheses. results/findings: quality of clinical use of blood was positively and significantly correlated with levels of awareness (r . ; p . ; df ) and accessibility (r . ; p . ; df ) to e-continuous learning among blood prescribing clinicians/nurses. there was significant difference in levels of awareness [f( , ) conclusion: today e-continuous learning has become a conditio sine qua non to effective and quality clinical use of blood. the higher the hdi level the better the awareness, accessibility and utilization of continuous education, both through e-learning and conventional programs. there is a better awareness among clinicians routinely prescribing blood as compared to others involved only incidentally in blood transfusion. accessibility of e-learning depends highly on the presence of a sustained societal infrastructure which is less guaranteed in the low and medium hdi countries; reliable power supply, maintenance of hardware tools and updated software programs, together with the necessary knowledge and skills of e-technology are prominent factors. the results are used for policy and strategy recommendations to improve knowledge and clinical practice through continuous e-learning programs eg, starting at undergraduate medical and nursing schools and continuing at postgraduate vocational medical specialization institutes, principles of clinical transfusion practice should be comprehensively included through appropriate and timely curricula; creation of a technical climate to guarantee access to e-learning courses and materials; stimulation of national and international exchange of e-learning programs focused on continuing education; creation of an e-learning mentoring network through professional societies, associations and education institutes. background/case studies: transfusion medicine (tm) didactic teaching materials for pathology residents are not widely available to share among residency training programs. the advancing blood knowledge (abo) leaders project is a novel approach wherein education materials are created collaboratively through a community of practice (cop). educational theorist etienne wenger defined cops as groups of people who share a concern or passion for something they do and learn how to do it better as they interact regularly. study design/method: as a pilot project, junior faculty co-investigators from west coast institutions each had months to create a minute powerpoint presentation on a fundamental tm topic, after which other members had months to review and edit. therefore, each member created and reviewed presentations (three total steps). during each step, members wrote multiple-choice questions for those particular topics. in the end, each topic would have quiz questions to assess learning. at completion, evidence-based, peer reviewed presentations would be available for all members to use for teaching pathology residents. three methods were planned to measure effectiveness of these materials: ) pre and postlecture abo leaders exam using the questions made for each topic to assess learning; ) pre and post-lecture question validated examination (best collaborative) to assess learning; ) resident in-service examination trends specific to tm. results/finding: six presentations were developed as of the abo leaders members continue to participate in this cop for tm education. abo leaders and best pre-test results are shown in tables and . abo leaders pre-test data could not be obtained for institution b, and trainees declined to participate in the examinations at institution a. challenges experienced by the cop have included heterogeneity between institutionsÕ resident schedules, balancing time dedicated to the group given busy schedules, and difficulty in giving all presentations during the defined institution-specific teaching period. post-test results will be included when assessments are complete. conclusion: despite logistical and organizational challenges, it is feasible to create a multicenter cop for tm education. the impact of such a group on resident learning will be assessed and plans for growth will be evaluated. background/case studies: the traditional educational curriculum for the pathology residency program is primarily based on didactic lectures, casebased presentations, and discussion of on-call cases. the use of dramatic vignettes has proven to be an effective educational tool to illustrate complex and multidisciplinary topics in medicine. our goal is to use and evaluate the relevance of this approach in resident education. study design/method: a clinical vignette based on a placenta accreta case was written by a pathology resident during the transfusion medicine rotation. during a two-week laboratory management course, residents prepared for the dramatic vignette performance with a focus on transfusion medicine and laboratory management topics. each resident completed a question preand post-test on topics related to the vignette. several meetings for review and adaptation of the script, topic discussion, and rehearsals were held. there were several commonly encountered problems and deviations from the standard operating procedures that the residents in the audience were asked to identify prior to the performance. during the skit, each resident presented at least one major transfusion management teaching point. results/finding: the educational activity, including the minute vignette performance and the minute discussion, was completed with a focus on: communication between the operating room and the blood bank during surgery, maximum surgical blood order schedule, pre-transfusion testing, transfusion safety, informed consent, massive transfusion protocol, emergency release blood products, thromboelastometry interpretation, patient safety, adverse events, and root cause analysis. all performers significantly improved their scores in the post-test (mean %) when compared to the pre-test scores (mean %) ttest p< . . during the vignette discussion, residents together identified all the intended non-conformances and answered related questions. residents in the audience actively participated in the post skit discussion and % reported a satisfactory learning experience. conclusion: dramatic clinical vignettes can illustrate multidisciplinary complex interactions that are of pivotal importance in the daily activities and professional development of pathology residents. with specific structured goals, clinical dramatic vignettes can be used as a complementary educational tool to illustrate challenging topics in an integrative way that is enjoyable and easy to understand and remember. the skit performers benefit from the activity further by preparing and extensively studying the topics to deliver a multifaceted and coherent presentation with emphasis on the integral role of the laboratory and transfusion medicine in patient care. hannele sareneva*, susanna sainio, inna sareneva, tiia kivipuro and taru jaske. finnish red cross blood service background/case studies: the finnish red cross blood service (frcbs) is the nationwide blood service provider in finland, responsible for collection, testing, processing and distribution of blood products to all hospitals and health care providers. the frcbs serves as the national blood group reference laboratory and provides a wide range of other laboratory services e.g. tests for hemostasis and tissue typing for possible donors as well as patients waiting for organ or stem cell transplantation. frcbs also performs antenatal blood group and rbc antibody tests covering whole country. as a sole national operator we are providing educational services to ensure the safe use of blood products as well as accurate use of our laboratory services. study design/methods: we have performed customer surveys to healthcare professionals to assemble the needs for education. based on these results and continuous feedback frcbs provides hospital customers in blood banks and clinics the following additional services: * regular education * e-learning application of transfusion medicine * handbook for blood products on the web site * reports to hospitals for their use of blood products * annual national blood safety reports regular elements of our educational program are the practical, problem solving course for blood bank personal and safe transfusion training day for clinicians. for every education we collect numerical feedback as following: "how did the education responded your expectations" and "can you utilize the knowledge in practice". we also inquire "how likely you would recommend the training for your colleges" indicating net promoter score (nps). results/findings: more than healthcare professionals participate training days at frcbs annually. in addition our experts give tens of lectures at hospitals across finland. feedback from educations has been very good, varying between . to . (in the range of - ). nps varies between and . according to customer surveys frcbs provides appropriate education to healthcare professionals. this score has increased - from . to . . conclusion: feedback, nps scores and surveys ensure that education and training program of frcbs responses to customer needs. hospitals can utilize annual courses of frcbs in their own initiation programs. together with clinical contact persons in hospitals our aim is to ensure patient blood management (pbm) and to optimize use of blood products. we also have plans to increase e-learning applications and the courses of transfusion medicine for nurses and medical students. educational outreach and effect on reporting septic transfusion reactions kathleen m grima* , anne eder , beth a. dy and mary o'neill . american red cross, georgetown university background/case studies: hemovigilance programs to monitor adverse events after transfusion depend on clinicians' ability to recognize and report reactions to the blood center. about in , apheresis platelet donations are implicated in septic transfusion reactions (strs), but this could underestimate the risk because of the difficulty in recognizing delayed or mild reactions. a large blood center designed an educational outreach program to increase awareness of strs and assessed its effect on the rate of str reporting to its national hemovigilance program. study design/method: in dec. , a large blood center developed a web based course on strs for cme/ceu credit. letters were sent to , hospital customers about recognizing and reporting strs, and alerting them to the availability of the course. blood center physicians and staff in sales and marketing also engaged hospital customers directly in discussions about recognizing and reporting strs, using the online educational content. the physicians tracked their interactions. the blood center's national hemovigilance program compared the number of strs reported in the months before and after launching the educational outreach. results/finding: the web based course was completed by more than participants; were physicians. based on a review of the evaluations, the course was highly valued with % of participants rating it excellent or very good. the blood center physicians gave over presentations to hospital customers. reporting of suspected strs in increased by % compared to the prior year. the increased reporting came from specific regions. the total number of strs that met the hemovigilance definitions for definite (culture-confirmed) and probable strs in the nationwide system increased but did not change significantly compared to the previous years. the educational initiative was designed to deliver a consistent message on the risks, recognition and reporting of strs. while the number of reports of suspected strs in two regions increased, there was no meaningful change in the overall reporting of suspected or confirmed strs across the national blood system. this finding could reflect that hospitals already recognize and report medically significant reactions or that the target audience was laboratory personnel and physicians in transfusion medicine, but not the clinicians closest to patient care at the bedside. more targeted educational efforts provided by personnel who interface with hospitals could be used to address identified professional practice gaps in transfusion medicine. implementation of subscription-based cgmp e-learning laurie mcgraw*, courtney saphier, helene belton, sallie bittner and ward scott. gulf coast regional blood center background/case studies: previous cgmp e-learning courses we developed required - minutes for learners to complete. while feedback was positive, manufacturing areas struggled to schedule time for staff to complete courses within their assigned schedules. at the same time, a shift in design trends suggest that subscription-based learning is more effective (thalheimer, .) subscription-based e-learning utilizes - minute modules, delivered at regular intervals. this changes the learning process from a singular event to a regular interaction that reinforces learning and keeps the content at the top of the learner's mind. study design/method: we began developing cgmp subscription-based elearning in by selecting our first five series topics: equipment, personnel, labeling, sops, and records. the first topic, equipment, was divided into modules on selection, validation, calibration, quality control, and maintenance. these modules, and pre-and post-quizzes for the equipment series, were developed and assigned to employees in manufacturing-related jobs using our learning management system. the pre-quiz was assigned to employees in june , with a new equipment module assigned each month for the following five months. the series concluded in december with the post-quiz. results/finding: using surveys, assessments and incident reports, we evaluated the training effectiveness using three of the four kirkpatrick levels. while our previous cgmp courses received good ratings from learners, the equipment series received the highest rating of . on a -point scale. of employees who completed all versions of our cgmp courses, the majority preferred the equipment series over all previous courses combined. comments clearly demonstrated that learners preferred the short, subscription format over the previous courses with positive and negative comment. level : learning the average score of users increased % from the pre-test to the posttest, with the greatest improvements noted in the scores from laboratory employees. a two-sample t-test determined the result to be statistically significant with a t-critical value of . and a t-stat value of . . level : results while equipment-related errors decreased by % after training, there is not enough data to demonstrate a statistical significance. conclusion: our level and evaluation data validated that the subscription approach was effective. knowledge increased from the pre-to postquiz, learners reported that they appreciated the shorter training, and they completed the modules without special scheduling requirements. as a result, we are continuing development of the remaining series. background/case studies: the interdisciplinary nature of transfusion medicine requires the collaboration of multiple work units for efficient patient care, but departmental "silos" impede collaboration between transfusionrelated care teams. we hypothesized that regular educational meetings would improve knowledge and awareness of each department's role, so in october , a multidisciplinary educational meeting called friday blood conference (fbc) began as a collaborative, interprofessional forum involving frontline staff of our transfusion practice. during these monthly meetings, which are also broadcast online for those unable to attend in person, presenters from different work units share background information and patient cases before opening the floor to constructive discussion. study design/method: a survey was sent to fbc participants (n ) to retrospectively capture the effect of fbc on interdepartmental collaboration. the survey was structured to obtain formative feedback using the published interprofessional collaborative practice competencies (icpc) as a guide. these core competencies target maintaining a climate of mutual respect, communicating within and between departments, fostering teamwork, and understanding everyone's role in patient care. results/finding: our survey response rate was %. of those, % endorse that fbc creates a climate of respect within our transfusion practice, % believe it has improved communication between work units, and % feel that fbc leads to increased understanding of interdepartmental processes. notably, laboratory scientists and transfusion nurses have the highest attendance rate. furthermore, those attending via the online broadcast report the lowest satisfaction, with only % responding positively. the main reasons individuals attend fbc are to increase knowledge about transfusion medicine, interact with and learn from other departments, hear about patient case studies, and understand the "big picture" of one's role in patient care. suggestions for improvement include preparing questions to help initiate discussion, increasing representation of other areas for broader perspectives during interdepartmental dialogue, and posting recordings of fbc for later viewing. conclusion: the application of icpc in transfusion medicine was an effective lens to assess the value of interprofessional collaboration. although there is room for improvement, the results support that fbc has contributed to better communication between transfusion-related care teams and has increased understanding of interdepartmental processes within our transfusion practice. novel approach to curriculum development: demystifying transfusion medicine ritcha saxena* and ananya saxena. all saints university school of medicine background/case studies: transfusion medicine is an essential element of education required for the future physicians in various disciplines like surgery, internal medicine and anesthesiologists to work effectively with the blood bank personnel. transfusion carries considerable advantages as well as risks. consequently, educational initiatives are required to identify the particular knowledge deficits in transfusion medicine and subsequently, bridge the gaps. and the challenge is to update the undergraduate medical curriculum to reflect the latest enhancements in transfusion medicine. study design/methods: students of undergraduate semester and students of semester participated in the study. self-directed learning resources combined with modules of interactive instruction were implemented in a tbl course design. five education modules focusing on quality management, blood collection, transfusion reactions, precise utilization of blood products and innovations in component safety were designed for the students. the students' reaction to tbl in transfusion medicine was evaluated using qualitative and quantitative assessment tools to analyze knowledge attainment and critical thinking development along with team continuity. the participants were first assessed with readiness assurance testing (rat) to guarantee that they understood the concepts and their application followed by case study based test questions. results/findings: students' reaction to tbl was primarily positive, with % of students giving a positive feedback. evaluation through readiness assurance testing (rat) illustrated improved team knowledge acquisition in implementation of effective quality management systems over knowledge acquired through individual study. students grasped a conceptual knowledge of principles of transfusion medicine and achieved confidence in dealing with transfusion-related complications. anecdotally, students significantly attained perception in blood component preparation, storage and their optimal utilization along with developments in safety techniques in blood donation. conclusion: our study suggests that reforming the medical curricula for undergraduate medical students, with specific educational modules designed to focus on blood banking and blood transfusion principles and latest advances in transfusion medicine, is much required in the interest of patient care and safety, by the future physicians. tbl is an interesting and efficient way to deliver the key aspects of transfusion medicine to the students. results/finding: open house attendees were given tours of the bb, led by a bb attending, bb residents, bb supervisor, or bb quality coordinator. the patient blood management nurse was also in attendance to answer attendee questions and educate about patient blood management. light refreshments were offered to the attendees in the bb break room. the first bb open house was held on wednesday, / / from - am. there were attendees, including a second-year medical student, four regular blood donors at the hospital blood donor center (who were also employees in facilities management and the university office of admissions, respectively), a hospital senior vice-president, six apheresis nurses, two clinical laboratory staff, two medical laboratory science students, and one additional staff member from the university office of admissions. the second bb open house was held on thursday, / / from - am. there were attendees, including regular blood donors (who were also employees in the office of international affairs and supply chain, respectively), a hematology/oncology fellow, and surgical residents. background/case studies: simple, partial, and exchange transfusions are routinely performed in patients with sickle-cell disease (scd) with the goal to increase the oxygen carrying capacity of the blood and reduce the relative percentage of sickled cells. it is essential for clinicians to be able to rapidly estimate the effects of the available therapeutic modalities using clinical information to minimize the risk of red blood cell exposure. given that the formulas for these calculations are complicated, we developed and validated an online calculator to assist physicians with such tasks. study design/method: a web application was generated (www.phamcalcs.com). the performance of the simple transfusion and partial manual exchange calculators were validated by comparing the predictions to clinical data. the performance of the automated and depletion rbcx calculators was validated using the terumo bct (lakewood, co) calculator up to a fraction cells remaining (fcr) % as patients with fcr % may benefit from delaying the procedure for performance in the future. validation process included ( ) a deming regression to globally assess the predicted vs. actual results and ( ) an individual comparison wherein validation was contingent on the (predicted-expected results)/(expected results) demonstrating |d| %. validation was performed for hematocrit (hct) and hemoglobin s (hgbs) level post-transfusion for simple and partial manual exchange and volume of replacement fluid for automated and depletion rbc exchange. results/finding: see table background/case studies: with the focus on new technologies the modern medicine requires more expenses. despite the increase in the target impact on patients there is still a risk of adverse reactions to medical treatment. the issues that are currently under discussion: the use of standardized or personalized approach, for doctors -being multidisciplinary or having a narrow specialization, integration of new technologies, the need for more trainings resulted from knowledge deficiency. in russia, the development of insurance medicine creates the demand for more intensive and cost-effective treatment programs. as a multidisciplinary approach, pbm optimizes the transfusion practice reducing the risk of adverse effects and improving the financial performance of a health care institution. however, the prosperous implementation of pbm also requires supplemental medical competencies that provide harmonization of dialogue logistics: administrator -clinician -transfusiologist. study design/method: at the medical simulation centre of hospital there has been a unique opportunity to launch an educational program for the medical specialists practicing blood components transfusion. the main innovative features of the training course are an interdisciplinary approach, intensive learning performance, comprehensiveness of learning methods. during days ( academic hours) the trainees can attend lectures, discuss the methodical materials, participate in seminars, interactive clinical discussions, a master class and a game that presents the modelling of working processes. since initiating the project in june, with the group capacity a transfusion vol. supplement s of up to people the number of medical specialists who have attended the training is nearly . results/finding: the medical competencies gained: knowledge of modern recommendations on the use of blood components the ability to interpret all parameters of the haemogram, coagulogram and tromboelastogram the ability to unveil the indications and contraindications for urgent and scheduled blood component transfusion personalization of the blood transfusion risks using a personalized approach on selecting the type and the dosing of transfusion habitat predicting the efficacy of transfusion the correction of anemia and hemostasis system malfunctions using the medicinal treatment performing the macroscopic assessment of blood component before the transfusion procedure performing the differentiated diagnostics and ability to prescribe the adverse effect treatment ability to carry out the auditorial check of health cards conclusion: the launch of the program "guidance for safe and effective blood use in adult patients of multi-field hospitals" is aimed to meet the educational and professional needs of medical specialists, develop the algorithmic thinking and a range of useful motivations in case of patient blood management and reach the compliance in practice. the effect of emergent situation drills on technologist teamwork and comfort levels abigail neils*, raeanne stensgard, rebecca wren, elisabeth greer, amy mata and camille van buskirk. mayo clinic rochester background/case studies: teamwork and composure are essential for technologists when dealing with emergent situations in a large hospitalbased blood bank where multiple situations can occur simultaneously. in an effort to reduce errors and improve emergency response, a group was formed to evaluate the effectiveness of emergency situation drills (esd). the esd were based on common emergent situations encountered in the lab and were run once per month per shift. the main goal of esd was to improve teamwork and comfort level during real emergent situations; therefore reducing the amount of unplanned standard operating procedure (sop) deviations. study design/method: prior to esd implementation, a survey was sent to all technologists to determine baseline comfort levels associated with various emergent situations. one year post esd implementation the same survey was sent to all technologists to reassess the comfort levels for the same situations. the surveys asked employees to rate satisfaction and comfort level on a grading scale of - ; being least satisfied/comfortable and being most satisfied/comfortable. the pre and post survey results were evaluated by calculating lab average comfort levels per situation and survey. in addition, unplanned sop deviations related to emergent situations were counted for one year before and one year after esd implementation. results/findings: out of total technologists, technologists took the pre esd survey and technologists took the one year post esd implementation survey. table shows the lab averages from the pre and post surveys as well as the percent difference. out of the employees who responded to the post survey, ( . %) answered "true" to the statement "esd have improved my comfort level with emergent situations." in the year prior to esd implementation there were unplanned sop deviations; in the year after esd implementation there were only deviations. conclusion: all but one area increased in comfort level post esd implementation. also most technologists agreed that the esd helped improve their overall comfort level with emergent situations. the goal of implementing esd has been met based on the unplanned sop deviation decrease and technologist satisfaction increase; therefore esd were deemed effective. monthly esd will continue to be run with the hope of continual improvement in teamwork, comfort levels and deviation levels. therapeutic background/case studies: category i indications for red blood cell exchange (rbc exchange) in children with sickle cell disease include following acute stroke and for stroke prophylaxis, as well as for iron overload prevention. as described in the first installment of this series about therapeutic plasma exchange (tpe), the challenges of access, volume management, and instrumentation persist, as along with the need to address the psychological and emotional well being of this population. rbc exchange is a complicated procedure to explain to adults and becomes an even more intimidating task when translating into the language of childhood. nevertheless, pre-treatment education is shown to decrease the anxiety associated with medical care. providing age appropriate specific treatment information to pediatric patients decreases negative behaviors, reduces stress and promotes faster recovery. a previous project explaining tpe to the pediatric population revealed the lack of age specific literature for apheresis procedures in general, including tpe and rbc exchange. study design/method: in collaboration with a child life specialist, an ageappropriate story-driven explanation of the rbc exchange procedure was adapted from a previously implemented project related to tpe. artwork was produced with the aid of a medical illustrator to complement the story-line. results/finding: the story board addresses why rbc exchange is performed, the steps involved in preparing for and performing the procedure, and strategies for coping before, during and after the procedure. the idea of long-term therapy is also briefly addressed, to prepare these children for the concept of ongoing therapy. the booklet is in production in concert with our hospital's medical illustrator and will be available on our hospital website for patient use. conclusion: using the previously illustrated story as a guide, an explanation of red cell exchange was created to provide education and reduce anxiety. this second installment continues the pediatric series helping to explain apheresis procedures to pediatric populations in the hopes of reducing patient stress and promoting age appropriate coping strategies. transfusion safety officer resource manual leonor de biasio*. it is also intended to be utilized by hospitals that do not have a formal tso position but which have delegated the responsibilities to other staff. the resource provides helpful information to assist with education in transfusion safety, adverse event investigation and reporting, product administration guidelines or monographs, and links to information about the equipment used for infusion of blood. the resource manual will serve as a useful reference tool to assist with a healthcare professional's transition into the tso role. turning on pathogen reduction: a case of flipping the switch kassandra poffenberger*, darla wendt, jennifer vrieze and james r stubbs. mayo clinic background/case studies: a critical aspect of implementing a new method in manufacturing blood products is to develop a training plan that adequately prepares staff but doesn't interfere with production or cause delays in patient care. with the implementation of pathogen reduction technology (prt) using interceptv r blood system for platelets it was understood that we would need more collections to make up for the loss of products, specifically our triple collections. our institution collects the majority of its blood products and supplements inventory from a major blood collection center. it was crucial for the component laboratory to maintain daily processing levels while learning the new method in order to sustain optimal platelet inventory levels without relying on purchasing additional platelets from external vendors. our approach in introducing prt for apheresis platelets was to "flip the switch" and process all products with the new method rather than a step wise roll out with a dual inventory. study design/method: it was essential to prioritize who would be trained first. collections occur monday through friday from to . the first group to be trained was those who would be performing training (a two person team) and product validation; they were trained by cerus deployment team. the second group was those who would process platelets on weekends and evening hours without direct management support. the last group was the technologists who would be working during normal hours with direct management support. prt processing for platelets in % plasma is broken up in to two days. on day platelets are treated with amotosalen and placed in a compound adsorption device to remove residual amotosalen for - hours. on day products are removed from the cad and modified into final product codes and labeled. each technologist was trained one on one, over a one week period. the trainers alternated training processing days for day and day . in the weeks following training it was important that each technologist rotated back thru prt processing to maintain proficiency. results/finding: of employees were trained in a two month time period. prior to "flipping the switch" the daily average of products collected was . for the two month training period the daily average rose to . conclusion: our "flip the switch" training plan for implementing prt platelets in % plasma has been highly successful for our laboratory; training while implementing the new technology did not create a bottle-neck in the process. it was imperative to prioritize who would be trained first to insure complete coverage during off hour shifts. technologists were able to become proficient with the new process while maintaining daily processing expectations and sustaining an optimal platelet inventory. accepted depending on each individual's conscience. due to these unique medical challenges, it is important for caretakers to have an understanding of their beliefs in order to provide optimal care. we describe the process of identifying jw in our hospital and communicating treatment needs to staff. study design/methods: proper treatment of jw requires the ability to identify the patient and his/her needs. when a jw is admitted to our hospital, our electronic medical record (emr) triggers several processes based on the patient's listed religion. one process creates an order that reminds caretakers to complete the declining blood consent (dbc) with the patient. the dbc contains language declining mabf and reviews the mibf with the patient to identify any that would be accepted. the emr order regarding the dbc provides educational links that include a bloodless policy, step-by-step instructions on obtaining the dbc, and information on alternatives to transfusion. a second emr process triggers a stop-gate to prevent the completion of any mabf order or mibf order for a product that the patient has declined. a third enrolls patients in the minimal blood volume labs protocol which uses microtainers, partial-fill vacutainers, and blood reservoir sets to reduce blood loss during draws. additionally, at registration, a bloodless packet is added to the patient's paper chart. this packet contains the dbc, a glossary of dbc terms, a bloodless sign to be placed over the patient's bed, a bloodless wristband to be worn by the patient, and two bloodless chart stickers that are added to the outside of the chart. these steps remind the caretakers of the patient's special requests. finally, the patient blood management (pbm) department receives emr developed reports which identify jw presenting to the hospital. these patients are followed by the pbm nurses and medical director during the duration of care. treatment plans to optimize hemoglobin, oxygen carrying capacity, and hemostasis are discussed with the bedside caretakers and implemented as needed. results/findings: nearly % of jw that enter our hospital have a dbc completed. this has resulted in increased education of the medical staff. in addition, patients have reported better communication with caretakers leading to a more inviting environment for the patients. conclusion: our hospital has found success by using an education-based team-oriented approach involving emr, pbm, and caretakers when caring for the jw patient. this approach has set up a foundation for treating other bloodless medicine patients. background/case studies: transfusion services should provide safe blood components from vein to vein with donors acting as suppliers and patients as final customers. this process involves labor-intensive activities, critical materials, human resources, facilities and highly coordinated processes. cost management has a great impact on technical processes guiding decisions upon supplies and technical staff. activity-based costing (abc) is a method to determine cost drivers within activities and determine process or product final cost allowing managers to take precise decisions. we demonstrate how an effective abc approach can result in financial savings without compromising process quality in a mid-size transfusion service. study design/method: materials costs can represent as far as % of an activity. in we had a central storage supplying satellite storages at each department and replacement was done independent of residual stock. purchases were performed on demand. at the end of we performed a supply inventory on all departments to plan future purchases and control residual stocks. in , we implemented annual purchases and satellite storages were supplied only to replenish programmed stock. cost drivers were defined upon activities on standard operational procedures (sops) resulting in a cost estimate. technical staff was involved in cost driver calculations to indicate possible changes to sops, supplier and deliveries. to minimize seasonal fluctuations we compared last quarter (q / ) with last quarter (q / ). in this work we present activity data from blood collections to illustrate abc method. results/finding: in q / blood bags were used compared to in q / , demonstrating an activity " . %. price negotiation resulted in . % readjustment. both indicated an estimated cost " . % with a possible impact of over us$ , . we have identified a real cost # . % in q / , representing an overall # . % and us$ , . (r$ , . ) savings. conclusion: economy had deteriorated in our country in with higher inflation and exchange rate variations, directly impacting imported materials, most of them critical. even with adverse economy, abc showed to be an effective tool that allowed cost decrease without significant changes in critical materials and processes. cost drivers calculations demanded review of sops and suppliers by technical staff resulting in optimization of activities. also, staff involvement reduced discharged materials since costs were wellknown to area supervisors and satellite stocks were reviewed briefly. automated verification of immunohematology results and the impact to donor testing barbara j bachman* , candace williams , carmen meyer , paul lamonby , anne cleverley and silke milbradt-pohan . bio-rad laboratories, diamed gmbh title: automated verification of immunohematology results and the impact to donor testing background/case studies: staffing challenges in today's blood banks require instrumentation with minimal operator intervention. technology advances have developed where every immunohematology result does not necessarily require operator visual review. this study evaluates the impact of automated result verification on the bio-rad ih- tm immunohematology system through the ih-com tm data management system (dms) for donor processing laboratories. study design/method: a multi-center study was performed on donor samples as shown in table a evaluating two of the most commonly used ih-system gel cards available in the us. workflow data was analyzed using process modellar app (ipad). this study focused on post-analytical steps of result verification, evaluating with and without automated result verification to determine the impact on quality (# operator touchpoints, visual result review occurrence), result accuracy, and speed (time from result interpretation to lis data transfer). operator touchpoints during the post-analytical phase are only required when doing visual result verification and are software defined. speed metrics were analyzed using minitab v , statistical the transfusion team collaborated with multiple user groups to educate them regarding the new processes. a gap analysis was performed to determine the optimal delivery process for blood products, with key stakeholders invited to review the options. the use of the pneumatic tube system to deliver blood throughout the entire campus was investigated to determine whether it would be a viable option given the expanded size of the new campus. results/finding: user groups requested additional training sessions as questions arose regarding use of the ehr for blood ordering. because the pneumatic tube system would be heavily used, and due to concerns that blood products could become "lost", it was decided this would not be the best route for delivery of blood. department educators requested support to create job aids specific to workflow changes impacting their departments, such as how to order rh immune globulin, a cord blood workup, etc. conclusion: leadership was challenged to provide a stable and positive environment during a complex set of changes. the simultaneous hospital move/merger and implementation of a new ehr constituted an arduous task that would not have been possible had substantial preparations not been initiated a year in advance. training is essential to the success for a scope of change this big and should not be minimized. while training was thorough prior to the move, gaps were nonetheless discovered following the move. abstract conclusion: strategic development partners funding and support based on newly developed government strategy on blood service with commitment of the government has brought a positive impact in establishing sustainable and safe national blood service program in ethiopia. even though the identified positive impacts mentioned are achieved, the bts remains with multiple challenges and needs continuity of funding and more partner support and government commitment. pilot implementation of a comprehensive hybrid performance management system at national blood service zimbabwe blessing mukwada*, judith j parirewa and tonderai mapako. national blood service zimbabwe background/case studies: the national blood service zimbabwe (nbsz) introduced its first performance management system (pms) in . in the - nbsz strategic plan it was noted that the current pms lacked objectivitety and there was no relationship between perfomance and remuneration. in order to revise the pms, the nbsz set up a three membered committee at the executive management level to spearhead the revamp of the nbsz pms. the aim of the new pms was to achieve a shared vision of the purpose and objectives of the organization, helping each staff member to understand and recognize the contribution to the strategic plan. in this paper, we share how nbsz revamped and implemented its new hybrid pms that derived its inputs from established pmss and nbsz monitoring and evaluation (m&e) process that have been linked together. one-selected departmental results for one quarter are shared to demonstrate how the system works. study design/method: pms committee developed and shared with executive management a pms conceptual and implementation framework. consultations including field visits were done on three established pms to assess suitability for nbsz adoption. a hybrid pms was adopted for nbsz and a pilot application for one quarter on selected department was done. review of policy, procedures to including hybrid pms templates and forms were done. pms committee trained all staff on how to implement an integrated scorecard, how to conduct appraisal, how to develop scorecards, how to measure performance using the new pms, how weighted performance reward systems based on all layers of performance for bonus payments works using standardised tools. throughout the process risk assessment were done. results/finding: the nbsz hybrid pms is based on five levels of planning namely strategic, departmental, branch, sectional and individual. the fourcoloured traffic light reporting system is central in uniformly assessing performance at all levels. the levels of accountability were properly defined for each level of planning. a weighted overall integrated individual scorecard (iis) is determined based on % individual and % for the other four levels ( % for each). the bonus (%) is calculated based on the iis as follows; category a: % (iis > %), category b: % (iis: -< %), category c: % (iis: -< %) and category d: % (iis < %). on the pilot implementation, the individual scores for staff ranged from % to %. the iis were % to %. the number of staff in each bonus categories were , % (category a) and , % (category b). conclusion: the new hybrid pms was generally accepted by all staff and it was easily implemented at various staff levels. this provides a basis for the full implementation of the new pms and this simplified pms can be easily be adapted in similar settings to ensure all staff contribute sufficiently and objectively to the realisation of the organisation strategic vision. rare donor engagement with american rare donor program (ardp) margaret c manigly* , deborah r fludd and sandra j nance . background/case studies: rare donors are defined as a blood type occurring in less than in people in a given population. these donors are discovered by testing new donors in a random or targeted way and require testing many donors to find one rare donor. once found, if the facility is a member of the american rare donor program (by being an aabb accredited or american red cross accredited irl), the donor is registered in the ardp database as a rare donor. in , there were , active rare donors in the ardp. with the mobility of the population in the usa, it is important that as donors relocate, that they are recognized as a rare donor when they donate and their unit can be identified and used for a patient with a rare blood need. in addition, when recruitment is needed for a patient need, correct contact information on the donor is required. study design/method: the ardp procedure for ardp members requires that donors be contacted every six months to ensure that ardp (or the facility) has their latest contact information. the timing is determined by the postal service time limit of six months to forward mail to a new address. this contact ensures that if recruitment is required to obtain blood for a patient with a rare blood need, the donor can be contacted by the collecting center to donate. this contact is achieved by ardp sending a contact card by postal mail twice yearly to all donors for whom the ardp has address demographics. results/finding: the ardp reports on the information obtained from the contact cards returned in the ardp annual activity report to the ardp members at the aabb annual meeting. of the ( . % of total active donors) returned contact cards alerting ardp of changes in calendar year , ( . %) were donors moving from one ardp facility to another, ( . %) were donors no longer eligible to donate, and an additional ( . %) were address changes. other changes were ( . %) reactivated donors and ( . %) donors who we were notified were deceased, or did not want to be listed in the ardp. in , new rare donors were submitted to ardp for registration. the number of donors that could potentially be lost to follow-up in was ( ), which would be . % of the new donors submitted. conclusion: with nearly a % response rate for donors receiving the mailed contact cards, it is clear that rare donors (and their families) are responsive to the ardp contact card, and inform ardp of address changes and changes in their health status that affects their ability to donate. this is evidence of the importance of the card in ensuring correct donor contact information. in , donors changed their addresses which often are not known to the collecting facility until the donors donates again, after their move. the ardp contact card is effective in retaining the relationship with the ardp registered donors and keeps the address information of rare donors current. workflow comparison of two gel analyzers in a large transfusion service j peter pelletier* , barbara j bachman , mike leamy , susan olson and candace williams . university of florida college of medicine, bio-rad laboratories background/case studies: vendor-assisted workflow studies are becoming more popular as analyzer choices and capabilities vary in the market. the purpose of this study was to evaluate the provue (ortho clinical diagnostics) against the ih- (bio-rad laboratories, inc.) in a large volume transfusion service using lean process flow. study design/method: twenty-two ( ) runs of one to six ( ) samples per run were observed for two ortho provues alternating testing at a large transfusion service performing , types, screens, type & screens (t&s) annually. the workflow patterns observed were then repeated on the ih- and compared. each process was mapped in detail by direct observation using process modellar app (ipad). the evaluation started at sample centrifugation completion and ended with results sent from analyzer to lis (lab information system). each was evaluated for quality (testing process steps, biohazardous exposure episodes, and maintenance tasks), speed (operator/analyzer time) and cost (testing/maintenance personnel hours recaptured). time studies were analyzed using minitab v , and statistical significance was assessed using the paired t-test, with p values of < . considered significant. regardless of quality or speed metrics evaluated, the ih- demonstrated a significant reduction (improvement) in process steps and associated times when compared against the ortho provue (p < . ). ih- process steps and time studies addressed in the table below did not account for the ih- reagent storage capacity. in reality, the improvements would be greater than what was displayed here in a real-life operation. evaluating the total number of maintenance tasks required annually, as well as the times associated with maintenance performance, there was a significant reduction on the ih- ( % reduction, a difference of hours/year). conclusion: this study verified the ih- provided significant efficiencies and cost avoidance over the ortho provue for a large volume transfusion service. workflow comparison of two high volume, high throughput analyzers aaron samson* , kimberly monnin , barbara j bachman , kyla warren , susan olson and candace williams . clinical pathology labs, bio-rad laboratories background/case studies: few workflow studies have been performed on high volume, high throughput blood bank analyzers in large volume testing facilities. the purpose of this study was to evaluate the galileo v r neo (immucor) against the ih- tm (bio-rad laboratories, inc.) using lean process flow. study design/method: a total of separate test runs of or samples per run were observed over a three day period on the galileo neo at a reference laboratory annually performing approximately , type & screens (t&s). the workflow patterns observed were then repeated on the ih- and compared. each process was mapped in detail by direct observation using process modellar app (ipad). the evaluation started at sample centrifugation completion and drop-off in testing area and ended with results sent from analyzer to lis (lab information system). each was evaluated for quality (process steps, biohazardous exposure), speed (operator/analyzer time) and cost (testing/maintenance personnel hours recaptured). time studies were analyzed using minitab v , and statistical significance was assessed using the paired t-test, with p values of < . considered significant. results/finding: detailed process steps, biohazardous exposures, and published analyzer maintenance tasks were evaluated/compared (table, part a) . time studies focused on operator time, analyzer time, and maintenance time (table, part b). regardless of quality or speed metrics evaluated, the ih- demonstrated significant reduction (improvement) in process steps and associated times when compared against the galileo neo (p < . ). evaluating the total number of maintenance tasks required annually, as well as the times associated with maintenance performance and downtime, was a significantly reduced on the ih- (difference of hours/year). conclusion: this study verified the ih- provided significant efficiencies and cost avoidance over the galileo neo for high volume/high throughput testing facilities. workflow impact of automated result verification for patient and donor blood typing barbara j bachman* , candace williams , carmen meyer , paul lamonby , anne cleverley and silke milbradt-pohan . bio-rad laboratories, diamed gmbh background/case studies: immunohematology facilities face many challenges including standardization, process control, productivity, staffing and patient safety. to alleviate these challenges, the ih- tm instrument and complementary ih-com tm data management system (dms) were designed to provide lean automation to enhance blood testing facility workflow. the purpose of this study was to focus on the lean functionality of automated result verification on the ih- and ih-com dms and determine its impact on workflow. study design/methods: internal and external studies using the ih- with the ih-com dms were performed with patient and donor samples. assays included abo/rh blood grouping and antibody screening (abs) as shown in table a . workflow data was analyzed using process modellar app (ipad). the evaluation focused on post-analytical steps of result verification, evaluating with and without automated result verification to determine the impact on quality (operator touchpoints, visual result review occurrence, result accuracy), and speed (time from result interpretation to lis data transfer). operator touchpoints during the post-analytical phase are only required when doing visual result verification and are software defined. speed metrics were analyzed using minitab v . statistical significance was assessed using the paired t-test, with p values of < . considered significant. results/findings: using automated result verification, only . % out of , samples evaluated for abo/rh testing would require visual verification, resulting in a % reduction in operator touchpoints (p < . ) and a labor saving of minutes ( : hh:mm) for abo/rh testing. for , antibody screens, automatic validation of results would result in . % reduction in operator touchpoints (p < . ) and a labor savings of minutes ( : hh:mm). no false positive or false negative typing results or false negative screenings occurred with results auto-verification. (rbc) has remained, and in fact is proportionally increasing while blood usage has notably declined in the era of patient blood management. over the past years a steady increase in demand for o neg rbc compared to other blood types has been observed at our blood center. utilization metrics for hospital customers are monitored monthly for overall trending and forecasting and the data shared with them during regular visits. despite heightening awareness, percent o neg rbc sales continued to rise by % annually and peaked at % in mid . to better understand this increased demand a survey was conducted to gather insight for improved utilization. we speculated that during the survey an observer effect, or change in the staff behavior, would result in reduction of o neg rbc sales. study design/methods: a tie tag was designed as a survey tool and attached to each o neg rbc distributed to hospital customers for an week period in late . hospital transfusion service staff were asked to record the final disposition of the o neg rbc (transfused, wasted, returned) on the tie tag. information on the survey objective and instructions for tie tag completion were communicated via customer meetings, emails and reminders sent by blood center drivers. completed tags were returned to the blood center. customers are allowed to return rbc units with greater than day shelf life remaining. units with tie tags attached were in hospital inventories for up to months due to the shelf life of rbc. return rates and percent of net sales (gross sales minus returns) by abo/rh type were tracked monthly before, during and after the survey. results/findings: participation was % of the hospitals surveyed. mean percent o neg rbc gross sales for a month period before, during, and after the survey was . %, . % and . %, respectively. mean percent o neg net sales during the -month survey fell to . % compared to an average of . % in the months prior. during the -month survey period o neg rbc monthly return rate increased to an average of . % compared to an average of . % in the months prior. for the months after the survey the average o neg rbc return rate further increased to . % while mean percent o neg rbc net sales trended slightly upward to . %. when customer hospitals were queried whether any process changes occurred, no major changes to policy or inventory levels were reported. conclusion: during and after the survey percent o neg rbc gross sales was fairly constant indicating that target inventory levels and transfusion service staff ordering practices remained unchanged. however, during the same period the increase in o neg rbc return rate and corresponding decline in percent net sales suggests improved o neg rbc utilization. increased awareness from participating in the survey and staff knowing they were being observed likely played a role in the lowering of percent o neg rbc net sales. tracking of monthly metrics will provide ongoing review to determine if the effect is transient or sustained and identify other opportunities for improving o neg rbc utilization. acoustophoretic separation of platelets from whole blood: a relevant and practical alternative to centrifugation pierre bohec* , jeremie gachelin , veronique ollivier , thibaut mutin , xavier telot , benoit ho tin noe and sandra sanfilippo . aenitis technologies, hôpital bichat, inserm u background/case studies: shear-induced platelet activation is an unwanted side effect of the centrifugation-based procedure currently used in blood banks to prepare platelet concentrates. transfusion of partly activated platelets could indeed increase the risk of adverse transfusion reactions. aims: here we evaluated the effectiveness of an innovative acoustic-based fractionation device by carrying out a qualitative and functional in vivo analysis of isolated human platelets. study design/method: whole blood was obtained from donors and fractionated using an acoustic-based device. platelet recovery and purity were determined by quantifying blood cell subpopulations in the microchannel outlet samples. quality of isolated platelets was evaluated using the surface expression of two activation markers (p-selectin, pac ) using flow cytometric methods while their procoagulant ability was investigated using in vivo experimentation. platelets isolated using a soft-spin protocol, were used as inactivated control. results/finding: fractionation using the acoustic-based device led to a red blood cell clearance ratio from whole blood greater than % (p< . ) and a purity of platelets close to . %. we did not find any difference in terms of quality and functionality of platelets from the same donors isolated using the acoustic device versus the soft-spin protocol. conclusion: this acoustic-based blood processing method led to excellent preservation of platelet quality and functionality providing a novel promising technique for whole blood fractionation in clinical settings. automation in blood bank processing: where we go? robert fernandez, lluis puig, pilar ortiz, joan ovejo, nuria martinez, elena valdivia and susana g gomez*. banc de sang i teixits background/case studies: nowadays, blood banking is requiring new strategies to manufacture blood components, due to the increase on their production. at banc de sang i teixits (bst), we have implemented during the last years automation manufacturing, including lean management methods, to be able to process our needs of over . blood donations for an area with more than million people. study design/method: the automation of blood donations process, bst has done different changes on the equipment. in , orbisac (terumo bct) was the equipment to obtain buffy coats and from this product, we got platelets concentrates. it was in , when we moved from this equipment to atreus c (terumo bct), to get red blood cells, buffy coat and fresh frozen plasma. then we did some updated on atreus; in we changed to atreus c (terumo bct) and finally in , we moved to reveos system (terumo bct). since the changes in , our blood components were red cell concentrate, plasma, platelets and a leukocyte residue. while all these changes in processing equipment, we added also some automation in our registration (donation id, weight and temperature) and labeling steps, implementing two homemade robots. and finally, to get better results and more efficacy in our production, in , bst incorporated an engineer to introduce lean manufacturing methods. these methods are based on the identification and analysis of problems, and then chose all these activates that add some value to the procedure. results/finding: once all these changes have been updated, we have evaluated the quality of blood components, such as red cells and platelets, also the number of donations that we were missing and working hours that were necessary to process our blood donations. this evaluation was done for processes during and . conclusion: with these results, it's obvious that automation in blood banking makes more efficient the manufacturing of blood components, getting better quality of them and also in a cheaper way. we encourage maintaining lean philosophy in order to keep improving our methods and identifying those activities that add value to our processes and get rid of those ones that are not necessary. in a globalized and industrialized world, where everything changes very fast, these improvements are necessary to be on top of the field and be a state of the art blood bank. background/case studies: the laboratory envisioned an automated blood product delivery system that extended blood access to the bedside through the use of remote blood allocation devices, or "smart blood refrigerators" to improve patient safety, provide timely access to blood products, and potentially reduce laboratory workload. as part of this initiative, bloodtrack haemobanks (hb) (haemonetics, braintree, ma) were installed and interfaced to the existing safetrace tx (haemonetics, braintree, ma) laboratory information system. one hb was installed in the methodist hospital (rmh) campus which includes a busy outpatient infusion therapy center (itc). study design/method: an assessment of the current blood supply chain revealed improvement opportunities for both nursing and blood bank staff. frequent daily trips to and from the blood bank take nurses away from the patient beside and can create congestion at the blood bank window during peak times. for the itc, with a daily outpatient volume of - patients and an average, round-trip travel time of approximately -minutes, even small delays waiting in line at the blood bank window would produce profound ripple effects. itc nurses faced the additional challenge of maintaining nurse-to-patient ratios and providing timely patient care. about % of patients in the itc have same-day transfusion orders, adding to the blood bank workload and creating unpredictability in workflow. often for patients in the itc, nurses had to repeat pre-transfusion vital signs because too much time had elapsed between gathering vitals and obtaining the blood. these inefficiencies resulted in longer patient wait times and, ultimately, a longer stay in the itc. results/finding: hb devices allow nursing staff to access red blood cells (rbc) for the majority of their patients at the point of care. since implementing in november , the hb has significantly improved the turnaround time of rbc issue -from -minutes to less than -seconds-and helped maintain nurse to patient ratios and reduced traffic at the blood bank issue window. prior to hb implementation, blood bank staff at rmh were issuing approximately rbc per month out of the window for non-surgical patients. this has been reduced to approximately rbc per month, a % average monthly reduction. conclusion: having the hb located in the itc has helped to expedite the care of patients and more easily manage blood products for patients with same-day orders. the use of hb devices has not resulted in a reduction in blood bank fte, but rather a shift in workload; from issuing products to monitoring inventory and restocking. consists of registered paramedics that are pararescue specialists and helicopter personnel. when in combat, the squadron conducts personnel recovery operations and rescues downed airmen. when stationed in the us, they mitigate in state emergencies and perform aeromedical evacuations. in , they supported a civilian medical emergency and the patient needed a transfusion in the field. they procured blood products from a distant air force base with adjacent medical facility. at the debriefing, members of the st rescue squadron ( rqs) decided to find a local civilian blood supplier. the master sergeant contacted our blood center and set up a contract for blood supply. study design/method: blood center representatives met with the rqs master sergeant in january . we asked what rqs's order and delivery expectations were. he said sporadic use and the blood order would be rbcs. we wrote a procedure for consignment and packaging, using standard blood transport boxes. we developed a communication template for staff to anticipate the rqs needs. staff was trained based on data from january meeting. we contacted the rqs in september to perform a trial run. at that time, we learned the master sergeant was shipped out to military theater. we invited his replacement to the blood center. this pararescue senior airman had just returned from syria and was assigned to civilian duty. he had no prior knowledge of the rqs association with a civilian blood center. based on his field experiences, he changed the blood order from to rbcs. he introduced blood transport containers, used in military operations, saying they were easier to carry during water and land pararescue missions. we rewrote the procedures, incorporated his transport containers, and made a pictorial job aid to assist staff on packaging blood using these containers. the blood center and rqs performed a mock run on october , and we felt prepared for any future events. results/finding: on november , , the rqs was deployed to a civilian aeromedical evacuation. we anticipated a rbc order. the actual order was rbcs and ffp. staff was preparing frozen ffp to ship, as was their norm for filling hospital orders. realizing that they could not thaw plasma in flight, we contacted the rqs and offered liquid plasma instead, which they accepted. product was consigned and picked up at : am by the rqs. the patient was transfused in the field and then taken to a nearby hospital. at our joint debriefing on november th , we established a maximum blood order of rbc and liquid plasma, noting future orders may request fewer products, yet meet the preferred rbc; plasma transfusion ratio. conclusion: military personnel are adapted to instantly adjust to an ever changing environment. regulated blood centers are not as adaptable. with clear and comprehensive communication and anticipation on the blood center's part, we now supply civilian blood products to the air national guard. (table ). the highest mean fib concentration was mg/donor unit; lowest mean fib concentration was mg/donor unit. all sites had a mean fib concentration at least mg/donor unit above the fda minimum requirement of mg/donor unit. fifteen of blood centers completed the manufacturing process survey. one used a leukocyte reduction filter with ahf destined plasma. all blood centers manufactured single donor cryoprecipitate; manufactured pooled donor cryoprecipitate. most froze plasma in a - c or colder blood bank freezer. one froze plasma using dry ice, and one used a blast freezer. two blood centers method of thawing frozen plasma took longer than hours. conclusion: blood centers consistently met the overall fib minimum requirement with a mean of mg/donor unit, over double the fda requirement. however there is variability in fib levels amongst blood centers. in general, manufacturing processes were similar with a few exceptions. blood centers should inform their hospital customers of their average fib level in cryoprecipitate in order to most appropriately care for patients receiving this product. compliance & productivity improvement via engineered-staffing/ scheduling calculator application (app) mary deck, mark angelelli and kevin lee*. american red cross background/case studies: the healthcare industry, particularly the blood banking industry continues to experience tremendous pressures not only with ensuring patient safety and quality daily, but managing and maintaining an efficient operation with a cost competitive structure. applications of basic industrial engineering tools, coupled with lean-six sigma techniques such as time study analysis, bottleneck elimination & process standardization to transfusion reduce variation has been transformed into an application (for short "app"), which can be utilized to determine process and staffing optimization and provide flexibility to the dynamic nature of changing needs in blood banking. study design/methods: a time study analysis offers valuable data about the process requirements. once this baseline has been established, translating the data into a user-friendly app would enable ease and practical use to facilitate business decision-making as well as effectively manage daily operations. important concepts such as lean-pull production system, bottleneck elimination, work-load balancing together with basic development of the app using ms excel software will be demonstrated. results/findings: successful rollouts and implementations of the staffing/ scheduling calculator app across pilot facilities, then onto facilities nationwide, has yielded improved productivity together with a sustainable compliance scorecard. the app interactive-based approach, programmed via a commonly used software, ms excel, was used to analyze how to optimize staffing requirements together with staff-scheduling (i.e. match incoming volume/work content to staffing availability). the staffing/scheduling calculator app has been utilized by executives to evaluate "what-if" scenarios (sensitivity analysis) as well as a planning toolkit to proactively manage the changing demands of blood banking. conclusion: besides providing a key mechanism for increased productivity and sustained compliance -a top priority for blood banks -the staffing/ scheduling calculator app will highlight continuous improvement opportunities and spring-board to system-wide acceptance and standardization. all coolers were prepared in a walk-in refrigerator. two scoops of wet ice or two ice packs were placed at the bottom of large/medium or small coolers, respectively, with rbc units on top of the ice. a quality-controlled thermometer was placed on top of the rbc units. a control thermometer was place at the interface between the ice and the rbc units in one large and one medium cooler. the start temperature was recorded and then the temperature was recorded every minutes for a hour period or until the temperature exceeded c. results/finding: the temperature recorded from the thermometer on top of the units in all five coolers reached > c in minutes as shown in table . the control thermometer recorded temperatures maintained at - c for the entire hour observation period in both the large and medium cooler. conclusion: when units are placed on top of the ice in a cooler, the temperature is not reliably maintained at - c for more than minutes. these data support a policy of wasting units that are returned to the blood bank with rbc units on top of the ice. background/case studies: an fda draft guidance has highlighted the need to reduce the risk of bacterial contamination of platelet components (pc) via pathogen reduction (pr) or secondary rapid testing (rt). hospitals must understand the cost implications that may result. our objective was to create an interactive model to analyze the budget impact for different pc types across the range of existing us hospitals. study design/methods: an excel model was built and populated with base case costs and probabilities identified through literature search as well as through a survey administered to us hospital transfusion service directors. the model was reviewed and refined by a panel of seven transfusion medicine physicians. the model allows base-case assumptions to be overwritten with values specific to the institution. three scenarios were generated to compare annual costs of plt acquisition, testing, wastage, dispensing /transfusion, adverse events (ae), shelflife, and reimbursement for a hospital that purchases all of its pcs: % conventional (c-pc), % pr-pc, and mix of % c-pc/ % pr-pc. the model predicts a modest ($ %) cost increase for pr-pc compared to c-pc depending on the degree of pr conversion; this takes into account cost offsets such as elimination of bd and irradiation, decreased waste due to increased shelf-life, and outpatient reimbursement. the effective pc shelf-life is potentially increased with pr due to elimination of bd, and is dependent on nat turnaround time. benefits not captured by the model include transfusion-transmitted infectious risk mitigation from emerging pathogens, which may impact cost/benefit analyses. future iterations of this model will also enable hospitals to consider scenarios in which rt is used. this model can serve as an important tool for hospitals considering pr adoption. in january . a report was created to identify donors previously classified as rare according to the american rare donor program (ardp) criteria. donors are classified as rare by meeting one of the following: highprevalence antigen negative, multiple common antigen negative, or iga deficient. the new process utilized the report and involved sending a letter to the donors notifying them of their rare donor status and encouraging them to continue to donate. a database was created to track the letters sent to rare donors. in august , inventory reduction efforts were implemented to gradually decrease the number of allogeneic red blood cells (rbc) collected to minimize unit age at transfusion. the inventory reduction occurred in phases and was completed by january . a study was performed to determine the impact of the inventory reduction on the number of rare donor donations. study design/methods: the total number of allogeneic rbc donations, rare donor donations, and number of rare donor letters sent was analyzed from to (see table) . the percentage of rare donor donations per year was calculated. background/case studies: blood centers (clients) often carry low inventory of blood and blood components. laboratories performing donor screening therefore, have limited time to determine the presence or absence of infectious disease within these products. in order to measure and ensure expedited donor screening we implemented a daily performance metric consisting of upload time goals for release of results to clients. in , zkv-nat testing was implement for travel deferral donors (july), followed by universal individual donor screening in september and november in response to the fda recommendations for "reducing the risk of zkv transmission by blood and blood components". per the fda guidance we implemented mandatory zkv testing for clients with proximity to areas with locally acquired mosquito-borne cases of zkv within weeks (sept. phase ) and nationwide within weeks (nov. phase ). zkv testing is performed on individual samples, unlike all other nat tests that are performed in minipools ( -donations). therefore zkv testing has a disproportionate impact on the turnaround times for testing, which we analyzed in this study. study design/method: within two regional testing labs, participating in the same clinical trial, lab had % and lab had % of clients requiring universal zkv testing. we evaluated a -month test result upload performance period to determine the impact of zkv test implementation. results/finding: during , lab upload time performance ranged from % to . % from january to july; upload time performance fell between august through november, returning to . % performance in december. lab upload time performance ranged from . % to . % january to august. performance fell september through december . % - . %. lab experienced a low of % upload time performance during phase when there was a rapid implementation; % clients required zkv nat. improved performance was observed during phase , with a % increase in zkv clients. for lab : phase experienced a modest decline of upload performance ranging from . % to . % with . % of clients implementing zkv nat. performance was . % in phase , when an additional . % of clients implemented zkv testing. conclusion: with an unprecedented rapid implementation of zkv testing our laboratories experienced a short period of reduced ability to maintain our upload time performance metric. enhanced platelet bacterial screening in an eight-hospital system robin larson* and colleen a. aronson . advocate lutheran general hospital, acl laboratories/ advocate hospitals background/case studies: in response to two platelet-related septic transfusion reactions and the draft fda guidance released in march regarding bacterial risk control strategies for transfusion services, an eight-hospital system implemented the verax pgd enhanced platelet bacterial screening test in of the hospital transfusion services. the sites that did not implement the test arranged for fresh platelets to be rotated in from the blood supplier. the sites which implemented the verax pgd test perform testing on all day and day platelets to be issued for transfusion. this abstract summarizes the data collected for the first weeks of testing. study design/methods: platelet bacterial testing logs were reviewed over the entire time period studied for platelets tested on day , day , and those that were tested twice. inventory reports were reviewed for platelets issued on day or day that did not require testing, and for the total number of platelets issued over the time period studied. results/findings: in the month of february ( week of performing the test), . % of all platelets issued by the participating transfusion services were day or day platelets. in march that number dropped to . %. it is expected that this number will level off at some percentage at or below . % with further data collection. in february . % of platelets were tested twice prior to final issue from the transfusion services. in march conclusion: the percent of platelets issued fresh (day or day ) will likely level off at some number at or below . % due to inventory management from both the blood supplier and the individual transfusion services. testing platelets twice is undesirable. ideally, no platelets would be tested twice as this represents a high cost for both the test reagents as well as the staff time to complete the testing. in addition, of the sites performing testing are level trauma centers and need to have tested platelets available at all times. this will require some amount of double testing, but the goal is to have this number be as low as possible, so that the percent of tested vs issued platelets does not exceed %. as the transfusion service staff becomes more comfortable with judging inventory levels and performing testing, it is expected that the amount of double testing will decrease. background/case studies: in order to make up for the deficiency of the apheresis platelets in clinical application, and also to improve the comprehensive utilization of blood, we investigate the feasibility of preparation of pooled platelet concentrates(pcs) for providing a reliable source for clinical application. to speed up the storage research of pooled pcs in china, we evaluate the changes in platelet function after filtering leukocytes with leukocytes filter for pcs and the quality changes during storage in pvc-bthc blood bags. study design/method: pcs were prepared from ml virus free whole blood by platelet-rich plasma (prp) method. five or six bags of abomatched pcs were pooled and filtered with leukocytes filter for pcs(n ). the swirling phenomenon, ph, automatic blood count, platelet aggregation, hypotonic shock response (hsr), the extent of shape change(esc), cd p expression, atp level in platelet, glucose and lactate concentration were detected before and after filtering, and on days , , and of storage, respectively. results/finding: the platelet recovery ratio of a therapeutic dose of pooled platelet concentrates after filtering leukocytes was ( . . )%, relative change rate of hsr was ( . . )%, the residual leukocytes were ( . . ) . the ph, hsr, and the cd p expression of pooled platelet concentrates before and after filtering were ( . . ) vs ( . . ), ( . . )% vs ( . . )% and ( . . ) % vs ( . . )%. there is significant change for wbc after filtering (p< . ). during storage in pvc-bthc blood bags, the biochemical parameters of pooled platelet concentrates changed with increasing storage time, as shown in table . conclusion: storage in pvc-bthc blood bags for five days, the quality of pooled pcs met the requirements of chinese standards (gb - ) . it can be a complementary source for apheresis platelets supplement in china. evaluation of samplokv r segment sampler to obtain and measure samples from blood component tubing segments abbejane blair*. ajblair laboratory consulting background/case studies: current methods used to obtain samples from blood component tubing segments are cumbersome and present a significant risk for exposure to biohazards, sharps injury and cross contamination. itl biomedical has developed samplokv r segment sampler (ss), a device for obtaining measured samples from sealed tubing segments that is less cumbersome and offers improved safety, eliminating the need to manually cut and squeeze tubing segments. ss was evaluated with the goals of reducing the number of steps required to obtain a measured sample, and, reduce biohazards and sharps exposure. study design/method: ss obtains fluid samples from sealed tubing segments into a needleless syringe. it consists of two chambers with recessed internal needles located at the top of the device and a female port located at the bottom of the device. a needleless syringe is attached to the female port, the sealed tubing ends are then aligned with the ss chambers and, gently pushed onto the needles to pierce each end of the segment. the sample from the segment is then withdrawn into the syringe. the study was performed at rhode island blood center (providence, ri) using tubing segments from three bag manufacturers to demonstrate ease of use on the following processes: segment alignment over needles and piercing, ability to draw sample into syringe, ability to expel air bubbles from syringe, fluid leaks, ease of transfer of sample from syringe to tube and to collect user feedback. two lengths of tubing segments were filled to contain sample volumes of ml and ml. two users then evaluated the ss tubing segment types with ml or ml samples for a total of data points. samples were collected into the attached ml or ml syringe then a measured sample was transferred from the syringe into a test tube or microcentrifuge tube. results were tabulated as pass or fail. results/finding: a total of ss were evaluated by two users. all samples were successfully collected and transferred into tubes. insertion of the segment edge requires observation to ensure placement onto the needles. any air bubbles collected into the syringe could easily be moved to the top by background/case studies: the management of platelet inventory is crucial due to a number of factors including the day product outdate, the allocation of staff due to the lengthy donation process, the increasingly small donor pool, and the high cost of production (e.g. platelet collection kits, testing, product processing). the use of a platelet inventory management tool has the potential to enhance the understanding of units transfused, optimize inventory, increase efficiency, and reduce waste. the objectives of this assessment were to decipher if the platelet inventory management tool has reduced the amount of outdated platelet products, total cost of platelet production, and full time equivalent (fte) allocation. study design/method: in january , a platelet inventory management process was implemented which uses a spreadsheet based tool to predict the amount of platelet collection procedures needed to be scheduled each day. the tool uses daily historical transfusion data from the last five weeks. additional calculations are included to account for deferrals, no shows, incomplete collections, and product split rate. the number generated from the calculations correlates to how many platelet collection procedures to schedule for the specific day of the week considering testing release and historical daily transfusion trends. the effectiveness of the tool was verified by comparing platelet collections, platelet products outdated, and fte information for a one year period prior to the implementation of platelet inventory management to one year period following implementation. results/finding: by implementing a platelet inventory management tool, collections have been lowered or shifted to accommodate the transfusion needs. the staffing adjustments and targeted collections have lowered fte and outdate cost by %. the platelet outdate rates dropped after implementing the platelet inventory tool from % ( units) to % ( units); a % decrease. fte was able to be monitored closely with the donor schedule and lowered from a yearly average of fte to . fte, lowering fte by %. conclusion: considering historical transfusion data for potential platelet demand has had a positive impact on scheduling platelet collections. staffing requirements and outdating products have decreased since implementation of the platelet management spreadsheet tool, leading to less waste both in terms of staffing and platelets. given these positive results, we are beginning to develop a similar tool for our whole blood collections. identifying opportunities to right-size hospital inventory using compotrace radio frequency id inventory management system nanci fredrich* , jaclyn mckay , jennifer curnes and rowena punzalan , . bloodcenter of wisconsin, children's hospital of wisconsin background/case studies: the ability to track inventory of blood components in real time is challenging for both hospital transfusion services (ts) and blood centers (bc) using current blood bank information systems (bbis). in addition, determining if established par levels of individual components meet or exceed daily transfusion needs is difficult to ascertain. a pilot was designed to track and monitor all blood components from distribution at the bc to issue in a hospital ts using fresenius kabi compotrace radio frequency id (rfid) enabled inventory management system. the objectives were to determine feasibility of the compotrace system and analyze compotrace data for real-time usage and optimal inventory levels. study design/method: a month pilot was conducted at a pediatric hospital and its bc using both bbis and compotrace systems to track all adult-size blood components. staff were trained on use of compotrace system. upon receipt of order from pilot hospital, bc staff applied rfid tags to all component bags and scanned components into the compotrace system. components were transported and delivered to ts following established procedures. upon receipt at the hospital, components were scanned into inventory using both the ts bbis and compotrace systems. dual scanning of components occurred upon issue to or return from floor, component modification or return to bc. products for emergency use or at time of high demand were not rfid-scanned. a priori, the pilot would stop if the compo-trace system hampered current workflow, component issue was delayed or if ts errors increased. no inventory changes were made during the pilot. results/findings: real-time data from compotrace system provided actual usage for all blood components including component disposal and shipment to and from bc. average daily rbc inventory levels and usage for selected blood types is shown in table. lessons learned related to equipment and workflow: ( ) use of smaller irradiation canister may damage rfid tag, which was resolved by relocating tag, ( ) ts workflow and stat orders challenged consistent use of dual processes to track component status. however no increase in ts errors or delay in issue of components occurred. conclusion: use of rfid to track blood components from bc to final disposition is feasible. real-time data from compotrace system identified optimal inventory levels for rbc at the pilot ts. use of real-time rfid to track inventory and adjust target levels based on actual daily usage over time may reveal seasonal influences that affect target inventory. background/case studies: physicians expect blood to be available at all times. following a national appeal in july for donors based on a predicted summer shortage with high likelihood of extending into the fall, our transfusion service (ts) recognized a potentially dire situation given the institution's patient acuity. our hospital-based ts supports a full range of services: a level i trauma service; stem cell and solid organ transplant services; a brisk cardiothoracic surgical program; a high risk obstetrical service; and high acuity medical/surgical services. a regional donor center supplies our blood products. to insure appropriate response to patient needs, the ts created a management plan, with input from multiple stakeholders, to assist with product management in times of extreme shortages. the approach is described herein. study design/method: at the direction of the transfusion committee (tc), ts directors presented the concern for impending shortages to the hospital quality directors (qd) committee. the qd committee consists of clinicians and non-clinicians trained in health care quality/regulatory affairs who are responsible for institutional health care quality (hcq) activities. the qd recommended creation of a multidisciplinary team: "the blood shortage task force (bstf)", analogous to an existing task force started for management of drug shortages. results/finding: with hcq and tc support, the ts created the bstf and blood shortage management algorithm (bsma). standing members of the bstf include ts medical director (chair), senior vice president (svp) of hcq, svps of clinical services director of regulatory affairs, legal counsel, and representatives from ethics, social work, pharmacy, patient referrals, and communications. ad hoc members include those whose patients would be most impacted by the specific shortage. the bsma designed by the bstf provides a framework for ts's to conduct operational and therapeutic assessments of potential impact and defines criteria for convening the bstf. trigger criteria include: marked ts concern; essential product; high likelihood of inventory depletion; broad patient impact. once convened, the bstf is responsible for situational assessment and formulation of a management plan, with a goal of maintaining quality patient care. conclusion: faced with the potential for limited blood supply, the ts reached beyond the laboratory and engaged the tc and members of hcq to assemble a robust, multidisciplinary task force. this resulted in an inclusive plan which can be activated at any time to address shortages, and assist in management of impacted patients. abstract background/case studies: cryoprecipitate (for short "cryo") plays a critical role in clotting and controlling hemorrhaging, and is often used in the treatment of massive trauma and major diseases, including metastasized cancers, cardiac diseases, hepatic failures, and organ transplants. the collection process of cryo is particularly challenging; due to fact to be processed into cryo units, the collected whole blood has to be shipped to the production facility and be processed within -hours after collection. this tight hour time constraint between collection and production can only be satisfied with precision collection planning and extra courier services; which makes the collection for cryo units more costly than other products. study design/methods: the american red cross (arc), in partnership with researchers from the georgia institute of technology (gt), has developed a blood collection model to increase the amount of whole blood that can be processed into cryoprecipitate. after reviewing blood collecting and processing schedules, collection locations, and other factors, arc-cryo subject matter experts together with gt researchers were able to analyze the problem structurally with several analytic/dynamic programming properties, and developed a near optimal solution algorithm or mathematical model. results/findings: to facilitate implementation, a decision support tool (dst) was developed to systematize the selection of the collection sites; determining when and from which mobile collection sites to collect blood for cryo production and how to schedule the courier services such that the collection targets are met and the total collection costs are minimized. the implementation of the dst led to an increase in the number of whole blood units satisfying the tight -hour completion time constraint for cryo production (capacity expansion). in particular, during the th -quarter of , a blood processing region was able to process about more cryo units/month (an increase of %) at a slightly lower collection cost (cost avoidance), resulting in an approximately % reduction in the per unit collection cost for cryo. conclusion: by utilizing operations research toolkits, a mathematical model or near-optimal algorithm could be developed to optimize the cryoprecipitate collection process, ensuring the time constraints and product consistency levels are achieved. this interdisciplinary improving cryoprecipitate collections collaborative project has been selected as a finalist on the -the franz edelman award, recognizing outstanding achievements and practices in operations research. inventory management and transfusion practice before and after -day apheresis platelets sarah k harm*. university of vermont medical center background/case studies: the shelf life of apheresis platelet (ap) units stored in plasma may be extended from to days in the usa using an fda cleared rapid test (rt). in august , our hospital based transfusion service began using a rt on day and to routinely extend ap shelf life to days. this report describes changes in platelet inventory management and transfusion practice six months following routine use of -day ap. study design/methods: data were obtained for two study periods: september -february (pre-implementation) and september -february (post-implementation). the study periods were intentionally made to span the same months of the year due to seasonal variability in platelet transfusion rates in our region. the transition period from -day to -day ap inventory was excluded. the following data was collected for each study period: the total number of ap transfusion recipients, ap units transfused, expired ap units, ap units ordered ad-hoc from suppliers, inpatient admissions, surgical volumes, and average length of stay. results/findings: data are shown in the table. the number of ap transfusions decreased by % post-implementation while inpatient admissions and surgical volume increased by % and %, respectively. the hospital length of stay was similar for both periods. ap inventory decreased by % post-implementation and the outdate rate decreased from % to % (p< . ). ad-hoc ordering was not statistically different between study periods (p . ). the average number of ap transfusions per patient between pre-and post-implementation periods was not statistically different ( . and . , respectively, p . ). furthermore, a new "rejection threshold" for lipaemic products will be implemented. this threshold represents the tg concentration above which viral marker testing for donor screening will be affected. in kcbb abbott's prism assays are used for: hbsag, anti-hcv ab, anti-hiv ab, anti-htlvi/ii . results/finding: using data management system and file records in kcbb as regard discarding blood components due to lipaemia during the last five years ( ) ( ) ( ) ( ) ( ) , it was demonstrated that number of discarded rbcs due to lipaemia during the whole period was units. number of discarded different plasma, platelets, and cryoprecipitate components during the last two years due to lipaemia was , , and units respectively. the mean number of discarded rbc units of the five years of the study exceeds % of the tested ones. literature about guidelines on the management of lipaemic donations were reviewed in order to minimize donation loss, and establish an accurate rejection threshold for lipaemic donations. by reviewing sample requirements for viral marker testing in kcbb, the accepted level for tg in blood samples is below mg/dl, and so the rejection threshold for lipaemia is level equal to or more than mg/dl. conclusion: many blood product units are discarded needlessly in kcbb due to lipaemia in the last five years (including rbcs, plasma products and apheresis platelet units). in an effort to reduce the waste of potentially lifesaving products, the rejection threshold for lipaemic products is recommended to be changed from mg/dl to mg/dl which does not affect blood safety. a follow up study is recommended after applying the new threshold to evaluate the new policy. logistical management of the incorporation of pathogen reduced single donor platelets (pr-sdp) into inventory at a u.s. tertiary care medical center eric gehrie* , , rebecca ross , debra mraz , anne baker , zenna neal , melanie champion and edward l. snyder , . johns hopkins university school of medicine, yale university, yale-new haven hospital background/case studies: the approval of pr-sdp by the fda provided an opportunity to improve the safety of our platelet inventory across all patient demographics. we outline our approach and address issues we faced during the first months of pr-sdp availability. study design/methods: our nursing education team provided presentations to the nursing and clinical unit support staff. a company-sponsored trainer staffed sessions for the evening/night shifts on the clinical wards. presentations to physicians were made by the blood bank medical staff. information technology personnel created a new product type in the blood bank computer system, tested the abo/rh truth tables, and ensured that billing codes were in place. the necessity for transiently supporting a dual inventory of pr-sdp and conventional platelets led to consultation with the ethics committee and risk management, to confirm that pr-sdp and conventional platelets (c-plts) tested for bacteria ("safety measure" testing) could both be considered the hospital standard of care. we chose to not gamma irradiate any unit of pr-sdp, consistent with the package insert. results/findings: the ethics committee and risk management confirmed that informed consent was not needed for transfusion of pr-sdp. pr-sdp available from our blood supplier incremented monthly. over the first four months of pr-sdp availability, pr-sdp were transfused at our hospital (out of a total of platelets transfused). after months of scale-up, pr-sdp were approximately % of inventory. questions received during the nursing and medical conferences related to: the risk of bacterial contamination with c-plts vs. pr-sdp; toxicology of the pr process; scanning pr-sdp labels into the electronic medical record; and the need to irradiate pr-sdp. our use of a "safety measure" addressed concern over bacterial contamination of c-plts. published pr-sdp toxicology data comparing the content of psoralens in food products such as grapefruit ($ mg per g) to the content in pr-sdp (< ng per ml) addressed toxicology concerns. nursing/it allayed concern over scanning issues with a simple demonstration. finally, we ensured that all parties were aware that fda did not require irradiation of pr-sdp. presentations at the medical conferences were also used as an opportunity to provide transfusion-transmitted disease training and information on platelet utilization. company personnel did not present at medical or nursing conferences per institutional policy. no background/case studies: ensuring platelet supply capability represents a challenge in terms of donor recruitment and inventory management operations. in september , the apheresis collection process (acp) was completely revised to increase the number of products per donation by maximizing the rate of double-platelet donations (dpd). the process review has led to several changes, including the substitution of the pre-donation platelet (plt) count measurement before donation type allocation, in favor of the use of the donor's past donation records. multiple processing steps were eliminated, and the evaluation of plt concentration as a function of time, deduced from complete blood count (cbc) measurements, allowed the centralization of the analysis at the qc department. finally, introducing the concept of non-optimal donations has led to an increase in the proportion of dpd. study design/method: at the donation centers, whole blood (wb) from donors was collected in k edta tubes. plt concentrations were determined at the qc department using the coulter act diff hematology analyzer (beckman coulter). sample tubes were stored at - c and measured at , and hours post-collection. single platelet donations (spd) or dpd were collected using the trima accel. units were pooled and split in elp (extended life platelet, terumo bct) storage bags to mimic spd ( ml; n ) or dpd units ( ml; n ). plt pools were stored at - c under mild agitation for seven days except for dpd, which were split in two -ml bags after h. samples were taken on days and . ph, po and pco , hypotonic shock response (hsr), extent of shape change (esc), cd p expression, atp content, lactate and glucose concentrations were used as in vitroquality markers. results/finding: plt concentration as a function of time, determined from wb cbc measurements, showed no significant difference at h ( pltx /l), h ( pltx /l) and h ( pltx /l) postdonation. dpd can be stored in the same collection bag for h after donation without any significant impact on plt quality markers. plt concentrations were within the manufacturer's acceptable limits ( - pltx / l) before splitting. on day , lactate and pco concentrations increased, and po decreased in dpd. however, these values normalize to those of control units at the expiration day. conclusion: this project was approved by health canada and implemented in our organization in march . there are numerous operational and cost benefits from this process optimization initiative, without significant impact on safety and quality. post-implantation efficiency data will be compared to the targeted % increase in the targeted number of plt units per donation ratio. phased implementation of pathogen-reduced platelets in a health system elizabeth s. allen* , colleen vincent and patricia kopko . university of california -san diego, american red cross background/case studies: pathogen reduced platelets (prp) provide improved safety compared to conventional apheresis platelets, but collection and manufacturing are complex. early evidence shows only - % of double platelet collections meet requirements for pathogen reduction treatment. blood centers need hospitals to implement prp to start manufacturing, but hospitals may not wish to use prp until they can provide the product to all patients. scaling up manufacturing at the blood center and phasing in prp across patient populations meets both parties' needs. we evaluated this strategy at our university health system (transfusion volume: , apheresis platelets annually), which includes two hospitals ( inpatient beds) and an outpatient cancer center. study design/method: before initiation, approval and funding were obtained from the hospital quality council and administration, and stakeholder groups such as hematology/oncology were educated and consensus gained. live training was provided for nurses in the outpatient cancer center (week ) and the bone marrow transplant (bmt) ward (week ). an e-mail communication explained the change to all physicians and nurses. in phase , we implemented prp in the outpatient cancer center. these patients are immunocompromised and do not have access to the immediate advanced critical care of the inpatient environment should a septic reaction occur. in phase , we expanded usage to include the inpatient bmt ward. in phase , we lifted all restrictions so prp could be used throughout the health system, with the goal to reach % prp within months. results/finding: in phase (weeks - ), we requested prp products weekly, based on typical usage in the outpatient cancer center. our blood supplier provided an average of prp weekly (range - ), and prp constituted % of platelet transfusions in the cancer center. in week , excess prp inventory required use of prp in the inpatient bmt ward ahead of schedule, a practice which continued throughout phase . in phase (weeks - ), we formally expanded issuing of prp to include the inpatient bmt ward and requested prp products weekly. our blood supplier provided an average of prp weekly (range - ), and prp constituted % of platelet transfusions in the phased-in areas. in phase (weeks - ), we began issuing prp throughout the health system. our supplier provided an average of prp weekly (range - ), and prp constituted % of all platelet transfusions. scaling-up is ongoing. conclusion: phased implementation of prp by patient group prioritizes patients who stand to benefit most from the product, and allows time for the blood center to scale up manufacturing. background/case studies: maintaining adequate inventory of platelets without significant outdating and waste of product is a constant challenge for many institutions, especially for smaller community hospitals. our health system comprises hospitals including smaller community hospitals (sch) and larger tertiary care medical centers (tcmc). for several years, we have been using a limited internal process of platelet sharing between some of our institutions to successfully reduce platelet wastage. this encouraged us to analyze platelet usage throughout our health system and devise an expanded novel concept of platelet distribution, in partnership with our blood supplier that would allow us to maintain an inventory of apheresis platelet (ap) units at our smaller community hospitals without significantly increasing platelet waste and the associated cost. study design/methods: a "round robin" (rr) transportation system for platelet delivery and pick up was strategically developed with the regional blood center to align with routine delivery of red blood cell (rbc) standing orders. an efficient delivery system was implemented so that the regional blood center would realize reduced supplemental and emergency deliveries of blood components to our hospitals. platelets are transferred at the time of rbc standing order delivery based on a predetermined route schedule. each day, ap are delivered to the sch and the previous day's platelets retrieved (if not transfused), packed in blood center transport boxes, and then picked up by the blood center driver. these platelets typically have a hour shelf life remaining. the same process occurs at the next sch on the route. all retrieved platelets from the sch are delivered to the tcmc which is the last stop on the route. thus, the sch has adequate number of units available for regular transfusion and massive transfusion protocol. results/findings: review of our rr process revealed a significant benefit to our smaller community hospitals as we were able to routinely maintain an ap inventory for patients requiring urgent platelet transfusion. an additional benefit was further decrease in ap waste (table ) resulting in a cost savings of $ k. an additional cost savings of approximately $ k was noted due to decreased cost of emergent platelet transportation. conclusion: our novel rr process of platelet distribution has resulted in improved platelet availability at our smaller community hospitals while maintaining the reduced level of ap waste at our health system from our previous platelet sharing process. we anticipate additional decreases in ap waste as a transfusion vol. supplement s we further streamline our process. with the trending merge of health delivery systems, we predict that other health systems will adopt similar processes to improve platelet availability and reduce waste. post implementation adjustments of our pathogen reduction process jacqueline carlson* , james r stubbs , scott a hammel and manish gandhi . mayo clinic, mayo clinic-rochester background/case studies: the implementation of pathogen reduction for apheresis platelets using cerusv r intercept system for apheresis platelets was a substantial endeavor encompassing many different areas. as with any process change, adjustments and modifications can occur along the way. after implementing % pathogen reduction technology (prt) for apheresis platelets, we made two additional adjustments to our sampling processes to ensure accurate labeling/categorization/branding of our final products. study design/method: our prt validation consisted of apheresis platelet products. each product was tested pre-processing for white blood cell (wbc) content and platelet yield, along with post processing platelet yield. this data was used to calculate our yield and volume retention during processing. we anticipated products with preprocessing yields of . , . , and . x may end up below a . in the final storage bag and would need a post-processing sample to ensure the product met criteria at ! . x platelets. results/finding: during the validation, we discovered one collection was not leukoreduced and two collections started at a . yield but ended with a yield below . . these two discoveries led to adjustments in our prt platelet process. with the wbc failure, we reviewed the wbc count on the sysmex xe- d preprocessing report to see if it would alert us to a potential wbc failure. the review discovered that of results were . or . x / mcl with the exception being the wbc failure with a count of . . further monitoring of the wbc counts discovered a result of . which was tested on the adam r-wbc for wbc count and determined to not be leukoreduced. we decided all sysmex wbc results from the pre-processing sysmex report would be reviewed prior to processing and a wbc result of . will be tested on the adam to confirm a leukoreduced product. we also discovered of ( %) of the . preprocessing yields products ended with a post processing yield < . . we decided to increase the yields requiring post processing samples to include the . . conclusion: we are continuing to sample all collections for a post processing yield so we can be confident that we are releasing products into inventory with a yield of ! . x platelets and to have enough data to accurately determine our volume and yield loss during processing background/case studies: the university of kentucky medical center (ukmc), a large academic hospital with level i trauma center, is supplied with blood products by the kentucky blood center (kbc) on a consignment agreement-based contract. ukmc is kbc's largest consumer of blood products. as platelet usage can vary widely day to day platelet usage projections are provided to kbc by the ukmc blood bank, thereby allowing kbc to act accordingly with a given day's stock (i.e. import vs export). daily platelet projections are based on phone calls asking clinicians working in high-demand locations to estimate their needs. this process can be easily confounded by multiple factors and has undergone multiple adjustments to improve its accuracy. study design/method: daily platelet projection forms from / - / were retrospectively reviewed and compared to actual usage data over that same time. the prediction system used in the ukmc bb up to that time (estimated clinical need ) was evaluated for effectiveness based on: total number of days under-predicted, number of days with large underprediction, average number of units under-predicted, and average difference between prediction and usage. the prediction system was subsequently changed based on this data in ; the revised prediction method (estimated clinical need ) was then evaluated retrospectively using the same data sources covering / - / and then compared to the prior method. results/finding: the average number of platelets transfused from / - / was . u/d with a standard deviation of . u/d; the predicted amount was . u/d. the difference between the predicted amount and the number of units used was - . u/d. % days ( d/month) were under-predicted (average: u/d). % of days ( ) were under-predicted by ! u (average: u; max: u ( x)). the average number of platelets used from / - / was . u/d with a standard deviation of . u/d; the predicted amount was . u/d. the difference between the predicted and units used was a . u/d. % days ( d/ month) were under-predicted (average: . u/d). one day ( %) over this period was under-predicted ! u ( u). conclusion: review of clinical platelet usage over this time identified a relatively stable average daily clinical demand. adjustment of our prediction system to ensure that no, or as few as possible, days were projected for less than that average has markedly reduced catastrophic shortages ( % a %), reduced the number of days under-predicted ( % a %), and decreased the discrepancy on those under-predicted days ( . u a . u). these improvements in estimating usage allow for an increased ability to handle unpredictable events without suddenly straining kbc's supply flexibility or severely limiting ukmc clinical settings. rapid implementation of zika virus (zkv) nat blood donor screening joan dunn williams* , maria noedel , nancy haubert , kenneth hudson , larry morgan , robert shaw , tracy fickett , jamie jue , valerie winkelman , sally caglioti , german leparc , and phillip c williamson . background/case studies: on / / , fda issued a guidance document for "reducing the risk of zkv transmission by blood and blood components". in response, a plan was implemented for mandatory zkv testing for all clients with locally acquired mosquito-borne cases of zkv within weeks; nationwide in weeks. this organization performs testing for clients (blood centers, hospitals) across the country. we report on of manufacturers' (sponsor) provided investigational new drug (ind) protocols. a single project management (pmo) system was used to control all required processes. study design/method: project focus included: clinical trial requirements, client onboarding, lab operations (labs). our objectives were to implement zkv testing for clients within weeks, and an additional clients within weeks. to minimize the impact to labs a staggered implementation was used with tracked/streamlined communications from stakeholders: vendors, institutional review board (irb), it (client and lab based), client services and labs. results/finding: clinical trial requirements increased the complexity of implementing an unlicensed test. documents included donor notification, informed consent, protocol training, staff certification, deviation management, and result reporting. multiple irb documents were required. to ensure accuracy in ind commitments a principle investigator was assigned to labs with client sub-investigators. deliverables were multiple including client requirements, vendor responsibilities and labs. client onboarding included confidentiality agreements between client and sponsor. an immediate zkv based webinar provided materials and understanding of sponsor protocol, lab test system, and client/donor based responsibilities. to facilitate and ensure effective communication, twice weekly conference calls were held. clients sent questions which were facilitated by labs and directed to sponsor. specific to clients were irb documents, it updates/validation for zkv test ordering and result receipt. labs were multifaceted: vendor instruments, assay materials, package inserts, staff training. assessments included: zkv sample volume, throughput, instrument capability/capacity. work requirements included vendor installation, equipment, assay and reagent qualifications, staff training, competency assessments, result reporting. all clients were provided with zkv testing within required timeframes. conclusion: the success in meeting a rapid implementation of zkv testing was largely due to a centralized pmo system which provided a controlled process for sponsor, client, vendor and labs. within lessons learned strength was found in a multi-client onboarding process. a weakness was in understanding instrument test volume capacity throughput which was exceeded during the -week period but overcome during the -week cycle. red blood cells baby units traceability and discard in kuwait central blood bank and five hospitals marwa moemen al deeb* , hala samuel boules , fatemah saleh al matroud , rabab hussien ali dashti , hanan alawadhi and reem al radwan . kuwait central blood bank, kuwait central blood bank, kuwait central blood bank background/case studies: ill children are more likely to receive red blood cells (rbcs) transfusion than any other patient age group. rbcs are the component most often transfused during neonatal period. small volume aliquots are used to limit donor exposure, prevent circulation overload and decrease donor related risk. traceability is the ability to trace each individual unit from donor to recipient or disposal. blood component should be fully traceable from collection to final disposition. the kuwait central blood bank (kcbb), is preparing baby units and distributing it to all hospitals all over the country. kcbb, being accredited by the american association of blood banks (aabb), is following the aabb's regulations in tracing every component. study design/method: this is a retrospective study to assess final deposition and the percentage of discard of prepared packed rbcs baby units in the kcbb and five hospital blood banks (hbb). also, to assess the levels of traceability as a reflection of the improvement in the efficient use of these blood products. methods: a total of rbcs baby units were randomly chosen to be traced to their final deposition from the year till . half of them ( units) were traced in kcbb. tables showing the numbers of the chosen units were distributed to the five governmental hbb ( units for each year of the study period). results/finding: preliminary results show that the tracing of rbcs baby units in the kcbb is % efficient. results from other hospitals are under process. statistical analysis of the traceability will be done as soon as the data is collected. the study will analyze the usage of the baby units in different departments and the percentage of discarded units. the traceability of rbcs baby units in the kcbb is excellent, this is due to good management and training of the working staff and the use of an electronic system in registration and issuing. most of the kuwait governmental hospitals are using electronic systems, so the traceability should be up to the recommended levels. the percentage of discard of the baby units in the hospitals is very high. this may be due to the practice of using fresh blood (< days of donation) and the reservation of the baby units of the same donor to the same baby to reduce the hazards of multiple donor exposure. the creation of a national policy for using rbcs baby units is highly recommended to reduce the discard of such units. we also calculated the number of false positive results. the study traced all products through mid-march . results/findings: a total of products were tested. fifteen units ( %) had a false positive result and could not have their life span extended. of the fifteen reactive units, two repeat donors were identified and their charts were marked to not test subsequent donations. cross-reactive antibodies were identified in all by the vendor and none were true positives by re-culture. of the units that were successfully tested, were tested again on day for use on day ( %). there were platelets transfused ( %) and expired after day ( %). the cost to test the products including controls was $ , and our calculated cost to produce products would be $ , . if we had needed to import products to meet needs, the cost would be roughly $ , without shipping costs which are estimated at $ , . . we averaged expired platelet products per month (range - ) before verax testing and (range - ) after implementation. conclusion: using verax point-of-care testing saved platelet products from discard. the cost savings were $ , . from importing and $ , from producing a replacement for those products. the average discard rate per month went from to after verax implementation. extending platelet shelf life to days more than paid for the cost of testing and ensured products were available for patients who needed them. secure text messaging in transfusion medicine: can texting decrease wastage? melanie estrella* and elsie lee. george washington university hospital background/case studies: secure text messaging in hospital settings allows for quick, easy, and hipaa compliant communication between members of patient care teams. it works on a mobile phone or computer, and provides read-receipt confirmation and a temporary record of team communications. secure texting has potential to be a useful management tool in transfusion medicine in reducing blood product wastage. for example, it provides a relatively low-burden means for busy clinicians to provide feedback to the transfusion service about scenarios of potential wastage. this information can be used to identify areas in which management strategies could be developed. it also allows for personalized educational opportunities between clinicians and the blood bank about usage guidelines and how to reduce future wastage. the goal of this study is to use secure texting to investigate wastage, evaluate the responses from clinicians, and evaluate the potential effects on reducing wastage. it is hoped that the results will identify secure texting as a useful management tool in transfusion medicine. study design/method: wastage records that were investigated without the assistance of secure texting from july to december were reviewed to identify the most common scenarios of preventable blood product wastage. wastage records from january to april were reviewed, and wasted products that were considered preventable were investigated using secure texting to communicate with the ordering physician. results/finding: for data, units were investigated without the use of secure texting. of these, units were identified as preventable wastage, and wasted units were considered beyond the control of the clinician. the categories for preventable wastage were defined as follows: ) product not released after procedure/ or when patient stabilized ( ) ) product returned outside of appropriate temperature range ( ) ) clinician unaware product was assigned ( ). thus far in , wastage records have identified units of preventable wastage. secure texting was used by a transfusion service physician to investigate. twelve responses provided useful feedback for future management strategies, responses thanked the transfusion service for the information, and in instances, the message was read with no reply. conclusion: secure text messaging has the potential to improve communication in transfusion medicine. it is easy to use, hipaa compliant, and helps identify strategies for reducing wastage by improving communication and allowing personalized educational opportunities between ordering physicians and the transfusion service. sequence of reagent adding for cryopreservation freezing solution guoling chen*, xu zhao, andrew tiss, sasha turner, devin emerson, manijeh shemirani, sharon novak, david garvin, john eng and wanxing cui. medstar georgetown university hospital background/case studies: dimethyl sulfoxide (dmso), plasmalyte-a (plas-a), human serum albumin (hsa) are widely used to prepare cryopreservation freezing solution. some use autologous plasma instead of plas-a and hsa. this study is to identify the choice of reagents and the optimal sequence of adding these reagents when making freezing solution. study design/methods: materials: . % dmso, plas-a, % hsa, autologous plasma extracted. containers: transfer pack (bag) and polystyrene tubes. the freezing solution recipe used in this study is (volume ratio) . %dmso: plas-a : %hsa : : . plas-a and hsa are kept at room temperature ( - c, rt) and refrigerated at c, plasma at rt (to simulate the end-of-centrifuge temperature), dmso at rt (due to high freezing point . c). different combinations of the reagents choice, storage temperature, adding sequence, are tested with photo taken. total tests. at least minutes cooling after dmso, before adding the next reagent. see table: ( ) after directly adding . % dmso alone to bag, the bag turned from transparent to white, so dmso should not add first. ( ) in tube, autologous plasma first, dmso next, powder-like precipitates. ( ) in tube, dmso first, hsa next, precipitated instantly, a layered appearance. ( ) & ( ) in tube, plas-a first, then hsa, dmso at last, precipitates formed; rt plas-a and hsa combination formed a thicker precipitate than those kept at c. ( )&( ) in tube, hsa first, dmso next: precipitation formed heavily, sculpture shape. precipitation in the c group is slightly milder/slower than rt group. so hsa should not be added first. ( )&( ) trace of hsa(< ml) was mixed into the plas-a bag ( ml). in tube, such "hsa-contaminated" plas-a was added first, then dmso, small fragments of precipitates formed, so dmso should not add last. background/case studies: maintaining a robust blood product supply is an essential requirement to guarantee optimal patient care for all major hospitals. however, daily blood product use is difficult to anticipate. platelet products are the most variable in daily usage, have short shelf lives, and are also one of the more expensive products to produce, test, and store. due to the combination of absolute need, uncertain daily demand, and short shelflife, platelet products are also frequently wasted due to expiration. sophisticated data analysis has the potential to accurately predict hospital wide platelet needs and therefor reduce wastage. study design/method: we have investigated platelet usage patterns at our institution, and specifically interrogated the relationship between platelet usage and aggregated hospital-wide patient data over a recent consecutive -month period. using a convex statistical formulation, we have found that platelet usage is highly dependent on several factors. these include day of week, number of abnormal cbc, location-specific hospital census data, and other less important factors. we exploited this relationship to develop a mathematical model to guide collection and ordering strategy. results/finding: this model minimizes waste due to expiration while never allowing for a shortage; the number of remaining platelet units at the end of any day never drops below in our model. compared with historical expiration rates during the same period, our model reduces the expiration rate from . % to . %. with an annual platelet usage of approximately , units, this reduction equates to approximately units saved from expiration annually. depending on platelet pricing in different regions, this accounts for annual savings between $ , to $ , , per institution. conclusion: to our knowledge our research is the first such use of hospital wide data to inform real-time donor recruitment strategies based on anticipated patient demand. thawed plasma implementation: signficant cost savings and decreased plasma wastage morvarid moayeri* , russell thorsen , rosaline ma , antonio g insigne , amy decourten , florence panganiban , patricia mckean , cyril jacquot , sara bakhtary and ashok nambiar . ucsf health, children's national medical center background/case studies: plasma (ffp, pf , pf rt ) stored at - c outdates hours after thawing. if collected in a functionally closed system, it may be relabeled as thawed plasma (tp), extending expiration to days from the thaw date. although coagulation factor levels decrease over this period, they remain above hemostatic levels. as tp can be safely used for the vast majority of patients requiring coagulation support, we implemented use of tp in our multi-site tertiary care system, with the aim of decreasing costs and minimizing wastage. study design/methods: the massive transfusion protocol at our instiution already allowed the use of group ab tp. following a review of literature and practice at other large centers, the transfusion committee extended the approval of tp to all patients. neonates (< months), patients undergoing plasmapheresis and those with factor deficiency or other disorders for which we also noted a significant decrease (not quantified) in technologist time and effort, as less time was expended on the following: thawing units, printing inventory reports and reporting/record-keeping for discarded units. conclusion: in many large facilities, providers frequently order more plasma units than are ultimately transfused, leading to high plasma wastage rates due to limited ( hr) shelf-life. tp has an extended shelf-life, and can be used interchangeably with ffp and pf for most patients. implementing tp in a multi-site tertiary health care system resulted in sustained decrease in plasma wastage, saving thousands of dollars and helping conserve a precious resource. the merging of immunohematology reference lab's (irl) inventories-using technology to create advanced search functions alexander delk and richard gammon* . oneblood, oneblood, inc. background/case studies: immunohematology reference labs (irls) must maintain diverse inventory of antisera to aid in antibody identification, antigen type rbc units, and meet regulatory requirements. when our current organization was established, two irl sites had independent inventory management systems. although the purpose of maintaining the antisera inventory was the same, organization, storage, & access to instructions for use (ifus) were not. our irl developed a synergistic method to organize and store antisera coupled with in-house designed custom excel spreadsheet to organize and search antisera and view ifus. study design/method: a list of similarities and differences was constructed. best practices of both methods were identified. we determined that our antisera could be broadly classified/organized into two main categories: rare and bulk for screening. sequential lab assigned numbers were given to antiserum for each category: s (rare sera) and b (bulk sera). a dynamic/static freezer box storage system that was inter-box static and intra-box dynamic was determined to be best option to combine two inventories while conserving elements of each allowing for library growth. antisera assigned to a box remained in that box, but may be moved within the box. the box itself may be moved among freezers. to track boxes, location and movement within the box, a custom excel spreadsheet was created. its location tracking feature allowed for two different storage methods to function in one spreadsheet. the spreadsheet had a tab for s and b antisera categories. abo group, desired and unwanted antibodies filters allowed quick search for appropriate antisera. the spreadsheet also had hyperlinks to scanned ifus. results/finding: sequential lab s and b numbers were assigned to new additions using a dynamic/static storage system. an excel spreadsheet with scanned ifus (hyperlinks) was used. pre-merger systems, it took on average - minutes to choose an antiserum and obtain the appropriate ifu. post-merger system was reduced to on average - minutes. (table) conclusion: the merging of two irl's antisera inventories resulted in a need for innovation to create an inventory management system with an advanced search function and hyperlinked ifus. this process saved valuable technologist time and organized the antisera more efficiently. abstract continue to flash until they are removed from shelf and their status updated in our database. 'units allocated' tab includes truncated patient name (to protect privacy), unit number, component type, allocation and expiration date/time, and time since allocation, with a flashing alert for units expiring in < hours. the xm/hla platelets tab provides patient names and status of units allocated. a 'trxn/xmplat' tab lists pending transfusion reactions and platelet cross match reports. dashboard eliminated the printing (several times/shift) of lengthy computer-generated reports, simplified thawed plasma inventory management and helped decrease plasma wastage (from % to %). conclusion: using in-house talent and minimal capital expenditure, we designed and implemented a dynamic web-based dashboard for managing blood product inventory across a multi-site transfusion service. the dashboard is stable, customizable and requires little maintenance. initially built to optimize inventory display for thawed plasma implementation, the dashboard was expanded to include all allogeneic blood products. over the past year, this tool has replaced manual processes for monitoring and rotating inventory and directly helped decrease plasma wastage. use of deglycerolized red blood cells for hospital transfusion service inventory management ronnie l. hill*, jason corley and lizabeth ostiguin. us army background/case studies: regional blood shortages have been documented across the united states during the winter holiday timeframe. deglycerolized red blood cells (drbcs) have been shown to be an effective alternative though more expensive to manufacture. this study looks into the fiscal and inventory efficacy of using drbcs to meet the needs of transfusion services during times of blood shortage. study design/method: on three separate occasions, a medium sized dod donor center used its frozen blood inventory to produce type o drbcs to meet the needs of two regional transfusion services. all frozen red cells were manufactured by an offsite facility with the haemonetics acp- using the low glycerol ( %) freezing method and frozen at - c within six days of collection. thawing occurred in a c water bath in the following order: o positive and o negative on january ; o positive and o negative on february ; and o negative on february . deglycerolization occurred on site using the acp- with all units passing internal qc requirements. drbcs were shipped the same day to a hospital transfusion service, allowing for days of shelf life prior to expiration. results/finding: during the three events, all supported transfusion services and the blood center were below minimum inventory requirements for standard type o red blood cells (rbcs). o positive rbcs were only available through nbe at $ - and had the limitation of arrival on the next business day. collection and processing time of liquid rbcs takes approximately two days including: donor screening, phlebotomy, component processing, testing, and labeling. drbcs cost the dod on average $ per unit to produce and distribute. drbcs have a shorter shelf life, days versus the days for other rbcs, but are washed during deglycerolization and thus produce fewer transfusion reactions. one tech can operate up to four acp- 's and deglycerolize four units at a time. in january and february , it took one tech four hours per iteration of eight units to include thawing, labeling, and packing for shipment. conclusion: while not as readily available as traditional rbcs, drbcs can be an effective product to bridge the inventory gap when small numbers of units are needed due to reduced inventory. collection and processing of whole blood into components takes approximately two days, but can produce greater numbers of units in that timeframe. based on this, drbcs can be ready faster than freshly collected units of blood. there is an increased cost associated with manufacturing frbcs which is compensated for by the longer available shelf life of years. having a small contingency supply of frozen red cells and deglycerolization equipment has been effective on three occasions in ensuring availability of type o red blood cells for hospital transfusion services. validation of a human anti-tetanus toxoid immunoglobulin assay performed on the abbott c izekial butler* , karen leighton , scott jones and rachel beddard . qualtex laboratories, biobridge global background/case studies: plasma fractionators require anti-tetanus quantitative testing to be performed on plasma samples collected from individual donors or plasma production pools. this testing serves as a quality control test and helps estimate the antibody potency of the product. the binding site, human anti-tetanus toxoid immunoglobulin liquid reagent kit is for use on a turbidimetric analyzer. the aim was to optimize and validate the human anti-tetanus toxoid immunoglobulin liquid reagent kit for use on a photometric analyzer. study design/method: experiments were performed in order to determine the optimal amount required of reagent buffer and latex reagent from the anti-tetanus toxoid immunoglobulin kit utilizing the abbott c instrument. precision of the new assay parameters was determined by testing replicates of a panel of samples at three concentrations of tetanus antibody in a single testing run. the panel samples were created by spiking appropriate amount of a who tetanus antibody standard into sodium citrate plasma. accuracy was determined by testing a series of samples ranging from iu/ml to iu/ml of tetanus antibody. the samples for the accuracy study were created by diluting an appropriate amount of a who tetanus antibody standard with sample diluent from the reagent kit. linearity regression was determined by using the accuracy study values within the range of . to . iu/ml. stability of samples was determined by testing samples stored at - c and - c in triplicate at various time intervals. results/finding: the %cv for the optimized anti-tetanus assay for all antibody levels determined in the precision study varied from . to . . so, precision was acceptable since the %cv for all samples tested was %. the mean values for the samples tested in the accuracy study were all % of the expected value which was much lower than the acceptance criteria which was % of the expected value. the linearity of the assay was acceptable with a r ! . %. the linearity study established that the known tetanus concentration was a statistically significant predictor of the observed concentration. the sample stability studies demonstrated the ability to quantitate tetanus antibody concentrations in samples stored up to days at - c and up to month at - c. conclusion: the data presented shows the successful optimization of the human anti-tetanus immunoglobulin reagent kit for use on a photometric analyzer. validation studies of this optimized assay demonstrate excellent accuracy, precision and linearity using samples stored for days at - c and stored up to one month at - c. a deep dive audit of intravenous immunoglobulin use for immune thrombocytopenia: is its use inappropriate? jiajia liu*. university of toronto background/case studies: intravenous immunoglobulin (ivig) is a generally safe and effective therapy for immune thrombocytopenia (itp) but is only suggested for scenarios when a rapid increase in platelet count is desired or as first line therapy if steroids are contraindicated. due to concerns regarding adverse effects, cost and resource availability, an ivig request form was implemented in our jurisdiction in to track utilization and appropriateness. a recent audit of these request forms from four academic institutions found a lack of compliance with form requirements and inadequate documentation of efficacy which led the authors to conclude that the use of ivig was broadly inappropriate (shih et al, ) . as such, we aimed to conduct an extensive chart review of patients who received ivig for itp at our institution to assess appropriateness of use. study design/method: we conducted a retrospective chart review of all patients with itp who received ivig in our institution from april , to march , . local research ethics board approval was obtained. results/finding: patients received ivig for itp at smh over the study period for a total of unique ivig infusions. the most common indications for ivig within currently accepted guidelines were: active bleeding ( , %), pre-operative or antepartum care ( , %), a platelet count of less than and contraindication to corticosteroids ( , %). additional indications that still fell within accepted guideline recommendations included: patients with arterial/venous thromboembolism or risk thereof requiring initiation of antithrombotic therapy; and patients requiring myelosuppressive chemotherapy. indications that fell outside of guidelines included: use of ivig as a diagnostic challenge where the etiology of thrombocytopenia was unclear and use prior to international travel for patients with difficult-to-treat chronic itp despite a platelet count between - x /l. patients received ivig for a likely diagnosis itp while a transfusion being investigated for alternative explanations for thrombocytopenia. three patients were refractory to all other therapy for itp and were dependent on regular ivig infusions. / ( %) of infusions consisted of g/kg over days; the remainder of infusions consisted of g/kg. of those who received g/kg, of patients ( %) had evidence of partial remission after a first g/kg dose. ivig was generally well tolerated and infusion reactions were mitigated with use of corticosteroids, antipyretics and/or antihistamines. conclusion: we found, at our institution, that use of ivig for itp was generally appropriate and carefully considered even in cases that did not meet current guideline recommendations. we believe that ivig remains an important treatment for itp particularly in the aging population where prevalence of conditions complicating bleeding risk is increasing. detailed utilization/ knowledge data inquiries are required to develop tools and policies to enhance appropriate ivig use in multiple settings. we believe that there is an opportunity to promote administration of a single g/kg dose to minimize unnecessary utilization of ivig amongst hematologists who manage itp. a process for improving crossmatch bench ergonomics janet dornfeld*, sheng-chung cheng, ann eggebrecht, beth greer, savannahsue rondeau, brian rognholt and beth taylor. mayo clinic background/case studies: a mission of our institution is to reduce the risk of work-related injuries. accordingly, each year an ergonomic survey is undertaken as a component of a general department of laboratory medicine and pathology safety audit. our survey identified potential musculoskeletal risks that suggested a redesign of our crossmatch benches. study design/methods: a seven item ergonomics survey of the working environment was sent to staff members in early february of . twenty-two technologists responded for a % response rate. the below table below reports the survey items and responses. results/findings: the most problematic area was the available workspace. of the respondents, % indicated that workspace size was insufficient and % that the chairs at the fixed height benches were problematic. problems noted were difficulty with climbing up into a chair and backing down and with the chairs holding the chosen height. our laboratory lean team operational support group was tasked to aid with the bench redesign and to choose products for improving the workspace. our goals were to design a layout to streamline testing workflow and better utilize lab space, including our plasma thawing and sink space, eliminating dead space. the configuration of the new workspace was guided by the survey findings. adjustable height workstations were recommended to replace our fixed height bench. we worked with our facilities design contactor to purchase adjustable benches and plan add-on cabinet shop work. the benches were assembled off site, which allowed a bench top layout to be determined and installation of cabinet shop add-ons of a drawer for supplies and a pull out breadboard as a writing surface. the opportunity to assemble off site streamlined the process of installation, resulting in minimal disruption of testing. conclusion: the survey was effective in identifying working areas for improvement. employee comments have been positive for the new workstations. an effectiveness assessment will follow, using the original survey, to assess the success of the project. a retrospective study of emergency department initiated type and screen testing: were patients transfused after testing? sandra lamm* , neil bangs and kimberly sanford . vcu health system, virginia commonwealth university background/case studies: type and screen (t&s) testing is often ordered on patients presenting in the emergency department (ed). if the patient does not have a historical type, a second sample is drawn with an additional phlebotomy for type confirmation. if the patient does not need a transfusion of red blood cells (rbcs), the testing and second phlebotomy is an inefficient use of resources and time. study design/method: as part of a performance improvement initiative in transfusion medicine, we performed a retrospective study of all t&s orders that were initiated in the ed from / / to / / to determine if testing was subsequently followed by transfusion of blood products. patients were stratified by ed department, time from t&s draw (tsd) to transfusion (< hours, > hours < hours), and if a second sample was required. results/finding: a total of t&s orders were initiated from the ed in this time period. ( . %) patients were not subsequently transfused any type of blood product within hours of tsd and ( . %) patients were not subsequently transfused any type of blood product within hours of tsd. a total of ( . %) patients required a second sample. of these patients requiring a second sample, ( . %) were not subsequently transfused any type of blood product within hours of tsd and ( %) were not subsequently transfused any type of blood product within hours of tsd. conclusion: routine ordering of t&s testing is not an efficient use of resources and time as many patients are not subsequently transfused. ultimately unnecessary t&s and second sample collection and testing for those patients not subsequently transfused within hours of tsd amounted to an estimated $ , in unnecessary patient charges and approximately . nursing hours for phlebotomies in a six month period. anti-d from alloimmunization versus rh immune globulin: detective work in the blood bank and transfusion medicine services (bbtms) margaret diguardo* , debra berry , yunchuan delores mo and gay wehrli . university of virginia health system, children's national medical center background/case studies: the institute for healthcare improvement triple aim incorporates enhancing patient satisfaction by providing high quality, safe care. towards these goals the bbtms is charged with communicating to obstetric physicians (obs) a patient's antibody specificity with associated hemolytic disease of the fetus/newborn risk. thus, when anti-d is detected in a female of childbearing age, it is critical to determine whether this represents rh immune globulin (rhig) or alloimmunization (alloanti-d). review of a patient's electronic health record (ehr) helps quickly identify rhig administration, but if this documentation is missing, then it is easy to assume presence of alloanti-d. rhd alloimmunization impacts mom, fetus, newborn and future pregnancies. therefore, without a national, comprehensive health information exchange (hie) system, it is imperative to investigate beyond the on-site ehr whether a patient received rhig at an outside hospital (oh). we report an irb approved (exempt) case series where detective work revealed rhig administration at ohs. study design/method: over a two month time period, anti-d was identified in four pregnant women. review of their ehrs did not reveal a history of abstract rhig administration; nor did subsequent direct communication with their obstetricians (ob) reveal a history of rhig. based on each patient's home address, the bbtms of any nearby ohs were contacted as was a primary care physician if listed in the ehr. results/finding: investigations beyond the ehr and obs revealed each of the four patients received discontinuous prenatal care with presentations at multiple sites. through phone calls to the bbtms of ohs, a history of one or more rhig administrations within the preceding three months was found for each patient. our bbtms records and ehr were amended to reflect the presence of a passive anti-d due to rhig, rather than alloanti-d. the changes were also directly communicated with the ob caring for each patient. conclusion: when a new anti-d is identified in a pregnant female, investigation is required to determine whether it is passive rhig versus alloanti-d. when neither the ehr patient history or ob reveal a rhig history, it remains in the patient's best interest to investigate further. through phone calls to oh we revealed a history of rhig administration in four patients. finding and communicating this critical information helps enhance the quality and safety of patient by ensuring subsequent rhig administrations when indicated, at our institution. future strategies for avoiding similar situations include expanding our national hie for critical information such as bbtm history and allergy history and expanding use of wallet-size patient identification cards with rhig and alloantibody histories. auditing massive transfusion protocol colleen a. aronson* , elizabeth halperin , sharon breining and mona papari . acl laboratories/ advocate hospitals, advocate health care, itxm background/case studies: a large midwest hospital system with level i trauma sites evaluated how to audit the massive transfusion protocol (mtp). the possibility of real time audits is impractical due to the unpredictability of these events. a search of the internet found an example from new zealand for post process evaluation. this was shared with a team as a starting point and then adjusted for system specific priorities. to start the audit, the initiation of the mtp needed to be determined as events are often started as a verbal request but then followed up with either downtime or computer orders. study design/method: the transfusion service (ts) was determined to be the source of truth for all of the mtp events. a tracking sheet was created to capture the patient demographics, start and stop time, number and type of products issued and wastage. this was then passed onto nursing quality staff that used the tracking form and the patient chart to enter an event into the error management data base as a focused event. the focused event was built to include patient demographics and other information from the tracking form as well as where the event was called (surgery (or), emergency (ed), labor and delivery (l&d), etc.), type of event, use of tranexamic acid (txa), calcium chloride (cacl), temperature monitoring and pre/ post lab results. a trial was started and months of data were evaluated that contained events. results/finding: there was an equal number of events that were initiated in the ed and the or ( ). male patients were involved % of the time and % of time the patients expired. trauma of some type was the majority of the cause but . % of the cases involved gi bleed and only . % were obstetric cases (see chart). the lowest hemoglobin (hgb) was found to average . with the post hgb average of . . ratios of : for red blood cells (rbc) to plasma as well as rbc to platelets (plt) and cryoprecipitate (cryo) were also determined with a target of : . it was found that the rbc: plasma was . : , rbc: plt was . : and rbc to cryo was . : . use of txa was only . % and cacl was utilized in . % of cases. conclusion: although this data is for a short period of time it has pointed out several opportunities for improvement. the use of mtp in gi cases was not previously understood but opens up a new group of people for which education and understanding of the mtp process is needed. the low use of txa needs to be evaluated and already has started conversations about how this drug should be stored and accessed for the mtp process. the product ratio numbers were suspected of being off but now that data is available, it is much easier to speak to this issue and look for improvement. the process will now be expanded to the level ii trauma sites in the system and routine evaluation will be shared with all sites. automated report significantly reduces turnaround time for rbc antibody alert jessica l dillon* , jody a barna , donald e ulinski and nancy m. dunbar . dartmouth hitchcock medical center, dartmouth-hitchcock medical center background/case studies: clinically significant antibodies should be promptly and clearly communicated to the patients' healthcare team to avoid potential transfusion delays in blood availability or complications of incompatible transfusion. at our institution, all newly identified clinically significant antibodies are immediately resulted in the electronic medical record (emr). an interpretative comment is also entered by the transfusion medicine service (tms) physician after the antibody work-up has been reviewed (this may be up to weeks after the antibody is identified). this comment describes the antibody(ies) identified, indicates the need for crossmatch compatible blood and alerts clinicians of possible delays in providing crossmatched units. since clinicians may not always review these results, the tms physician also simultaneously adds an "allergy to red blood cells" alert in the patient emr at the time the interpretive comment is entered. study design/methods: in july , we implemented an automated report to reduce the turnaround time (tat) for entry of the allergy alert. the report contains all detected red cell antibodies in the prior hours and is provided to the tms physician during daily morning rounds (monday through friday) for manual entry of allergy alerts. this study describes a three month comparison both before and after the automated report intervention, to evaluate the tat for allergy alert entry into the emr. age ( abstract results/findings: between august and november (pre-implementation) , newly identified clinically significant antibodies were resulted for patients compared to patients between the months of august and november (post-implementation). the tat for allergy alert entry for both periods is shown in table . we observed that % of allergy comments were performed within hours in the post-implementation period versus only % pre-intervention (p . ). using the new process, nearly all of the alerts were entered into the emr within hours of antibody resulting and none of the entries were missed. conclusion: there was a significant improvement in the tat for allergy comment entry following implementation of an automated report. this project illustrates how information technology can be leveraged to facilitate timely communication of antibody identification. blood bank verbal tool implementation for cardiovascular surgery rita louie* , shailesh macwan , nancy nikolis , arline stein , janelle richardson , manju bagu , lennart logdberg , alexander indrikovs , vishesh chhibber and sherry shariatmadar . north shore university hospital, northwell health background/case studies: our institution is a tertiary care facility performing over cardiovascular surgeries (cvs) in , an increase of % after the healthcare system cvs integration in . transfusion support of these patients includes preoperative preparation of prbcs according to a maximum surgical blood order schedule. additional blood components are issued as orders are placed. until december , the additional written orders were submitted to the blood bank via the pneumatic tube system without further communication. after reported events in q that resulted in delays in blood transfusion, we examined our process very closely and identified opportunities for improvements. in collaboration with cvs, the blood bank implemented a new workflow process to enhance communication with the cvs team, reduce turnaround time and improve patient safety. study design/method: . open discussions and collaboration between blood bank and cvs nursing teams . mapping the process using flowcharts for additional blood orders from cvs. . identify bottlenecks and brainstorm solutions. . a verbal cvs order process and form was implemented to improve communication between cvs and blood bank, which solidified communication by including the time of the order, patient identifiers, caller identification, ordering prescriber, staff receiving order, the quantity and kinds of products ordered, the mode of order delivery, and anticipated future orders. a read back was also documented for verification of the order. . the blood bank staff immediately processes this order while waiting for the written order to arrive. upon receipt of the written order the blood is issued to the or. . follow plan-do-check-act. the transfusion safety officer reviews each order for the following parameters: number/type of products, turn around times (tat), wastage/returned products and overall efficacy since implementation of this process. results/finding: a significant improvement was noted in communication and tat after implementation of the process described above. for the period / / - / / the blood bank has received verbal orders with varying product combinations. the table below represents average turn-around times to issue blood products: conclusion: the introduction of the verbal order tool for cvs has streamlined the blood ordering process leading to increased efficiency and lower tat. effective communication between the or team and transfusion service is the key to timely provision of blood products for these critical patients. challenge of blood type testing for multiply transfused sickle cell disease patients jayanna slayten* , tracie ingle and heather vaught . indiana university health, indiana university health (iu health) background/case studies: we report our midwestern, university transfusion service challenge of obtaining the correct blood types in rbc exchanged sickle cell disease (scd) patients tested by our primary testing method, solid-phase red cell adherence analyzer echo (immucor. norcross, ga). the echo operation manual in chapter - and appendix d it states: "warning: the galileo echo cannot reliably detect hemagglutination reactions that are graded as or less in tube methodology. the galileo echo does not generate as interpretation of mixed-field. such a mixed-field reaction will be interpreted as positive, negative, or equivocal." we report of a challenge with this analyzer limitation which impacts the assignment of the correct blood type for multiply transfused scd patients. study design/method: two scd when initially tested by the echo as o, d negative; however, each patient was historically o, d positive. both patients had received a rbc exchange transfusion with - o, d negative red blood cells over days previously. repeat testing of the samples was completed by the vision (ortho clinical diagnostics. raritan, nj), neo (immucor. norcross, ga), and by standard abo/rh manual testing (anti-a, anti-b, anti-d series , anti-d series , a cell and b cells. immucor. norcross, ga). the repeat testing was compared to verify the patient's abo/rh typing and the results were entered into the computer system to allow for assigning the patient's abo/rh typing and electronic crossmatch. results/finding: table summarizes the initial and repeat testing with the two patient samples. although the echo failed to interpret or flag the blood type as mixed-field, the other methods identified the transfusion of o, d negative blood with the detection of mixed-field in the d typing or by failing to interpret the abo/rh as not type determined (ntd). the vision and manual abo/rh typing yielded the easiest mixed-field to interpret macroscopically. conclusion: our results agree with the findings of summers et al (trans-fusion ; : - who reported the challenge detection of mixed-field with the use of the echo compared to improved detected with automated gel column agglutination. when the samples were tested by multiple automated and manual abo/rh methods, the expected mixed-field was detected. the failure of the echo to detect the mixed-field is acknowledged by manufacturer, but there is a risk that a facility may mistype the abo/rh when there is not a historical abo/rh to compare. to avoid this risk, it may be appropriate to re-type first time scd patients by other methods rather than the echo to avoid this challenge. consistent with summers do not account for regional distribution. many large hospitals acting as regional hubs for redistribution may appear to have optimized inventory based on odr and bsr, but we hypothesized that these are crude key performance indicators (kpis) requiring redevelopment. study design/method: kpi redevelopment occurred in a large tertiary care hospital blood bank in canada, responsible for % and % of transfusions in the region and province respectively. rbc supply, inventory, and disposition data were retrospectively assessed from february -june as the baseline period. a "demand-driven inventory planning policy" (ddip) was instituted to assess and implement the optimal rbc reorder quantity based on the difference between the historical maximum and minimum rbc inventories during weekdays; that would not lead to blood shortages. shelflife inventory (sli) was chosen as the main surrogate marker for the assessment of efficiency of the supply chain process, calculated by the differences between age of blood transfused (abt) and received (abr). iterative simulation modeling (r statistical software) was then performed to optimize sli in a post-implementation period from june -october . results/finding: modeling predicted observed rbc disposition. through simulation, optimization of sli was found to occur by optimizing a set of kpis for each abo blood group (table ) . this led to a reduction in observed overall sli ( . . days vs . . days, p< . ) and odr ( . % vs . %). the bsr was not significantly increased during the postimplementation period. conclusion: optimization using simulation modeling of multiple factors other than bsr and odr led to further efficiency gains in a large tertiary care hospital blood bank. hospital blood banks should use an integrative approach with a set of kpis to optimize the supply chain. this approach requires validation in other blood banks and jurisdictions. ( )) requires that the hospital make reasonable attempts to notify the patient (or the patient's physician), counsel the patient, and offer testing. the hospital must maintain records of this lookback notification as part of the patient's medical record. paper records of lookback notifications are less accessible than electronic records and are at greater risk of being damaged or lost. to facilitate the lookback process and reduce paper documentation we sought to use the electronic medical record (emr) to perform and document notifications. study design/method: representatives from transfusion medicine (tm) and information technology (it) worked together to define minimum and optimal emr solutions. minimally, a completed paper packet could be scanned into the emr. this solution had no advantages in terms of ease of use, process control, or transparency. desired optimal functionality includes the ability to send letters in the emr, document control so that original communications may not be altered, opportunity for patient's physician to electronically sign and return responses, letter and form templates that can be individualized, and the ability to track when and by whom notifications were sent and received. the emr system at our institution, epic (epic systems corp., verona, wi), has a function called "letters" with the capacity to do all these tasks. a series of five templates were developed: hiv and hcv letters to physicians, response forms for physicians to return to the transfusion service, and a blank letter template to be used for specially tailored letters. templates are opened within the patient's emr and demographic information is automatically populated by epic (eliminating many possibilities for clerical errors), the blood product transfused (e.g. rbcs or plasma) is selected from a drop-down menu, and the date of transfusion is manually entered by the sender. the completed letter is then routed to the patient's physician; it shows up automatically (and instantly) in their electronic in basket as well as in the patient's emr. physicians may electronically complete and return the response form within epic, or print it and return the form by fax. results/finding: between january and december thirty-five ( ) notifications were sent to physicians using epic letters and of those, fourteen ( ) responded to the epic notification and five ( ) used the provided electronic response form. for these cases the time to mail or handdeliver paper notifications was avoided. the remaining cases required follow-up paper notification, but the electronic letter remains as permanent, easily accessible documentation of when the transfusion service first notified the physician. conclusion: lookback notifications within the emr makes compliance with government requirements more transparent and records more accessible to caregivers, patients, and assessors. secondarily, efficiency may be improved by reducing the need to print and mail/deliver letters. evaluation of ordering practice in the operating rooms and its impact on product wastage alexandra budhai* , denden benabdessadek , annu george and alexandra jimenez . westchester medical center, new york blood center background/case studies: blood product wastage is an issue that many hospitals aim to address. the or was identified to have the highest rate of wastage within our hospital. in this study, we assessed the appropriateness of the product order and utilization by the or to understand its impact on wastage. study design/methods: data on product orders, issue, and return for two months were analyzed. the hospital cpoe and product requisition forms were used to collect this data. the surgical procedures and number of ordered units were compared to the hospital's maximum surgical blood order schedule (msbos). trends for inappropriate orders for products by physicians were evaluated. results/findings: a total of orders were reviewed. approximately, % of these products were issued to the or. we found that the physician orders were within the guidelines of the msbos for most cases ( %), but of the issued products, all were returned to the blood bank in % of cases. we observed that the percentage of products ordered and used compared with the products ordered and returned in cardiac surgeries are nearly equal. in addition, all of the products ordered for c-sections were not used; albeit ordering frequency being significantly lower than for cardiac cases. conclusion: the data analyzed demonstrates that the majority of surgeons are adhering to our institutional msbos guidelines. it was noted that surgeons are requesting products be issued for invasive procedures where rapid exsanguination is possible. our analysis revealed that the hospital's msbos does allow for an excess in blood ordering for some surgical procedures. the msbos should be updated to reduce the suggested maximum product order. in general, the data does not imply that the blood product wastage in the or is due to the ordering practices of the surgeons. a larger period of surgical blood ordering practices should be analyzed to detect blood product ordering, utilization and wastage trends in other subspecialties. background/case studies: the visionv r and visionv r max (ortho diagnostics, raritan new jersey) are id-mts tm gel card-based automated immunohematology analyzers marketed for small to medium, and high-volume [> type and screens (t&s) per day] blood banks , respectively. our laboratory which serves a large -bed multispecialty academic hospital and receives - t&s specimens per day needed to replace three provue analyzers prior to the availability of the visionv r max. we implemented three visionv r analyzers to work with our existing neov r and echov r (immucor inc, norcross georgia). a recent multicenter field application trial of the visionv r reported a mean turnaround time (tat) for t&s and abo, rh typing (abo/rh) of . . and . . minutes , respectively. the objective of this study was to determine visionv r tats under routine daily high-volume practice. study design/methods: one visionv r was in operation during a five-week period (phase i), and then two additional analyzers were brought into service (phase ii). tats are defined as the time when the order is received by the instrument to when the test is completed and available for review. three-cell screen and abo/rh tats, and number of visionv r antibody panels were collected for a nine-week period. the tat for the screen was used as the tat for the t&s because the screen is the rate determining step. all testing was performed using in-service analyzers on routine patient samples by trained technologists. samples were not deliberately batched but were placed on the analyzer based on the volume and flow of work at the time. results/findings: under the high volume conditions of our laboratory with three visionv r analyzers, the mean t&s tat was % longer and had a larger standard deviation (s.d.) than the published trial result of . . . transfusion vol. supplement s abstract during phase i visionv r performed panels. during phase ii visionv r performed of the visionv r panels. conclusion: our visionv r analyzers are used under high volume conditions more suitable for the visionv r max. when balanced with the testing menu, including ability to perform select cell panels, our tats using three analyzers were satisfactory. the large standard deviation indicates that opportunities remain for improving tats through workflow improvement. from west nile virus to the emergence of zika virus: a nationwide survey of how regulators are keeping the blood supply safe and available falisha atwell* , john roback , ronald arkin , michael bartlett , robert geiger and jaxk reeves . university of georgia, emory hospital background/case studies: with the emergence of zikv in the united states, it is important to assess the fda's response time in providing guidance to ensure the safety and availability of blood products in the face of newly emerging infectious diseases. this research compares the responsiveness of the fda during west nile virus (wnv) and zika virus (zikv) outbreaks to evaluate our current preparedness. study design/methods: the literature review was conducted to analyze fda's response time during the wnv crisis and determine if it was effective and efficient. the research survey was performed to determine if the donor history questionnaire (dhq) adequately screens donors for zikv as the sole preventive method (as per the february guidance for industry: recommendations for donor screening, deferral and product management to reduce the risk of transfusion-transmission of zika virus) and to determine if the current regulatory practices (including the august guidance for industry: revised recommendations for reducing the risk of zika virus transmission by blood and blood components) are perceived to be effective and efficient in the face of the current zikv outbreak. survey monkey was used and participation was anonymous. over , emails and web-links were sent to members of aabb, scabb, seabb, and personal network with a % target response rate. participants self-selected or deselected based on the inclusion and exclusion criteria listed in the consent letter. results/findings: the literature review revealed that the fda's response was slow during the wnv outbreak, while the zikv response is efficient thus far. a total of participants responded to the survey ( . % response rate). statistically, participant agreement with fda's decisions was performed by "t" test (with n- - df) of the null hypothesis that the mean vs. the alternative that the true mean is> . overall participants had favorable opinions of the fda's decisions. statistically, whether participants in different levels of the demographic variables (region, profession, and years of experience) answer significantly differently, one way anova models were used with likert-scale question responses as if they were continuous. the f-statistic and p-value are for the null hypothesis that all levels of the explanatory variable have the same mean for the response variable. there were no significant differences in the years of experience and profession variables for participants. region was determined to be unreliable due to undefined states for each region listed. conclusion: the research revealed that industry experts conclude that the current system of dhq and fda guidance documents, if issued timely, are adequate. background/case studies: when evaluating a new instrument solution for pre-transfusion testing, it is important to consider the operational impact of the system on the lab. there are a variety of operational, performance and system metrics that can be evaluated to determine this impact including: test workflow, hands on time, and automation time. study design/methods: the study involved a current state to a future state comparison of testing processes with an instrument ortho provuev r (pv) and manual testing vs. an instrument ortho visionv r (ov). data collection methods included direct observation, time studies, and interviews. the pv bench performs type & screens (ts) on the pv and manual abid/selected cell panels in the gel test. all other testing; cord blood(cb), dat, unit confirm(uc), patient type confirm(pc) and crossmatch(xm), etc. are done manually in tube. the future state incorporated the ov. ts, abid and uc were evaluated in both states. cycle time(ct) was averaged based on run cycles. ct was comprised of metrics; instrument time(it), standby time(st) and labor time(lt). st may be comprised of components, time that could be utilized as "walkaway" time or vigilant time (vt) which requires operator presence but not operator action. for automated instruments, vt for each cycle was measured as instrument access unavailable. instrument daily maintenance (dm) ct was evaluated as well. similarly, timing of manual tube test processes used these metrics. for repetitive activities within a process, such as uc or xm, a time per individual process was captured and then multiplied per unit. results/findings: table provides details about the metrics of current state and future state processes. tube based test timing is as follows: pc ( : ), xm ( : ), cb ( : ) and dat ( : ). by implementing the future state, an average $ . min. lt and vt is saved on each sample loaded for ts equating to a % labor reduction over the current state. a % improvement in tat on the ts was achieved in the future state. moving from manual abid to automated processing resulted in a % lt reduction. on average, a min. continuous walk-away time is achieved for each automated abid. uc had less impact on labor time with minimal difference however allowed for focus on consistency and quality metrics. conclusion: based on the metrics evaluated and compared between current state and future state, the ov has demonstrated improvement in lab operations to both the labor required and result tat delivery. opportunity exists to automate workflows on other tests that are still manually performed. background/case studies: high throughput and efficient automation of serologic tests is crucial in the workflow of a blood bank that tests $ type and screen samples per day. the erytrav r (grifols) is a fully-automated walkaway analyzer utilizing -column gel cards for pretransfusion testing. the blood bank validated and implemented the use of erytrav r for abo/d typing, antibody screening and identification of patient samples as a replacement for a solid phase testing platform. the blood bank also validated automation of donor unit retypes. the instrument has bidirectional interface to the blood bank lab information system (lis), hcll tm (hemocare life line, mediware). instrument validation and implementation were done in conjunction with the software version upgrade of hcll tm and an interface system change to maestro tm . study design/method: correlation testing of the erytrav r results with the manual tube testing (peg iat; reference method) was performed on patient samples for abo/d typing and antibody screening; of which at least had a positive antibody screen. out of the , had known antibody specificities. forty-two rbc units were also tested for abo/d confirmation; of which were d(-) and were d( ). calculations of concordance, sensitivity, and specificity were performed. precision studies were also done. interface testing of erytrav r , hcll and the hospital's information system using the maestro tm interface system was performed and validated. results/finding: concordant results between both methods were obtained in all of the patient and donor samples tested ( % concordance). all samples with positive antibody screens were obtained by both methods. all clinically significant antibodies were detected by both systems. erytrav r gave % sensitivity and specificity. the precision studies showed that both methods gave the same type and screen results for samples at different testing events. after validation of the lis upgrade and interface system change, a bidirectional interface with hcll tm was established. the instrument has been operational in our lab for over months. conclusion: erytrav r was found to be reliable and accurate and can handle the high workload of our lab. users found the instrument easy to use; hence training, proficiency, and competency of the users are achievable and manageable. the validation of the the instrument is straightforward. the major challenge and delay in the implementation experienced by this blood bank were attributed to the concurrently occurring lis upgrade and migration of the data integration system. a post-implementation workflow assessment would be ideal to perform to ensure that the instrument is being used at its full potential. implementation of a system-wide platelet inventory report optimizes platelet utilization and reduces unit wastage elly landolfi* , craig fletcher and peter millward . beaumont hospital, beaumont health system background/case studies: a sufficient number of blood components should be available to meet routine and emergent hospital needs. this must be assured while minimizing outdating of scarce and expensive blood components -an inherent challenge with platelet units which have a short -day shelf-life. we report the results of a quality improvement project implementing a custom computerized platelet inventory report designed to mitigate the most common cause for platelet wastage at our institution: high platelet outdate rates. the report includes blood type, product code, unit number, respective product attributes, supplier and availability status of all platelet units for each hospital location. all system blood banks receive a morning fax of the report which facilitates transfer of units prior to expiration and adjustments are more readily made for product orders to the supplier. study design/method: the study was conducted in the hospital-based blood bank and based on available platelet inventory and wastage quality data. the report went live october and quality data was reviewed from august to december . the collected data was then analyzed using descriptive statistical methods. results/finding: data from indicates platelet wastage comprised % of total received platelets and % of these wasted platelet units were due to expiration. other reasons included failed visual inspection, blood dispensed but not used and wasted on the floors, potential tube station problems or short-dated units transferred into our blood bank from another facility. the mean of monthly wasted platelet units months preimplementation of the report was units, compared to units months post-implementation and units months post-implementation. wastage rates improved from % (wasted yearly platelets/total received yearly platelet units) in , the year of report implementation, to post-implementation rates of % in and % in (see table) . importantly, this occurred despite a greater than % increase in platelet inventory between and and resulted in cost savings of over $ , in this period. conclusion: study limitations included restricting data collection to one campus. the option to transfer expiring platelet units to another blood bank was available to all participating sister hospitals. it would have been interesting to see the effect of the report on those hospitals which have lower transfusion rates and different ordering practices. aside from lowering platelet wastage within years of implementation, additional benefits to the report included facilitating ordering from the blood supplier. cornerstones of a successful inventory management plan include daily inventory monitoring and, ideally, coordinated system-wide efforts to share platelet units. we have shown achievement of this end is facilitated by a customized daily platelet inventory report -an efficacious and easily adaptable tool with demonstrable gains. valerie halling* , lisa marie button , lori scanlan-hanson , karen koch , janet finley , deepi goyal and camille van buskirk . mayo clinic-rochester, mayo clinic, mayo clinic rochester background/case studies: transfusions in the emergency department of a level i trauma center were ordered using a handwritten order form. the transfusion lab's (tl) management team and medical director met with emergency department (ed) leadership and it resources in to define the needs of a successful electronic blood transfusion system. the handwritten order forms had several potential error sources which could lead to a delay in filling the order pending correction (in the best of circumstances) or could lead to transfusing the wrong patient or the wrong product if the error was not detected. the potential error sources included clerical errors involving the patient's name or medical record number (mrn), writing two different names on the order form (because there were two locations to record patient name), two product types ordered on one form when the requirement is for one product type per order, no priority indicated (stat or routine), or not including the prescriber call-back information. the number of ed reported transfusion related events in and were / (events/ed transfusions - ). study design/method: electronic ordering for the ed was implemented march st . any transfusion orders generated from the ed are now electronic, unless in the case of electronic downtime. the system electronically fills in the patient's name and mrn, controls for the type of blood product being ordered, requires an order priority and provides service contact information. it was designed to accommodate transfusion ordering needs for adults, pediatric patients < kg and pediatric patients > kg. a transfusion orders had three critical fields identified that are required for the order to be processed including patient weight, product volume, and infusion rate. the electronic system was designed so that an order cannot be submitted unless all critical fields are completed. results/finding: the electronic ordering system has been in place for years (april -march ), and during that time there was instance of blood being ordered for an unintended patient . % ( / ). this was because a previous patient's medical record was accessed rather than the intended patient's medical record. there have been no instances of clerical errors (name misspelled or mrn transposition etc.), missing service contact information, missing order priority information, more than one product type ordered on a single order, or two patient names on one order. electronic ordering also provided a place for the transfusionist to chart against, leading to increased transfusion documentation compliance. prior to electronic order implementation, in , / ( . %) units were transfused in the ed but not charted in the patient's medical record. in , / ( . %) transfusions were not charted. however, in , the first full year of electronic transfusion order capability, only / ( . %) transfusions were not charted in the patient's medical record. conclusion: electronic ordering in the ed has essentially eliminated ordering errors in this area resulting in less rework for both technologists and physicians. it allowed the order to be processed more quickly by tl, resulting in a faster turnaround time. improvement in the overall quality of transfusion ordering through electronic ordering reduced the influence of human factors in order placement and provided an added benefit of having a specific order to chart against. implementation of blood bank automated attendant lok tse*, gerald motta and maria aguad. brigham and women's hospital background/case studies: the blood bank receives numerous nonemergent phone calls on a daily basis. these calls not only occupied valuable time but also made the lines unavailable when a real emergency occurred. the hospital is categorized as a level trauma center, with over inpatient beds and over operating rooms. a proposal to implement a blood bank automated attendant was recommended to decrease phone calls, minimize errors due to distraction from phone calls, free team members to perform other duties and have a direct line designated for requesting trauma coolers, massive transfusion protocol (mtp) and emergency release of blood products. study design/method: the first step was to categorize the types of phone calls received by the blood bank by creating a phone log. data were collected and analyzed for four weeks. the blood bank collaborated with nursing, hospital administrative staff and telecommunication team to evaluate the possibility of implementing an automated attendant to minimize phone calls. it was very important to maintain patient safety and quality of service at the same time. the automated attendant consist of: option (urgent) for trauma, emergency release, mtp and obstetric hemorrhage emergency release; option (verbal) for verbal orders and coolers set up; and option (staff) to speak with staff member. instructions were also given for specimen inquiry and product availability in the hospital information system. results/finding: the data in table showed that most of the incoming calls fall into three categories (specimen inquiries, product order inquiries, and other inquiries). most of the calls were from nursing staff inquiring about the length of wait time for blood products and specimen availability. there was an overall decrease in phone calls by % with the implementation of an automated attendant. conclusion: with the implementation of an automated attendant, the blood bank team was able to identify and respond accordingly and efficiently to urgent requests and verbal phone orders. the decrease in phone calls freed up team members to perform other critical tasks in the department. improved detection of wrong blood in tube errors: implementation of a two-sample blood type verification process ariana king* , steven zibrat , geoffrey wool and angela treml . university of chicago medicine, university of chicago background/case studies: our organization used a blood bank identification (bbid) band system for pre-transfusion testing and detection of wrong blood in tube errors (wbit). additionally, type & screen results were compared to patient's historical records; the specimen was retyped by a second technologist if historical results were not available. the bbid bands were prone to clerical errors and excessive specimen rejections, and believed to miss some wbit errors. in , blood bank accounted for % of all rejected clinical laboratory samples, yet comprised only % of total laboratory volume; % of rejected blood bank samples were due to bbid band issues. the wbit error rate detected by bbid-based system was . %. study design/method: a multidisciplinary workgroup was formed to review data and best practices. the decision was made to discontinue bbid bands and implement a two-sample verification process, in keeping with standards. a new laboratory test order was created in the emr system and embedded into the existing t&s order. providers are prompted to order the abo verification test only when no previous abo/rh typing results are found. education was provided to all clinical staff in the form of in-services, emails, and annual competencies completed electronically. the new process went live in september . results/finding: in the five months following implementation, four wbit errors were detected with the second sample. these may have been missed using the bbid band system. improved detection revealed a wbit error rate of . %, three times the national average of . %. under the new system, rejected blood bank samples decreased from an average of % to % of all rejected laboratory samples, a % decrease. implementation of the new process produced a net savings of $ . k. conclusion: replacing the bbid band system with two-sample verification successfully improved our ability to detect wbit errors among patients who lacked historical blood bank results. additionally, discontinuation of the bbid system decreased the incidence of clerical errors and unnecessary specimen rejections, and also saved money for the organization. next steps are for blood bank and laboratory quality leaders to partner with nursing leadership to drive down wbit error incidence. a addendum with the final culture results. we used a student's t test to determine whether there was a statistically significant difference in the mean tat for result addendum entry in the post-implementation period compared to the pre-implementation period. results/findings: in the pre-implementation period, we cultured residual products for suspected str. the tat for final culture result entry into the patient's emr was - days (mean days, sd ). in the postimplementation period, we cultured residual products for suspected str. the tat for final culture result entry into the patient's emr was - days (mean days, sd ; p . ). there were no positive cultures during either study period. conclusion: our study demonstrates that tat for documentation can improve with the use of information technology to notify the transfusion medicine physician when results are available for documentation in a patient's emr. improved turnaround time of type and screen samples michaelene hultman* , marcus holme , johnathan bakst , gunta musa and angela treml . university of chicago medicine, university of chicago background/case studies: the primary test performed in the blood bank with regard to pre-transfusion testing is the type and screen (tys). the current target for this institution's blood bank is an minute turnaround time (tat). in april of , the blood bank was forced to move to a temporary location due to building construction, which necessitated a switch from automated solid phase methodology to manual gel method. the average number of outliers increased %. tat analysis of a representative one week sampling per month showed an increase in outliers from per month to per month. average monthly tys samples performed is . these numbers did not improve even upon returning to the original facilities. study design/method: two ortho clinical diagnostics visionv r analyzers (raritan, n.j.) were purchased for the blood bank. the instruments were set up with a bi-directional interface allowing for samples to be continuously loaded without manually ordering the tests. batch testing was eliminated allowing samples to be run as received. the results were auto interpreted, and transmitted to the laboratory information system (lis) based on predetermined rules. only results in need of manual review or interpretation were held back. final verification of results was performed by the technologist within the lis. reagents and other needed consumables could be preloaded on the instruments eliminating the need to repeatedly load consumables with each sample run. key quality indicators including tat continued to be monitored throughout implementation. data was monitored for significant changes and improvements in patient care. the go-live date was / / . results/finding: the average number of outliers decreased % from per month to . further benefits include a reduction in the number of technologists needed to perform tys testing. additionally, reduced waste due to better utilization of supplies by the instruments along with less repeat testing has resulted in projected cost savings of $ , for fiscal year . conclusion: the use of gel technology, in combination with a two way interface and a continuous load instrument can result in a significant decrease in tat over manual gel method. improvements in the timely reporting of final product culture results in the patient's emr. barbara a. hewitt*. dartmouth hitchcock medical center background/case studies: in certain transfusion reactions it is required that a culture of the returned blood product be performed. these cultures are reported in our cerner operating system but those results do not cross over to the patient's emr . the finalized product culture results are entered into the patient's emr as an addendum to the transfusion reaction clinical note. a review of the transfusion reaction database revealed that there were occasions when the final product culture results were not entered into the patient's emr in a timely manner. it is important to the patient's care for the transfusion medicine service and the patient's primary provider to know if a transfusion reaction is related to a contaminated product or the patient's general overall health. this information is also crucial to the supplier of the product to determine if others have received components of the affected unit and to possibly determine if there are any quality control issues at the donor facility. study design/methods: a review of a specific month period revealed that the timeframe in which the finalized product culture results were entered into the patient's emr ranged from - days with a mean of . days. it was determined that this was not in the interest of improving patient care. in collaboration with laboratory information services a report was created in which once product culture results were finalized an email would be generated notifying the medical director and the transfusion safety officer that results were available. results/findings: data was collected for months following the implementation of this report and it was noted that timeliness of finalized product culture results being entered into the patient's emr improved to a range of - days with a mean of days. conclusion: improvements in patient care require diligence and timely reporting of finalized culture reports to determine potential causes of transfusion reactions. this process can be made easier when the correct tools are used. omer ilyas* and randy levine . northwell health, lenox hill hospital background/case studies: transfusion of non-irradiated blood in patients with hematologic malignancies and those receiving cytotoxic chemotherapy can result in life-threatening graft versus host disease (gvhd). after noting several instances where non-irradiated blood was transfused in patients requiring irradiated blood, we designed a quality improvement project with educational sessions involving the oncology unit and blood bank. study design/method: the project was separated into three parts. in the first part, data on transfusion practices was retrospectively collected over a four month period on the oncology unit. the variables collected included date and time of transfusion, pre-and post-transfusion hemoglobin, patient diagnoses, and whether or not blood was ordered to be irradiated and if so, whether or not irradiated blood was issued by the blood bank. all patients with hematological malignancies and all patients receiving cytotoxic chemotherapy were candidates for irradiated blood. the second part of this project was an educational intervention. residents, oncology floor nurses, and blood bank staff were given lectures on the importance of transfusing irradiated blood on the oncology floor. residents were also instructed to order irradiated blood for all patients on the oncology unit. in the third part of this project, repeat data was collected over a two month period to assess whether the intervention was successful. results/finding: pre-intervention, units were transfused on the oncology floor with units ( %) requiring irradiation and only of those units ( %) ordered as irradiated. since the blood bank occasionally issues irradiated blood without a specific order, additional irradiated units were issued ( / ; %). post-intervention, units were transfused on the oncology floor with units ( %) requiring irradiation and all of those units ( %) ordered irradiated specifically to prevent gvhd. eight additional irradiated units were ordered with no requirement for irradiation; thus of the ( %) total units were ordered as irradiated. again, additional irradiated units were issued ( / ; %) without a specific order by the blood bank. the results are summarized in the accompanying table. conclusion: this quality improvement project demonstrates that educational intervention can succeed in changing clinical practices. continued monitoring of ordering practices will ensure that compliance continues. we plan to expand the quality improvement project to other settings, including the emergency department and surgical floors. we expect that adherence to transfusion guidelines in this patient population will reduce the incidence of adverse events. samantha ngamsuntikul* , charlotte van dyke , dina garza van hoose and rachel beddard . biobridge global, south texas blood and tissue center background/case studies: at our blood center, apheresis platelets and red cells are collected on trima accels and double red cells on haemonetics s. in addition to routine quality control (qc), qc is performed for instrument flags on collection instruments. quality control for apheresis platelets includes: volume variance and rwbc; quality control for apheresis red cells includes: product volume, volume variance, hemoglobin and red blood cell mass. study design/methods: during the period of january , to april , , , total collections were flagged for additional qc by our trima accels and haemonetics instruments. quality control at our center is tracked by our quality control software management system, hematerra's hemacomply which allows the ability to track and retrieve this information. the majority of products flagged for instrument qc pass and are released for distribution. a small percentage, however do fail qc leading to loss of product. quality control data can be retrieved and monitored for trends using a quality control software management system. background/case studies: in , the centers for medicare & medicaid services (cms) rolled out a plan for implementing iqcp (individualized quality control plan) as a new quality control option based on a risk management plan for clia laboratories performing non-waived testing. this plan was meant for clia approved tests, but serves as a good tool for labs performing non-traditional and traditional tests on non-traditional samples. study design/method: clia clinical laboratories can either follow traditional clinical clia qc requirements according to the regulations or implement iqcp. while we perfrom traditional qc assessments on all the tests we perfrom on our cellular products we did decide to implement the iqcp program within in our quality control laboratory. we followed the iqcp process for assessing some of our qc tests used to assess the safety, purity and potency of our cell based products. one test in particular where we applied this tool was in the review of our qc sterility testing method and found it to be a very useful in improving the overall process. the tool walks you through three process requirements: ) risk assessment, ) quality control plan and ) quality assessment for the preanalytical, analytical and post analytical phases of testing. abstract conclusion: the integration of the iqcp into the quality control laboratory was determined to be a success. the iqcp tool was successful in identifying gaps within the sterility testing process. this tool will be used on additional quality control tests and manufacturing processes. the implementation of the iqcp program ensure regulatory qc requirements appropriate for testing performed. we were able to revise our procedures, reeducate those involved in the process and hopefully minimize potential sources of error. objective performance of massive transfusion protocols at a single institution gustaaf de ridder* , rachel jug , kimberly ingersoll , nicholas bandarenko , nicole guinn and jessica poisson . duke health pathology, duke university hospital, duke health anesthesiology background/case studies: hemorrhage is both a leading cause of mortality in trauma patients and morbidity in non-trauma patients. using a balanced : : transfusion ratio (tr) for massive resuscitation is recommended based on trauma data. objective performance during massive transfusion protocol (mtp) activations is poorly studied and there may be differences based on site or medical service of mtp initiation. with the impending release of a unified, redesigned exsanguination protocol (exp) at our institution, we established baseline performance characteristics for our existing mtp and obstetric massive transfusion protocol (obp). study design/method: following institutional review board approval, we performed a retrospective study on blood product utilization and outcomes of mtp and obp activations from july -december . data were manually collected from transfusion service paper records, electronic (safe-trace) records, and an automated data report from the electronic medical record (epic). conclusion: we observed considerable variability in transfusion practices during acute hemorrhage depending on the service and location of activation. trauma activations demonstrated the sharpest deficit in platelet transfusion, whereas all groups lagged somewhat in transfused plasma relative to packed red blood cells. los and mortality varied among groups, likely reflecting underlying medical conditions and indications for massive transfusion. we have identified an opportunity for improvement in mtp transfusion ratios observed in trauma cases, the specific environment from which the : : ratio was derived, and in which the impact of protocol-driven blood resuscitation is most efficacious. patient identification improvement strategy to help reduce unacceptable specimens arline stein* , nancy nikolis , linda benison , ruthmire thelusca , renee liberty , sherry shariatmadar , alexander indrikovs and vishesh chhibber . north shore university hospital, northwell health background/case studies: our blood bank (bb) processes approximately , specimens per year. bb specimens are unacceptable when they are unlabeled, unsigned or missing necessary documentation. in such cases, a new specimen is requested to be drawn as per protocol. our investigation of unacceptable specimens previously included generation of a report by the blood bank staff that was subsequently submitted to the bb supervisor for completion. following completion, the report was sent to the nurse manager of the patient care unit (pcu) for follow-up and investigation with the staff members involved. this process was cumbersome, taking a few days before the staff member of the pcu was alerted to the deviation in protocol. at times, residents or float staff involved were difficult to identify and it was often challenging to track down the staff and do the necessary investigations and in-services. study design/method: in june , a patient identification improvement strategy was implemented jointly by the department of nursing and the bb to address mislabeled, unlabeled and unsigned specimens as part of a patient safety initiative. currently, following this strategy, when an unacceptable specimen is received, the nurse manager (nm) of the pcu is immediately notified by bb staff. the nm promptly initiates a debrief process with the staff involved in drawing the specimen. a debrief form (tool) was created to guide the discussion. this process is followed / . the nm will also engage other available staff in a huddle to review the incident and reinforce the policy. the debrief form is then submitted to hospital qa and the bb with preventative actions included. we believe in using the just culture model to help us understand the reasons why the staff did not label the specimen according to policy. just culture helps promote shared accountability to ensure we have the proper systems and processes in place to deliver high quality care. results/finding: the table below represents the percentage of unacceptable specimens identified by the bb since the second quarter of . the implementation of this new process has led to a decrease in the number of unacceptable specimens up to % quarterly following its implementation. the opportunity for direct intervention by the nm with the staff involved has risen from % to %, due to the immediate debrief process. abstract conclusion: the patient identification improvement strategy allows for real time engagement of the bb and pcu staff to promptly investigate and institute corrective/preventive actions when there is a deviation from policies related to specimen collection. the heightened awareness of correcting patient specimen labeling errors can only improve patient safety and the patient experience. platelet transfusion practices among pediatric oncology patients: a single institutional experience nicole m crews* , , morgan rockwell , joseph hagan , jun teruya , and shiu-ki hui . texas children's hospital, baylor college of medicine background/case studies: despite advances in adult platelet transfusion (ptx) literature, questions persist regarding pediatric transfusion thresholds, dosage and responses. therefore, ptx are commonly guided by local institutional recommendations (ir). the aim of this study was to determine the degree of adherence of ptx practice to ir at a pediatric tertiary institution. study design/method: retrospective review of ptx practices including transfusion thresholds, responses and dosages were collected. platelet counts within hours pre and post transfusion were evaluated. patients ( - years) receiving prophylactic ptx from july to december admitted to the oncology acute care unit with diagnosis of leukemia or lymphoma were included. for prevention of volume overload, the ir for ptx were < ml/kg for patients < kg and one apheresis unit (au) for patients > kg; therefore, patients were separated into groups: < kg and > kg. a significant proportion of orders for both < kg and > kg did not meet patient platelet threshold criteria (p< . ). conclusion: ptx threshold above ir for both groups were ( kg) and % (> kg). most common reason for above ir threshold was an invasive procedure or low molecular weight heparin therapy. greater than % of ptx dosage in both groups were above ir, however the platelet response did not increase significantly (p> . ) with a higher dose vs. ir dose. this study demonstrated that there were still considerable deviations from ir in ptx practice among pediatric oncological patients. in addition, the false assumption that a higher dose will yield a better response can put patients at increased risk for transfusion related adverse events. each institution should conduct a quality assurance review to determine ptx practice. pre-surgical sample process improvement to enhance patient safety and compliance lisa marie button*, stephanie saathoff, jered luedke, benjamin colvin, umalkair amare and james r stubbs. mayo clinic background/case studies: our institution provides the option for presurgical samples (pss) to be drawn up to days prior to surgery as long as the patient reports not being transfused with a blood or blood component containing allogeneic red cells and they have not been pregnant in the preceding months from the date of pss collection. when pss patients returned for surgery, the patient's service was required to ask the patient again about their transfusion and pregnancy history to determine if there had been any new opportunities for allogeneic red cell exposure, however, there was no process to capture the information the patient reported for the time between the pss draw to the day of surgery/possible transfusion. study design/method: an electronic fix was designed that was applied to the surgical intake process. a new set of questions was added to the a.m. admit questionnaire that must be completed prior to the patient's surgical procedure. the questions ask the patient if they have been pregnant or transfused in the preceding three months and if the answer is affirmative, the computer system runs a blaze rule causing an alert in the blood bank. the blood bank techs review the alert and inactivate the patient's pss based on the new transfusion/pregnany information. one year post-implementation of the electronic fix, transfusion lab performed a retrospective review of all pss alerts generated during a three-month period. results/finding: the results of the review were analyzed and are displayed in the table. it was determined that only . % of patients with a pss alert had an active sample requiring inactivation. conclusion: implementing an electronic solution that requires documentation about pss eligibility upon return for surgery has resulted in an estimated ( x ) pss alerts in the blood bank each year. of these alerts, it is estimated that approximately patients ( x ) per year are identified as no longer eligible for pss status. once this retrospective review was performed, it was shared with the project stakeholders to determine if the electronic questionnaire could be further tailored to patient's based on age, gender, and pss status. while the benefit of having fewer false positive pss alerts ( . %) was recognized as an ideal future state, it was not compatible with the institution's current it project of implementing a new electronic medical record (emr) system. the safety enhancement provided by the current electronic fix will remain as is and the improvement suggestions were shared with the team creating the parameters for the new emr with the intention of targeting only patients with an active pss in the blood bank, rather than all surgical patients. weill cornell medicine, columbia university school of medicine background/case studies: blood ordering is a complex, high-risk process with multiple steps that have the potential for errors and delays. risks associated with this process, from ordering through pick-up, require evaluation and strategies for mitigation. given the complexity and high-risk nature of blood ordering a proactive risk assessment (pra) for blood product ordering using the fmea methodology was conducted. the goal of the project was to proactively assess the effect of a redesigned electronic order set on the quality and safety of blood ordering study design/method: to evaluate the electronic blood ordering redesign process, a pra was completed using the fmea methodology. the team identified each step and sub-step of the electronic blood ordering process, all failure modes and causes, and then scored each by severity occurrence and detectability to determine the risk priority number (rpn). all rpns with a score above the threshold were reviewed and rescored based on mitigation strategies designed to address the failure mode. results/finding: the group scored the identified failure modes by categories used in root cause analyses. the electronic blood order process has internal logic and alerts that improve communication and reduce the risk score. several mitigation strategies that will reduce the risk of the identified failure modes include type and screen status within the rbc order, streamlined alerts when the order does not meet the laboratory threshold, a nursing task list for transfusion, and a change to the pickup process that is linked to the product ready status in the laboratory information system. a transfusion history will be available to providers when ordering blood products to further reduce communication risks. categories for failure modes included clinical,communication,equipment people,process and system. the average overall failure mode rpn was reduced by % with the communication category average rpn having the greatest reduction of %. conclusion: an fmea of an electronic blood ordering process can proactively improve quality and patient safety by preventing transfusion delays and errors in blood product administration. accurate and timely information in the blood ordering process has the potential to reduce risks associated with ordering,preparing and dispensing blood. reducing turn around time for type and screens in the blood bank kimberly ouellette* , karen king and joseph sweeney . rhode island hospital, lifespan academic medical center background/case studies: expeditious turn-around times (tat) in the blood bank are critical to provide fully tested and crossmatch compatible blood in a timely manner. the blood bank at rhode island hospital, a level trauma center and teaching hospital associated with brown university, was originally designed to accommodate tube testing by all technologists. the original setup of the lab was split into three sections allowing for preparation and issuing of units in the first section, bench testing in the second, and the receipt of components in the third. as technology changed, the blood bank adopted first the manual gel station and then the automated gel system (ortho provuev r ) but did not adapt the space. the second section of the blood bank contained the manual and subsequently automated gel stations with no other changes. the process of sample receipt through completion of testing and issuing of units remained segmented and inefficient. the average tat for type and screens was minutes. study design/method: the blood bank design was remodeled to make for a more open concept to allow for collaboration amongst technologists as well as the best use of space and technology. the first section of tables were removed and replaced with a center console to allow for movement about the entire front of the laboratory. a wall was constructed to separate the main work flow, automated gel testing and issuing units, from the area for complicated workups and inventory receipt. the third section remained, but was repurposed for teaching medical technology students and residents. in addition to the remodel, the blood bank retired the ortho provuev r for the ortho visionv r , which is considered a true continuous feed machine. although the inter-device tat is not significantly different ( minutes for the provuev r and for the visionv r ) the visionv r is built with a scheduler that effectively handles the system and processes samples efficiently. the visionv r is also equipped with two centrifuges to process samples, which further reduces tat when multiple samples are onboard. a bi-directional interface was designed to allow for test orders (type and screens) to go to the visionv r and test results to go directly from the visionv r to the lis without the need to manually order the tests or transmit the results. data on tat were collected and analyzed using independent t tests and chi square. results/finding: the mean tat pre-and post-reconfiguration and implementation of the ortho visionv r and a fraction of samples with tat over minutes are shown in the table. the results show a reduction in tat by minutes with a % reduction of tat greater than minutes. conclusion: a combination of new technology and space remodeling can lead to a significant reduction of tat of testing in the blood bank. caleb wei-shin cheng* , , lorna orengo , monique scott and christopher a tormey , , . yale university school of medicine, yale-new haven hospital, va connecticut healthcare background/case studies: the type and screen (t&s) is a fundamental laboratory test that allows the blood bank to provide compatible blood for patients. despite this, erroneous blood product administration may occur as much as in , blood transfusions. to prevent errors, adequate specimens such as those lacking hemolysis and those with proper specimen labeling are necessary; otherwise the specimen is rejected, leading to a second blood draw and a delay in medical/surgical management. hemolysis rates for t&s specimens are reported to be as high as % prior to interventions, but may potentially be reduced to as little as . %. however, there is little published data on non-hemolysis-related type and screen rejections. an initiative was undertaken to reduce the rejection rate in the blood bank to a sustained rate of < %, with a particular emphasis on non-hemolysisassociated forms of rejection. study design/method: a root cause analysis (rca) was performed over the preceding months to obtain a baseline understanding of the errors involved. t&s submission at our facility involves standard completion of the specimen label plus completion of a unique witness form to confirm the identity of the patient from whom the specimen was collected; specimen and witness form must be submitted simultaneously. when a specimen was rejected, we recorded the patient name, medical record number, and the reason for rejection. following rca, an intervention was created to resolve the most common issues documented that resulted in rejection. approval for the intervention was granted by the department chair, transfusion committee, forms committee, and the medical executive committee. after implementation, prospective data will be collected for several months in the same manner as before to determine the effectiveness of the intervention. results/finding: over the study period, the t&s rejection rate averaged . %. reasons for specimen rejection were divided into groups: ) hemolysis, ) blood bank witness collection form errors, ) quantity not sufficient, abstract ) duplicate sample, and ) specimen tube labelling errors. the highest percentage of rejections was due to improperly-filled witness forms (table ) . after multiple form redesigns and approval by appropriate committees the new form was implemented. preliminary data collected thus far demonstrates a . % rejection rate with only rejection relating to witness form errors. conclusion: rejected t&s specimens are an impediment to safe clinical care as it may delay medical/surgical management. rejection rates could be reduced through simplification of blood bank specimen collection forms. care providers have multiple tasks that need to be performed in a short amount of time, therefore, simplification is often times necessary to reduce human error. future quality initiatives will aim to simplify complex healthcare processes without compromising patient care. reduction of failed whole blood donor testing runs on the roche cobas s system christopher shahan* , christina dejesus , mosi mccall , fallon hampton , tangi herring , judy davis , anjali patel , sonya gomillion and bonnie maltby . qualtex laboratories, qualtex laboratories background/case studies: as part of our quality control program, we track the number of technician related failed runs observed on the roche cobas s system. this system is used to test whole blood donor samples for human immunodeficiency virus (hiv) rna, hepatitis c virus (hcv) rna, hepatitis b virus (hbv) dna and west nile virus (wnv) rna. technician related failed testing runs can cause the laboratory to report results outside of the contractual - hour turnaround time. failed runs also cause retesting which increases reagent utilization for the system. currently % of whole blood donor testing turnaround time delays are due to issues and failed runs on the s system and we have technician related failures per week. a lean six sigma approach for process improvement was utilized to identify root causes and develop countermeasures in order to decrease the number of technician related failures on the s system. study design/method: the number of technician related failed runs on the s system were tracked from / / thru / / . a pareto chart was used to determine that technician related errors was the largest controllable factor causing run failures. the whys were performed to determine root causes of technician related failed runs. a gemba walk was performed on all of the lab testing processes to help identify areas for improvement. the process improvement team talked, met, observed, and worked directly with staff that operate the s system. roche was also contacted to provide guidance on how to help decrease technician related failures. results/finding: the main root cause determined was that there was no current process flow map for whole blood donor testing using the s system. counter measures implemented included creating a two phase process map. one phase was related to the processes related to start-up of the system and the second phase was related to the processes involved in processing of samples. roche provided a job aid for the technicians which provides clear steps technicians should take when handling and cleaning up crashes and failed runs on the s system. after counter measures were implemented, the number of technician related failed runs decreased from to . failures per week, which was a % decrease. conclusion: a lean six sigma approach for process improvement was utilized to identify root causes and develop countermeasures in order to decrease the number of technician related failed runs on the cobas s system. this lean six sigma approach and counter measures significantly decreased the number of technician related failed runs by %. patients who were transfused for pre-transfusion hgb > g/dl with resulting post-transfusion hgb > g/dl were reviewed. demographics, medical history, provider identity, indication and transfusion complications were abstracted & compiled individually by volunteer internal medicine residents. group discussion for each case ensued before determination of transfusion appropriateness occurred. principal investigator/attending physician then made final determination of appropriateness of rbc transfusion. results/finding: patient charts were reviewed. were excluded for bleeding and cardiovascular instability. / ( . %) were determined to be transfused inappropriately. there was no difference in appropriateness of transfusion with respect to age or sex. patients with solid tumors ( . % vs . %, p . ) and anemia of chronic disease ( . % vs . %, p . ) were more likely to be inappropriately transfused. patients who had higher pre-transfusion hemoglobin were more likely to be inappropriately transfused (median hgb . g/dl vs . g/dl, p< . ). inappropriately transfused patients also had higher median post-transfusion hemoglobin ( . g/dl vs . g/dl, p< . ). moreover, lab evalutions revealed association with lower folate levels (median . nmol/l vs . nmol/l, p . ). / ( . %) patients were inappropriately transfused at least in part because they received more than one unit without an interval hemoglobin check in between. / providers were responsible for . % of all inappropriate transfusions. / appropriately-transfused patients experienced an fnhtr. deaths unrelated to transfusion occurred ( in appropriate, in inappropriate group). conclusion: physicians in training are interested in promulgating optimal rbc transfusion practice. this study identified patient factors (such as solid tumors and anemia of chronic disease) that correlate with a higher likelihood of receiving an inappropriate transfusion. beyond cpoe, educational intervention at individual level should be designed for specific providers responsible for more inappropriate transfusions. successful implementation of a blood bank information system in a small-scale caribbean blood bank: a structured step-wise approach. luigi sille* , willem martin smid and ashley john duits . red cross blood bank foundation, sanquin consulting services background/case studies: an important tool for complying with gmp quality standards is the effective use of a blood bank information system (bis). validation and implementation of a bis is described for centralized large blood bank and literature and guidelines are lacking for the nonautomated small scale blood bank environment. . small-scale blood banks face specific challenges for computerization in relation to economies of scale and existing processes requiring special attention. for the introduction of a bis at the blood bank of the dutch caribbean island of curaçao a specific procedure was designed based on existing guidelines and adapted to the local setting. study design/method: the red cross blood bank foundation curaçao is the sole provider of labile blood components for the dutch caribbean islands of curaçao, bonaire and sint maarten. after selection of the bis provider for implementation isbt and bcsh guidelines for validation of information systems in blood establishments were carefully analyzed to prepare the design of local procedures. these procedures were meant to evaluate and validate the features of a bis (e-delphyn, hemasoft america, miami, usa) before introduction. the outcome of the approach was entered in worksheets that were evaluated by the implementation team and management. from this the implementation plan was designed and implemented. an external auditor (sanquin consulting services, amsterdam, netherlands) was invited to evaluate the implementation and validation plan and its practical implementation. the evaluation was performed according to risk assessment of critical process steps. results/finding: based on the isbt and bcsh guidelines a process flow chart describing the relevant phases and critical steps for introduction and validation of a bis was designed. comparison of the current processes and procedures were compared to the bis characteristics making use of worksheets. with these worksheets the existing gaps with the bis procedures were carefully described. these gaps and the appropriate procedural changes for bis or blood bank were effectuated. the worksheets also provided the basis for staff training in a separate training environment before bis introduction. during the early validation phase all procedures and processes were audited by an external auditor. with the feedback of the expert several improvements were added for the validation and subsequent implementation processes. conclusion: with the use of existing international guidelines a validation and implementation plan was designed to prepare for successful introduction of a bis in a small scale caribbean blood bank. the program as designed seems well suited for small scale blood banks contemplating introduction of a bis. time and cost savings through implementation of a remote blood fridge jessica peters* , dee dee cassidy , jed b gorlin , and nancy l van buren , . hennepin county medical center, innovative blood resources background/case studies: rapid delivery of emergency release group o red blood cells (rbc) are vital to patient care. commercial remote blood fridge packages are available but have large upfront and maintenance costs. we implemented a remote blood fridge directly in our emergency department (ed) using an under counter fridge requiring id access, and a selfdeveloped ios application that scans, tracks and real-time alerts transfusion service (ts) to products used and to whom they were dispensed. prior to ed fridge implementation, rbc units were verbally requested and an ed blood runner would pick up and return the cooler. given that our ed is located in a separate building from the ts, this meant or more minutes may be required for transit of units often released in less than minutes. the net effect was that providers would routinely order products to ensure they were at the bedside for patient arrival as a precaution, only to return them when not required. implementing a blood fridge at bedside resulted in the predicted outcome of delivering emergency release rbcs more quickly, with the observed benefit of decreasing wasted staff time. study design/method: the remote blood fridge was implemented in july . data for rbc requests in coolers, rbc returns and rbc transfusions from the ed was collected and compared. baseline data included january -june , and post change included august -december . july data was excluded as it included both the pre and post processes. results/finding: baseline data shows that the ed requested an average of rbc/month in coolers. post change this dropped to rbc/month, thus less blood was requested from the transfusion service in coolers as units were being used from the fridge. baseline data also shows that an average of rbc/month were returned ( %). post change, the average rbc/ month returned was ( %), this represents an absolute % reduction in number of returned products. each rbc dispensed and returned takes approximately minutes to complete paperwork and transport, therefore this change saved an average of minutes per month. it was also noted that the average rbc/month transfused was for baseline and post change. this confirms that the decreased requests and returns were not due to decreased patient volume or severity. the fridge was also successful at decreasing delivery time of blood to patient bedside, as baseline delivery time of - minutes (estimated) was reduced to - minutes. conclusion: implementing a remote blood fridge and moving blood access closer to patient bedside ensures a faster delivery of blood to the patient. this change has an additional benefit of decreasing wasted time, and hence cost, by decreasing unnecessary product requests and returns. implementing a blood fridge can also be done at a reduced cost through homegrown processes. transfusions are everyday procedures and over patent-applications have been filed related to "transfusion medicine" and over related to "transfusion alarm", during the last years, employing numerous technical settings, aiming to support automated supervision of the mentioned actions. the aim of this contribution is to present a developed low-cost real-time individual intravenous blood-transfusion monitoring system, based on the internet of things. study design/method: the designed system is based on a commercially available pan-tilt-zoom (ptz) camera, employing an / inch color cmos sensor, providing effectively . mp, a . mm lens, ir-cut, day/night minimum illumination . lux/f and viewing angle. the camera is focused on the droplets and acts as vis/ir detector with a hz sampling-rate. custom-developed software supports droplets' ratemonitoring, causing acoustic alarm-signals if necessary (e.g. clotting, blood a transfusion vol. supplement s abstract or other suspensions depletion etc.) and enables, if necessary, wide-angle image-capturing. the video image-audio settings provide for compression h. , video frame rate (fps) - /s, refresh rate hz and audio input, through bidirectional built-in microphone. the acquires an ip-address, the connection mode is wireless, the network interface is wi-fi/ . /b/g, the supported protocols include dhcp, tcp/ip, upnp, http, smtp and p p is provided. typical v power-supply, sized x x mm and weighing g. client software is required. the ir range is - m; ir-cut filters, remote access, dual stream, motion detection, day/night and ir night vision distance of m are offered. two-way radio-link is provided, as well as, trans-flash (tf) recording and storage on a gb sd-card. pan/tilt-horizontal o and pan/tilt-vertical o movements can be performed. the system facilitates, if needed, also patient's position monitoring and readings of other monitoring displays, such as nibp, ecg, and spo , if present. results/finding: the system and is being presently tested in a laboratory (non-clinical) environment, by simulating the virtual patient, with a custommade "phantom", combining flow-rate, negative pressure and viscosity resistance regulation. conclusion: the system can measure infusion-speed with a deviation lower than %. the developed iot-system takes advantage of the existing hospital wi-fi networked environment and offers a low-cost solution, under $ for each monitoring-set. it allows for even multi-platform (ios, android, windows) smart-phone, short-range connectivity, for up to participants, for example nurse, physician etc. two potential approaches for the quality control of bact/alertv r culture media using various bioball tm organism preparations patricia rule*, michelle keener and christine crawford. biomerieux inc. background/case studies: the bact/alertv r bpa and bpn culture bottles are used with the bact/alert microbial detection system for rapid screening and detection of microbial contamination in leukocyte reduced apheresis platelets (lrap). recent changes in the clia quality control guidelines and aabb accreditation program will require additional quality control of manufactures media that is both lot specific and shipment specific to ensure recovery of bacterial growth. a study was conducted using commercially prepared organisms evaluating both a comprehensive organism panel as well as a streamlined method utilizing only two organisms from the panel. study design/method: the general protocol consisted of three replicates each of each organism inoculated into two lots each of bpa and bpn by two different analyst. the study was two part in that aspergillus brasiliensis, candida albicans, bacillus subtilis subsp. spizizenii, pseudomonas aeruginosa, escherichia coli, clostridium sporogenes, staphylococcus aureus and streptococcus pyogenes were prepared from bioball singleshot ( cfu), multishot cfu or highdose k organism preparations at a low level (< cfu) and evaluated on the same day of preparation as method validation. the second part of the study utilized escherichia coli and staphylococcus aureus prepared and frozen at a higher level and then evaluted over a day study as a stream line approach to routine quality control testing of the bact/alert culture bottles. inoculation preparations were enumerated in duplicate to confirm the level at each inoculation time point. inoculated bpa and bpn bottles were loaded into the bact/alert microbial detection system at c for automatic monitoring of growth. negative bpa and bpn bottles were included in duplicate at each day of testing. results/finding: escherichia coli, staphylococcus aureus, streptoococcus pyogenes and bacillus subtilis subsp. spizizenii were positive in both the bpa and bpn culture bottles. the aerobic aspergillus brasiliensis, candida albicans, and pseudomonas aeruginosa grew and were reported positive in only the bpa aerobic culture bottle as expected. while the obligate anaerobe, clostridium sporogenes was positive only in the anaerobic bpn culture bottles. bacterial cultures were positive in the bact/alert bpa and bpn bottles < days and the fungal organisms in < days. the overall agreement was . % in bottles tested here. no significant differences were observed in the time to detection between the different lots or between the different analyst. conclusion: the bioball prepared organisms demonstrated a reproducible method as both a comprehensive and streamline approach for the quality control of bact/alert bpa and bpn culture media. the method was simple and did not require additional microbial preparations or storage of live organisms by the laboratory. use of an electronic patient identification system for blood banking specimen labeling found to be superior over historical armband approaches annie newton* , diane schafer , debra brown , jesse cox , scott koepsell and sara shunkwiler . nebraska medicine, the nebraska medical center, university of nebraska medical center background/case studies: anticipating the implementation of the new ( th addition) aabb standard concerning the confirmation of patient abo blood typing of type and screen (crossmatch) specimens performed prior to the issue of crossmatched blood products, laboratory and organizational leadership evaluated the practical application of an electronic patient identification system to label blood bank specimen collections versus the traditional use of blood bank armbands. continued use of the armbands would require a second sample for abo confirmation of patients that did not have a historical blood type on file. concern was raised regarding the amount of increased workload of staff and delayed results availability based on the number of increased specimens that would be generated, as well the potential for increased iatrogenic blood loss and patient dissatisfaction. moreover, nd sample collection alone would not improve the rate of mislabeled specimens observed, which is of supplementary concern. study design/method: current organization employment of an electronic patient identification system for the labeling of other laboratory specimen collections made it feasible for applying this technology to the blood bank as well. an in-depth evaluation, including a failure modes and effects analysis (fmea) spanning several days, was completed to ensure that the use of the electronic system would produce comparable or superior safety results to its armband counterpart. an alternate process for specimen labeling and abo confirmation (which would satisfy the new standard) was established to support care areas that did not have the capability of using the electronic system. extensive education was provided to all staff (physicians, advanced practice providers, phlebotomist and nurses) to ensure comprehension as well rational for the new process. alerts were congruently built into the electronic health record (ehr) to supplement any information regarding crossmatch testing expiration that may not be readily available by the elimination of the armband use. results/finding: within days of implementing the new process (september , ), there was a noticeable reduction in the amount of mislabeled blood bank specimens received, totaling in months post implementation compared to in the months prior. in addition, the vast majority of specimens received into the blood bank are henceforth collected and labeled using the electronic system and thus have reduced the amount of potential nd specimen collections needed for abo confirmation. conclusion: use of an electronic patient identification system for labeling blood bank specimen collections in lieu of traditional blood bank armbands has proven to improve patient safety and department efficiency by substantially reducing the occurrence of mislabeled specimens and negate the need for nd specimen collections, reducing potential iatrogenic blood loss and improving patient satisfaction. background/case studies: based on a few small randomised controlled trials (rcts) performed in the late ' s and in early , intravenous immunoglobulin (ivig) use has been suggested as a potential treatment to avoid exchange transfusion (et) for rh hemolytic disease of the newborn (hdn). this treatment modality is now routinely used for rh-hdn and has been extended to hdn caused by abo incompatibility or by other red blood cell antibodies. however, larger rcts performed since have shown that prophylactic ivig did not reduce the need for et, the duration of hyperbilirubinemia, the maximum bilirubin levels nor the need for top-up red blood cell transfusions. the primary objective of this study was to describe the usage of ivig for hdn at a tertiary academic referral hospital. study design/methods: a retrospective chart review was performed of all neonates who received ivig for hdn in the neonatal intensive care unit (nicu) from january , to june , . data collected included patient demographics features and diagnosis, indications for ivig, neonatal laboratory results, treatment details, adverse events and patient outcomes. results/findings: ninety-seven neonates received ivig during the study period: % were female and % were less than weeks of gestational age. none had co-existing g pd deficiency, pyruvate kinase deficiency or spherocytosis. all neonates received phototherapy prior to ivig treatment. indications for ivig were abo-hdn ( %) and rhesus-hdn ( %). antibodies most often implicated in rh-hdn were anti-d ( / ), anti-d and anti-c ( / ) and anti-c ( / ). sixteen infants with rh-hdn had received intrauterine transfusions. the mean cumulative dose of ivig was g/kg (range from , g/kg to , g/kg). neonates received one to four ivig administrations. table shows the number of patients receiving ivig during two time periods. three adverse reactions were noted during ivig administration: cutaneous rash, hypotension and fever. of all neonates, required an et for rh-hdn and for abo-hdn. forty-five ( %) patients needed top-up transfusions during hospitalisation and until three months of age: with abo-hdn and with rh-hdn. the mean number of transfusions was three (range: to ). conclusion: although initially described for rh-hdn, abo-hdn is now one of the most frequent indications for ivig in neonates. the optimal use of ivig in abo-hdn needs to be better characterized. our study shows a wide variation of ivig dosing and a significant proportion of neonates requiring top-up transfusions. further research is required to evaluate whether anemia in abo-hdn might be exacerbated by hemolysis from ivig isohemagglutinins and if it is dose-dependent. background/case studies: background: one of the most serious adverse reactions to transfusion is the development of graft versus host disease. symptoms include the development of a characteristic cutaneous rash, enteritis often resulting in watery diarrhea, elevated liver function tests and ultimately pancytopenia. the clinical course is rapid with an over % case fatality rate. the patient population at risk is reasonably well-defined including patients who are immunocompromised due to disease process or therapy, the fetus and low birth-weight neonates, recipients of hla-matched cellular blood products and the recipients of cellular blood products donated by blood relatives. the basic etiology of ta-gvhd is the inability of the transfusion recipient to mount an effective immune response against donor t-lymphocytes. treatment options for ta-gvhd are ineffective, making it imperative that cellular blood components be irradiated prior to transfusion which virtually eliminates the risk of the complication. study design/methods: most transfusion service information systems have mechanisms to alert transfusion service staff to patients who have been previously identified as needing irradiated blood components. however, if these patients are not identified to the transfusion service at the time of the initial hospital visit or the time at which the qualifying diagnosis, these patients can erroneously receive non-irradiated blood components. following a "near-miss" situation, our hospital information department developed a -part program to minimize the risk that the transfusion service is not notified of patients newly requiring irradiated blood components. results/findings: our blood products ordering system has been redesigned to include specific queries to identify those patients who required irradiated cellular blood products. first, physicians have been notified to include the need for blood product irradiation in the patient problems list. once this is included in the list, the transfusion service will be notified of the need for irradiation on all subsequent transfusion orders until the problem list is modified by the clinical staff. second, if irradiated blood components have ever been requested on a patient, an alert will be generated for the ordering physician even if the requirement for irradiated products has not been included in the problem list. finally our system will automatically default to request irradiation on all cellular products ordered for children less than months of age to comply with local irradiation policies. conclusion: we believe that our approach can be further enhanced by including a list of specific diagnoses typically requiring blood product irradiation within our computer algorithm. we believe that this list will provide an additional level of safety in insuring that patients receive irradiated blood components when appropriate. using lab information system and a dynamic dashboard for labeling and tracking coolers russell thorsen, rosaline ma, peter suslow, gina giannarelli, sara bakhtary, ashok nambiar and morvarid moayeri*. ucsf health background/case studies: our tertiary-care transfusion service routinely issues blood products in validated coolers to high acuity areas such as ors, icus, cath-lab, etc. coolers are also used for emergency release and massive transfusion protocols, and for shipping products between our different hospital sites. a robot that can hold one cooler delivers products to locations not served by the pneumatic tube. on average, coolers are issued every day. cooler set-up is a multi-step, labor-intensive process. transfusion service staff track cooler location and elapsed time-in-use and notify clinical teams to return/recharge coolers to avoid product wastage. we developed a lab information system (lis)-based solution to manage cooler labelling and tracking more efficiently. study design/method: nine cooler test batteries were built; the batteries for rbc, plasma, platelet and cryoprecipitate ( each) are identical, whereas the final battery designated for the cooler delivered by robot (containing plasma and rbcs with variable expiration times) is slightly different. the second battery in each pair was built to avoid duplicate test cancellation by lis when a second cooler (for same component type) is being set up for the same patient. each battery consists of tests capturing the following information: cooler location, cooler id, number of units issued, and expiration. custom barcodes representing each test battery and different locations can be scanned from a 'quick-pick list', avoiding need for manual entry. when coolers are returned, a final entry is made in the test battery, updating lis. a dynamic cooler tracking dashboard with live-feed from lis displays data captured in the test battery. elapsed time, starting from cooler set-up (which is identical to time cooler battery is ordered in lis) is captured automatically. color codes alert users to coolers that have less than hour before expiration. a flashing alert pops up for coolers that have expired. results/finding: we replaced our manual process (hand-write patient information and expiration time on separate tags; affix one tag to cooler and retain second one to track cooler location and expiration) with a novel lis-driven labeling and tracking system. each time a cooler is set up, a test battery is ordered and resulted in lis by scanning the related custom barcodes. a single lis-generated label is printed and attached to each cooler. cooler expiration is defaulted to hours (per our current cooler validation) from the time the test battery is ordered during cooler set up. techs pay attention to expiration of each product they place in a cooler. if an individual product outdates before the cooler expiration time, this information is entered in the test battery and gets displayed on the dashboard as a cooler expiration time, distinct from the system-driven countdown. color-coded visual display and alerts greatly simplify cooler monitoring, and the elimination of some manual steps has improved staff satisfaction. conclusion: using lis for cooler set-up and deploying a linked dynamic dashboard to display cooler locations and expiration time makes cooler management more efficient. these tools reduce manual steps and decrease likelihood of wastage by aiding cooler tracking. improving cryoprecipitate collection operations using operations research and analytics-based methods american red cross, georgia institute of technology ap reduction in unnecessary use of type o-negative rbcs in a level i bellevue hospital-nyulmc our hospital is a level i trauma center serving a diverse predominately non-caucasian population. historically we stocked our trauma blood bank monitored refrigerator with o-negative rbcs. trauma requested that we stock additional rbcs to be able to initiate a mtp for multiple patients at the same time. believing that most of our trauma patients are male, elderly, or rh-positive, we agreed add type o-positive rbcs to the stock. rules for determining which units to use were established. o-positive rbcs are to be given to a) all adult males (am), b) women of non-childbearing age (wncba), and c) if both o-negative rbcs were used but not yet restocked, and o-negative rbcs are to be given to a) women of childbearing age (wcba) and b) children until the patient's aborh type are determined. we sought to assess the impact of this change on our usage and purchases of o-negative rbcs. study design/method: all patients issued emergency release trauma rbcs following the addition of o-positive rbcs were assessed %) would have needed to be o-negative. the addition of o-positive rbcs to our trauma refrigerator will enable us to markedly reduce our purchases of o-negative rbcs. ap saving apheresis platelets through use of verax point of care testing jennifer rhamy* and rebecca wride . st. mary's regional blood donor center, st. mary's regional medical center background/case studies: our rural hospital-based blood center serves hospitals and a diverse patient population including acute trauma. because of the varying need for platelet products (varies between and per day in ), we investigated the use of the verax point-of-care test to better manage our valuable inventory barrett lawson and jun teruya , . texas children's hospital, baylor college of medicine ap vision titers --easier or problematic? (table ) . results/findings: post intercept, t had volumes of - ml, with % hemoglobin (hb) recovery. t had -fold less extracellular protein than c. after days of storage t had higher atp and na than c while lactate and hemolysis were lower. hct, ph, k and glucose were equivalent between t and c on d . d hemolysis for t was . - . %, while for c it was . - . %. t and c atp was > mmol/g hb, the level of atp associated with effective rbc viability, throughout storage (table ) . hematocrit (hct, %) . . * . . . . . . hemoglobin (g/unit) not measured hemolysis (%) . . * . . . . * . . ph ( c) . . * . . . . . . total atp (mmol/g hb) . . * . . . . * . . k (mm) . . * . . . total tested total plts issued feb mar totals table: . resident reports to the intranet "drop box" increased from . % to . % to %, each over month time spans. conclusion: safe transfusion ordering requires a team approach to ensure the right information is available to the ordering provider at the right time. safe ordering prevented recurrent allergic reactions in our patient population. the tso plays a pivotal role in ensuring the full circle of communication occurs. processes that integrated the pathology resident improved with pdsa cycles and impacted the quality and timeliness of hand off. finally, the data provided from the residents enabled efficient participation in hemovigilance. decreasing results/finding: the main root cause determined was that there was no standard work process. sops were being followed but there was no standard work process that included the details so testing was not following the most efficient work flow. counter measures implemented included implementing a standard work process, visual cues were added to the work process, and a samples awaiting testing report was created for the batch release department. specific locations were identified within the work cells in the lab to place samples based on their phase/stage of testing. after counter measures were implemented, the number of exceptions decreased from . per day or , dpmo to . per day or , dpmo. this is a statistically significant difference since the p-value calculated was . . conclusion: a lean six sigma approach for process improvement was utilized to identify root causes and develop countermeasures in order to decrease the number of exceptions related to the testing of whole blood samples in the laboratory. this approach and counter measures statistically significantly decreased the number of exceptions seen in the whole blood testing process. background/case studies: our blood bank processes approximately , specimens per month. since , the requirement of having a second blood type on record was met by: . utilizing the historical blood type and the current specimen, or . having second type performed on same specimen by different technologist, and . each type and screen specimen signed by staff, one being a licensed practitioner attesting the identification of the patient was done accurately at bedside.to comply with the aabb standards th edition, # . . . a decision was made to change our practices. we considered challenges encountered at other hospitals and collaborated with nursing and it to create a streamlined and safe process. in april , the second specimen procedure was implemented addressing the following: ii. extensive education was provided to all involved in the process prior to implementation including a learning module prepared by blood bank and nursing collaboratively. results/finding: . there was a minor adjustment period with more phone calls made to blood bank to explain the process. . there was minimal impact on turn around times for release of components. . aborh retype workload decreased from to ( % to % of t&s volume) per month. . unnecessary blood draws minimized, improving patient experience. . no emergency release requests due to absence of a second specimen. the second specimen process with the conditional order has been beneficial to our blood bank as well as patient care services. overall feedback from staff on the process has been positive. our workload has decreased which results in cost savings and increased efficiency allowing us to devote more resources to the growing services at our institution. background/case studies: the hazards of transfusion are well recognized and in certain cases restrictive transfusion strategies compared to liberal transfusion strategies may be associated with better clinical outcome. with this in mind, aabb and others published guidelines for transfusion, but even with guidelines in place, rate of inappropriate blood transfusions is reported to be as high as to %. computerized provider order entry (cpoe), is a process of electronic medical order entry for medical practitioners with instructions and guidelines for treatment. the objective of this study was determination of transfusion practice quality by thorough chart and electronic medical record review, with measures in place to avoid inappropriate transfusion. additionally, factors associated with inappropriate transfusion were examined. study design/method: in our bed hospital, a retrospective chart review was performed ( / / - / / ) on hospitalized internal medicine patients. cpoe with hospital guidelines for rbc transfusions were in place. transfusion thresholds in different clinical settings were determined by a thorough literature review of studies analyzing restrictive transfusion strategy, and transfusion guidelines by various medical societies. charts for background/case studies: our midwestern university-based transfusion service (ts) evaluated the appropriateness the automated platform vision (ortho clinical diagnostics. raritan, nj) for prenatal titration studies. it has been established from previous publications that the micro-column assay, of which the vision is based, may lead to higher titer results compared to standard tube titrations. this study sought to evaluate the transition from manual to automated titer studies from a sensitivity as well as cost perspective. study design/method: twenty-three prenatal retention plasma samples were tested as part of the evaluation of titration studies of the vision. the samples were manually tested with a standard two-fold serial dilution. the titer was reported as the last tube to demonstrate a reaction by macroscopic observation. the titer studies were then repeated using the vision. the results of the manual and automated processes were compared and categorized as "< grade" or "> grade" difference between endpoints. this analysis is similar to the acceptable ranges used for evaluating college of american pathologists (cap) proficiency survey challenges. a cost analysis was completed based on the direct and indirect cost for each method, excluding the cost of an analyzer. results/finding: table demonstrates a summary of the samples tested by manual titer study and vision titration method. the vision titer results (mean, median, and mode) were higher than the manual tube titer results. less than half of the samples ( %) were > titer results higher, while the majority was titer results different ( %). the cost analysis is summarized in table . the indirect cost (labor) was significantly lower with the use of the vision. the reduction in pre-analytical technical time for manual preparation of the titration is eliminated with the vision completing the titration as part of the profile of the titer study of the analyzer. conclusion: with an estimated % decrease in the cost of a vision titer compared to manual tube method, the change in practice would clearly be a cost and efficiency measure in the blood bank. however, the vision demonstrated the expected increase in titer results compared to manual tube titer results. this would impact the critical values currently utilized. an impact assessment for clinical staff would be necessary to adequately implement the change in method. consideration must be given to changes in the computer logic for critical values on titer studies and training of physician and nurse obstetric practitioners for changes in the critical values. in addition, as part of changing to the vision an implementation period will be necessary to ensure that manual titers are compared to previous manual titers and not to vision titer results which would be higher and may be interpreted as a significant change for clinical care of the patient. what is the best practice for testing residual white blood cells in blood components for monthly routine quality control? janja pajk*. general hospital celje background/case studies: we wanted to discover what is the best routine quality control practice for testing residual white blood cells in blood components. our aim was to validate the adam device for counting thne number of residual white blood cells (wbc) in leucocyte depleted and in non-leucocyte depleted blood components (bc) and to compare with standard counting method by microscopy in fuchs rosenthal chamber (frc) used in ghc and with flow cytometry (fc). study design/method: after samples of red blood cells (rbc), platelets (plt) and fresh frozen plasma (ffp) (leucocyte depleted in top and top (t/ t) bags and non-leucocyte depleted in top and bottom (t/b) bags) were stained with propidium iodide (pi) on r-slides; adam -rwbc device was messured fluorescent images of stained wbc nucleus. data were analised by image analysis software and later compared with results of testing samples in frc by microscopy and with fc. samples of bc were microscopic tested in frc at department of laboratory medicine in ghc; another samples were measured with fc in ucc maribor. results/finding: samples ( rbc, plt, ffp-all leucocyte depleted and non-leucocyte depleted ffp) were tested in triplicates on adam and with frc once.coefficient of variation of (kv%) of samples measured on adam for leucocyte depleted bc varied for: rbc from , - , ; plt from , - , ; ffp from , - , ; and for non-leucocyte depleted ffp from , - , (table ) . samples ( rbc, plt, ffp -all leucocyte depleted and nonleucocyte depleted ffp) were tested in triplicates on adam and with fc once.kv% of samples measured on adam for leucocyte depleted bc varied for: rbc from , ; plt from , , ffp from , ; and for non-leucocyte depleted ffp from , - , (table ) .high percentage of kv was noticed in samples with low numbers of wbc (in leucocyte depleted bc; low percentage of kv in non-leucocyte depleted ffp, with higher amount of wbc was observed. conclusion: all samples tested with adam met expected criteria for wbc in bc in european union (less than x /unit for leucocyte depleted or x / unit for non-leucocyte depleted) and were comparable with those tested with fc; the correlation with microscopy in frc was worse.with use of disposable r-slides, the risk of exposure to the potential hazardous blood samples is grately reduced, the method is more precise and not time consuming.from january we changed our protocol for testing residual wbc in bc with adam device and we advise it as the best practice for monthly routine quality control.